Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate

27

Biochimica et Biophysica Acta, 1179 (1993) 27-75 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93/$06.00

BBAMCR 13462

Review

Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? L.R. Stephens, T.R. Jackson and P.T. Hawkins Department of Development and Signalling, AFRC Babraham Institute, Babraham, Cambridge (UK) (Received 16 March 1993) (Revised manuscript received 25 May 1993)

Key words: Phosphatidylinositol(3,4,5)-triphosphate; Intracellular signalling; Inositol lipid; PI-3 kinase; Growth factor; Protein tyrosine kinase; G protein

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Discovery of P13K and its translocation to activated protein tyrosine kinases . . . . . . . . . . . 2. Discovery of Ptdlns(3,4,5)P3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28 28 28 28 30

II.

The identity of the agonist-sensitive reaction in 3-phosphorylated inositol lipid metabolism . . . . . A. Metabolic inter-relationships between the 3-phosphorylated inositol lipids . . . . . . . . . . . . . . . 1. Hydrolysis of 3-phosphorylated inositol lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Synthesis of 3-phosphorylated inositol lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Synthesis of 3-phosphorylated inositol lipids in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Synthesis of 3-phosphorylated inositol lipids in intact and permeabilized cells . . . . . . . . B. The origin of PtdIns(3,4,5)P 3 in agonist-activated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The origin of PtdIns(3,4)P 2 in agonist-activated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The pattern of agonist-stimulated 3-phosphorylated inositol lipid metabolism . . . . . . . . . . . . . E. The origin of PtdIns3Pin cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30 31 31 34 34 35 35 35 36 37

III.

Receptor-coupling to increases in Ptdlns(4,5)P 2 3OH-kinase activity . . . . . . . . . . . . . . . . . . . . . A. How closely coupled is the activation of Ptdlns(4,5)P2 3OH-kinase activity to the activation of receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Implication of PTK activities in the regulation of Ptdlns(3,4,5)P 3 accumulation . . . . . . . . . 2. Implication of G proteins in receptor-stimulated accumulation of Ptdlns(3,4,5)P 3 . . . . . . . . B. Characterization of enzymes potentially responsible for Ptdlns(4,5)P 2 3OH-kinase activity . . . 1. Characterization of a PI3K activity which can transiocate to activated PTKs . . . . . . . . . . . . 2. Characterization of a Ptdlns(4,5)P2 3OH-kinase activity that might be regulated by G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37 37 38 39 40 40

How do agonists 'activate' Ptdlns(4,5)P 2 3OH-kinase activities . . . . . . . . . . . . . . . . . . . . . . . . . . A. Interactions between Ptdlns(4,5)P 2 3OH-kinase activities and receptor-regulated PTKs . . . . . B. Interactions between receptor-PTKs and PI3K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The role of tyrosine phosphorylation in the regulation of PI3K . . . . . . . . . . . . . . . . . . . . . C. Interactions between src-family PTKs and PI3K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Transforming src-type gFK-mediated accumulation of Ptdlns(3,4,5)P 3 . . . . . . . . . . . . . . . 2. Middle T antigen: c-src-PTK complex-mediated accumulation of Ptdlns(3,4,5)P 3 . . . . . . . .

44 45 46 47 49 50 51

IV.

41

Correspondence to: L.R. Stephens, Department of Development and Signalling, A F R C Babraham Institute, Babraham, Cambridge CB2 4AT, UK.

28

V.

3. Receptor-stimulated src-type PTK-mediated accumulation of Ptdlns(3,4,5)P 3 . . . . . . . . . . 4. Receptor-stimulated, G-protein and src-type PTK-mediated accumulation of PtdIns(3,4,5)P3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interaction between Ptdlns(4,5)P z 3OH-kinase activities and G proteins . . . . . . . . . . . . . . . .

52

How do agonists integrate Ptdlns(3,4,5)P3-encoded messages into their signalling repertoires? . . . A. Receptor and tissue specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Receptor-specific and tissue-specific engagement of signalling pathways by PTK-coordinated mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Receptor-specific activation of PI3K by PTK-coordinated pathways: the concept of a consensus sequence for PI3K binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Tissue specificity in the activation of PI3K by PTK-coordinated pathways . . . . . . . . . . . . . C. Specificity of signalling via G proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54 54

52 52

54 55 58 59

VI. Physiological function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Ptdlns(3,4,5)P3: an intracellular messenger? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Potential targets of Ptdlns(3,4,5)P 3 action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60 60 62

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. The assay of PI3K in vitro with exogenous lipid substrates . . . . . . . . . . . . . . . . . . . . . . . . . II. The origin of Ptdlns(3,4)P z in agonist-activated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The source of Ptdlns3Pin cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. 'Activation' of soluble lipid-metabolizing enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Ptdlns(4,5)P2 as a substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Nomenclature of signalling cascades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. SH2 domain:tyrosine phosphate interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 65 66 67 67 68 69 69

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70

I. Introduction I-A. Aims This article reviews research which addresses the metabolism and functions of a recently discovered family of m e m b r a n e phospholipids, the 3-phosphorylated phosphoinositides. It is possible to read the main body of text with minimal reference to the figure legends or appendices. These are provided, in many instances, to allow m o r e comprehensive justification or elaboration of certain points in the main text.

terized signalling pathway based on inositol phospholipids, in which an agonist-sensitive phosphoinositidase C (PIC) hydrolyses Ptdlns(4,5)Pz to release the 'seco n d - m e s s e n g e r s ' inositol (1,4,5)-trisphosphate and diacylglycerol (Ins(1,4,5)P 3 and D G ) [3,4], and is likely to be used by a large n u m b e r of diversely structured cell-surface receptors (including examples activated by cytokines, growth factors, small h o r m o n e s and antigens [1,2,5]. This section briefly reviews the origins of this idea and aims to provide a perspective for the discussion of m o r e recent work which has b e e n designed largely to address this concept.

LB. Background

I-B.1. Discovery of PI3K and its translocation to activated protein tyrosine kinases

T h e term 3-phosphorylated phosphoinositides (or 3-phosphorylated inositol lipids) is used to refer to a family of three lipids, PtdIns3P, PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 (their structures are described in Fig. 1). These lipids are f o u n d in all higher eukaryotic cells in which they have b e e n sought and are believed to be the in vitro products of an enzyme we will call phosphoinositide 3OH-kinase (PI3K) [1,2]. T h e available evidence suggests that both P I 3 K and the 3-phosphorylated inositol lipids are c o m p o n e n t s of a new intracellular signalling system, responsible for conveying information from activated cell-surface receptors to specific intracellular targets. This system appears to be i n d e p e n d e n t o f the previously charac-

T h e origin of the idea that P I 3 K may be involved in cell signalling is e m b e d d e d in the m a n n e r in which the enzyme was first discovered. P I 3 K was initially characterized as a PtdIns kinase activity (i.e., an activity that could phosphorylate PtdIns) which could be extracted from cells tightly attached to the virally e n c o d e d protein tyrosine kinases (PTKs) v-src or v-ros [6,7]. This PtdIns kinase was subsequently shown to be different from the P t d I n s 4OH-kinase(s) previously characterized in cells and to catalyze the 3-phosphorylation of purified PtdIns, P t d I n s 4 P and PtdIns(4,5)P 2 in vitro [8-10] (see Fig. 2; hence its name, 'phosphoinositide 3OH-kinase'). T h e relationship b e t w e e n P I 3 K and P T K s was clari-

29 that PI3K plays a relatively ubiquitous role in the cellular functions of these proteins. In view of the accepted critical role for PTKs in the control of mitogenesis, witnessed by the number of oncogenes identified as constitutively active forms of cellular PTKs, the potential implications of these discoveries for our understanding of the signalling pathways controlling cell growth and division were obvious and naturally moulded much of the subsequent development of this field around a molecular characterization of a PI3K activity that could translocate to activated PTKs. This work has culminated recently in the purification and cloning of a major PI3K activity in cells and the construction of a plausible mechanism by which it can translocate to activated PTKs (discussed in Sections III-B.1 and IV-B). During the progress of this work it became apparent that PI3K is a member of a family of activated PTK-associating proteins (which include PICT, ras-GAP, and GRB2/SEM5) which possess certain common structural features and are believed to generate the signals that ultimately elicit the effects of

fled by findings that it could also be recovered from cells attached to a variety of normal cellular PTKs, including c-src and the activity intrinsic to the PDGF receptor, but only if they were 'activated' in an appropriate manner, e.g., in the case of c-src, by transforming cells with polyoma virus middle T antigen (mT) [11] or, in the case of the PDGF receptor [9,12], by stimulating cells with its ligand. Thus, these studies served to define a PI3K activity which did not appear to be involved in 'conventional' inositol lipid metabolism (by virtue of the reactions it catalyzes, i.e., phosphorylation in the 3-position; see Fig. 3) but which could physically translocate to various types of activated PTK, including both receptor and src-type families [13]. We now know that many different growth factor receptors with intrinsic PTKs are able to gather PI3K in a ligand-dependent fashion (e.g., the receptors for insulin, EGF and CSF-1) and that a number of activated forms of src-type PTKs can be found physically associated with PI3K in ceils (e.g., v-src, v-yes and receptor-activated lyn, fyn and/ck) [1,5]. This suggests

< < < < < <

< < < <

o']=o

< = 0"'~= 0

--

0

0

o

o

0

0

0

i

i

I

f

P

J

I

Ptdlns(3,4,5)P 3

I

Ptdlns(3,4)P 2

I

Ptdlns3P

Fig. 1. The structure of the 3-phosphorylated phosphoinositides. Schematic representations of D-phosphatidylinositol 3-phosphate (Ptdlns3P or 'PI-3P'); b-phosphatidylinositol (3,4)-bisphosphate (Ptdlns(3,4)P 2 or 'PI-34P2') and D-phosphatidylinositol(3,4,5)-trisphosphate (Ptdlns(3,4,5)P3, 'PI-345P3' or 'PIP3') are shown (all known, naturally-occurring phosphoinositides belong to the D-series of enantiomers; i.e., their phosphodiester phosphates are analogous to that in D-Insl P). The structures of the water soluble head groups of the 3-phosphorylated inositol lipids have been assigned by processes based on the sequential application of selective enzymatic or chemical degradations combined with high resolution chromatographic identification of the products formed at each stage. This type of analysis has been used to rigorously characterize 3-phosphorylated inositol lipids present in various mammalian cells: astroc3rtoma cells, FMLP-stimulated human neutrophils, PDGF-stimulated mouse 3T3 cells and thrombin-stimulated human platelets [16,17,19,30]. In other cells, identifications of 3-phosphorylated inositol lipids, or their water-soluble deacylation products, have been based on evidence of co-chromatography with 'standards' a n d / o r the fact that their levels have been shown to rise dramatically upon appropriate stimulation. Some evidence suggests the fatty-acid moieties of the 3-phosphorylated inositol lipids in rat brain are similar to those of Ptdlns, Ptdlns4P and Ptdlns(4,5)P 2 [276]. The structures of the water soluble deacylation products of Ptdlns3P and Ptdlns(3,4)P 2 have been rigorously characterised in Spirodela (a duckweed) [177,278] and that of Ptdlns3P in S. cerevisiae (a budding yeast; where Ptdlns3P constituted 40-60% of the total PtdlnsP pool) [242]. Further, compounds chromatographically similar to the water-soluble deacylation products of Ptdlns3P and Ptdlns(3,4)P 2 have been identified in Chlamydomonas (a protozoan algae) [279], and to that of Ptdlns3P in Dictyostelium (a slime mould) (L.S., unpublished data). Despite substantial efforts, Ptdlns(3,4)P 2 or Ptdlns(3,4,5)P 3 have not yet been detected in Dictyostelium or S. cerevisiae; it is not yet clear, however, if this has resulted from their absence in these cells, their inefficient extraction, or the lack of appropriately activating stimulation.

30 the activated PTK. These analogies with known signalling-related proteins indicated that PI3K may play a role in the production of cell surface, receptor-triggered intracellular messages.

I-B.2. Discovery of Ptdlns(3,4,5)P3 The discovery of PtdIns(3,4,5)P3 in cells was independent of work proceeding in parallel characterizing a PI3K which could translocate to active PTKs. It was first noticed in human neutrophils as a uniquely polar phospholipid which appeared very rapidly upon stimulation with the chemotactic peptide FMLP [14]. The structure of this lipid was postulated, on the basis of the chromatographic properties of its head-group, to be PtdIns(3,4,5)P3 [14,15] (this structure has since been confirmed by an unambiguous assignment of the position of the mono and diester phosphate groups on the inositol ring [16,17]). These exciting observations were quickly followed by reports that the levels of a similar lipid, thought (and subsequently established) to be

A

Ptdlns -----=-Ptdlns3P

B

Ptdlns4P

C

Ptdlns(4,5)P2

--- Ptdlns(3,4)P 2 = Fffdlns(3,4,5)P3

Fig. 2. Pbosphoinositide 3OH-kinase activities in vitro. The reactions

A, B and C shown above can be catalysed in vitro by an enzyme which we shall refer to as phosphoinositide 3OH-kinase (PI3K). It utilises the y-phosphate of ATP to phosphorylate all of its known potential physiological substrates (i.e., D-series phosphoinositides) on the 3OH-moiety of their inositol rings [8,10]. However, PI3K will phosphorylate L-PtdIns (i.e., where the myo-inositol ring is linked to the diester phosphate via its D-3 position), uniquely amongst the PtdIns kinases that have been tested, on the D-5 and D-6 hydroxyl moieties [280]. A PtdIns kinase activity which could translocate to activated PTKs was shown to be different from previously characterized Ptdlns 4OH-kinase activities (see Fig. 3 for a description of the assumed role of Ptdlns 4OH-kinase in 'conventional inositol lipid metabolism') on the basis that it was resistant to inhibition by adenosine and sensitive to inhibition by supra-micellar concentrations of non-ionic detergents; this activity was called 'type I PtdIns kinase' (and PtdIns 4OH-kinases, types II and III) [13]. Type I PtdIns kinase was subsequently shown to catalyze the phosphorylation of Ptdlns in the 3-position and hence became known as 'PtdIns 3OHkinase' (or PI-3-kinase) [8]. Further work suggested that this activity could also phosphorylate PtdIns4P and PtdIns(4,5)P2 in the 3-position and this has been confirmed by the recent purification of a 'PtdIns 3OH-kinase' to homogeneity which can readily synthesize PtdIns3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 from PtdIns, PtdIns4P and PtdIns(4,5)P2, respectively [10,35,34]. Thus, the combined 'substrate-flexibility' and 3OH specificity of 'PtdIns 3OH-kinase' has lead to the perhaps more appropriate current label of phosphoinositide 3OH-kinase (PI3K; the use of the abbreviation 'PI' in this way is consistent with the use of the abbreviation 'PIC' for phosphoinositidase C). This name is meant to focus on the fact any of the three major phosphoinositides can serve as substrates and specifically avoids the idea of the enzyme being a PtdIns3K (i.e., only uses PtdIns as a substrate).

PtdIns(3,4)P2, also rose in these same situations [1517]. At about the same time as PtdIns(3,4,5)P 3 was discovered in FMLP-stimulated neutrophils, it was realized that a PI3K activity was present in cells which could translocate on agonist stimulation to a receptorPTK and which could also synthesize Ptdlns(3,4,5)P3, and PtdIns(3,4)Pz-like lipids in vitro. The coincidence of 3-phosphorylation of inositol lipids seemed to demand that these discoveries were related and, indeed, it was shown that PDGF could stimulate both the translocation of a PI3K activity to its receptor and the rapid appearance of lipids with the chromatographic properties of PtdIns(3,4,5)P 3 and PtdIns(3,4)P2 in the same target cell [10]. A number of studies have now correlated agoniststimulated translocation of PI3K activities to PTKs with the stimulated accumulation of 3-phosphorylated inositol lipids in various cells (discussed in Section III and Tables II and III). In addition, a number of agonists may be able to activate accumulation of 3phosphorylated phosphoinositides without causing an apparent 'translocation' of PI3K activities (FMLP may be a member of this group; discussed in Section III and Table IV). The pattern of changes elicited in the levels of 3-phosphorylated inositol lipids during all these forms of stimulation is remarkably similar across a wide range of agonists, receptor-types and cell-types; PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 accumulate rapidly on stimulation from negligible control levels and there is little change in the substantial control levels of PtdIns3P (see Fig. 4 and Table I). This pattern is not easy to reconcile with the apparent substrate specificity expressed by PI3K in vitro (where PtdIns is an effective substrate: this issue is addressed in detail in Section II). The distinctive manner in which some agonists can stimulate both translocation of PI3K to PTKs and the rapid accumulation of phospholipids that are potential products of PI3K activities, has convinced many workers in the field that PI3K may represent a new and independent signalling enzyme and that its 3-phosphorylated lipid products are its most apparent (although not necessarily the only, see Section VI) potential messengers [1,2]. The following sections of the review concentrate on addressing a number of issues which are central to this idea. II. The identity of the agonist-sensitive reaction in 3-phosphorylated inositol lipid metabolism The metabolic map describing the relationship between PtdIns, PtdIns4P and PtdIns(4,5)P2 in intact cells was defined in the early 1960s by Ballou and co-workers (A and B in Fig. 3 [18]). However, until recently, the analogous relationships between these lipids and the 3-phosphorylated inositol lipids (first described in cells in 1989 [19]) were still uncharacter-

31 ized, such that any of the alternative patterns depicted in Fig. 3 were possible and the source(s) of the PtdIns(3,4)P 2 and Ptdlns(3,4,5)P 3 that accumulated in agonist-stimulated cells was also unknown. Several enzymes capable of synthesizing 3-phosphorylated inositol lipids in vitro have been described (see below). One of these enzymes, PI3K, has already been mentioned and clearly possesses characteristics which are very persuasive of its role in at least some forms of agonist-stimulation (i.e., those where the agonists stimulate PTKs; see above). Despite this knowledge however, the precise reactions which these enzymes catalyze in vivo are still far from clear. The reason for this uncertainty is that the substrate specificities displayed by enzyme activities in vitro do not correlate with those of lipid metabolizing activities predicted to operate in intact cells, and probably result from the problems associated with appropriately reconstituting a supply of lipid substrate like that which exists in the cell (discussed below). This has formed the basis of one of the central issues in 3-phosphorylated inositol lipid metabolism, that of the identity of the primary agonist-sensitive reaction in the intact cell and its relationship to the 3-phosphorylated inositol lipid synthesizing activities defined in vitro. The following section considers work aimed at this question. The problem can be seen at a number of different levels; firstly, in the rigorous definition of the structures of the lipids that are present in cells (although some of their levels in unstimulated cells are low) - this amounts to a description of the list of potentially implicated metabolites (see Figs. 1 and 3); secondly, characterization of the metabolic

pathways in intact cells which interconnect these metabolites and thirdly, the identification of the agonist-sensitive reactions in vivo and the critical, potentially signal-generating enzymes responsible for catalyzing them.

II-A. Metabolic inter-relationships between the 3-phosphorylated inositol lipids II-A. 1. Hydrolysis of 3-phosphorylated inositol lipids The speed with which agonist driven accumulations of Ptdlns(3,4,5)P 3 and Ptdlns(3,4)P 2 can be cleared from cells, even in the continued presence of agonists (e.g., FMLP-stimulated neutrophils, see Fig. 4A [14,15]; or EGF-stimulated 3T3 cells and PC 12 cells [20,21]), suggests the rates at which these lipids can be metabolized are substantial. Not only does this rapid removal of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 support the notion they might represent signals, but it also raises the possibility that their degradative reactions, if agonistsensitive, might also have a role in causing their accumulation (see below). Precedent set by the metabolism of Ptdlns(4,5)P 2 and Ptdlns4P suggests hydrolysis by either PIC or phosphomonoesterase catalyzed routes could be responsible for the majority of the further metabolism of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3. Indeed, the fact that the head groups possess within them the potential to release Ins(1,3,4)P 3 or Ins(1,3,4,5)P4, respectively (i.e., inositol phosphates produced by activation of the PICbased signalling pathway; see Fig. 3), immediately prompted speculation that a PIC would account for • Ins(1,3,4)P 3

Inositol ",=- - - Inositol phosphates /

/

Ins(1,3,4,5)P 4 X\

/

Ins(1,4,5)P 3

/

t Ptdlns ~ P t d l n s 4 P

,\

~

Ptdlns(4,5)P2

/PiC = Diacylglycerol

X\

Ptdlns3P - - - ' "

Ptdlns(3,4)P 2

" Ptdlns(3,4,5)P

otential utes of pho6phorylated inositol lipid wmetabolism.

~

Fig. 3. The metabolism of 3-phosphorylated inositol lipids. The potential metabolic relationships between the polyphosphoinositides currently identified in mammalian cells. A 'skeleton picture' of the well-established phosphoinositidase C (often abbreviated PIC or PLC)-signalling system (which generates the 'second-messengers' Ins(1,4,5)P3, Ins(1,3,4,5)P4 and diacylglycerol on agonist stimulation [3,197]) is included to give a perspective with which to view the metabolism of the more recently discovered 3-phosphorylated inositol lipids and, in particular, to draw out the notion that phosphorylation of inositol lipids in the 3-position appears to define a 'separate category' of inositol lipid metabolism. The phosphomonoesterase and kinase mediated metabolism of PtdIns4P and PtdIns(4,5)P 2 (A and B) was originally described and established to operate in vivo by BaUou and co-workers (and has been reviewed [210,281]). The remaining reactions simply classify the possible interconversions between the lipids that have been rigorously established to be present in cells (see Fig. 1).

32 TABLE I The concentrations o f the polyphosphoinositides in control and stimulated human neutrophils

The concentrations of the polyphosphoinositides in human neutrophils were determined before and after 10 s in the presence of a maximal effective dose of FMLP [16]. Total concentrations have been corrected to give estimates of the local concentrations of the lipids in the plasma membrane. An estimate of the concentration of Ins(1,4,5)P 3 in the cytosol is also given to act as a comparison for these values. Lipid or inositol phosphate

Theoretical concentration at the inner leaflet of the plasma membrane Basal

10 s, FMLP

Ptdlns(4,5)P 2 Ptdlns4P Ptdlns3P Ptdlns(3,4)P 2 PtdIns(3,4,5)P 3 Ins(1,4,5)P 3

5 mM 3 mM 150-200/zM 10-20 IxM 5/zM 1/zM (in cytosol)

3.5 mM 2 mM 150-200 # M 100-200 # M 200 tzM 10/xM (in cytosol)

their degradation. However, estimates of the rates of accumulation of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 in thrombin-stimulated platelets suggest they could make, at most, an apparently trivial contribution to the formation of 3-phosphorylated inositol phosphates in this cell [22]. Moreover, there are now a number of examples where agonist-stimulated accumulation of PtdIns(3,4)P 2 and Ptdlns(3,4,5)P 3 is not accompanied by a parallel activation of PIC (e.g., stimulations by insulin,

GM-CSF or CSF-1 [23-26]). Finally, neither 3T3 cell or neutrophil lysates, nor purified preparations of the major species of currently characterized PIC can cleave any of the 3-phosphorylated inositol lipids to yield inositol phosphates [16,27,28]. Taken together these observations indicate that rapid metabolism of PtdIns(3,4)P2 and Ptdlns(3,4,5)P 3 by a PIC is unlikely [1,29]. In vivo studies of the rates and routes of hydrolysis of the 3-phosphorylated inositol lipids by putative phosphomonoesterase activities are rendered very difficult by the high rate of turnover of PtdIns(3,4)P2 and Ptdlns(3,4,5)P 3, the problems in establishing viable radioisotope-based pulse-chase experiments, and the lack of specific inhibitors of either steps in the pathway or of the receptors that drive it. As a consequence, nearly all of the data assessing the degradative pathways to which 3-phosphorylated inositol lipids are subject is based on in vitro assays with radioactively labelled exogenous substrates (see Fig. 5). However carefully these assays may be constructed, they will represent distortions of the intracellular environment in which the endogenous counter parts of the radioactive substrates would be degraded (the problems associated with in vitro assays using exogenous lipid substrates are discussed later in Appendix I). Hence data of this type should be considered as qualitative and supportive, rather than quantitative or definitive, evidence that a pathway operates in intact cells. With these corollaries in mind, PtdIns(3,4,5)P3 has been shown to be metabo-

TABLE II Agents which couple to PI3K stimulation and~or Ptdlns(3,4,5)P3 accumulation through activation of receptor-PTKs

The various agents listed are all thought to act via tyrosine kinase containing receptors (receptor-PTKs) and have been demonstrated to stimulate accumulation of Ptdlns(3,4,5)P 3 in intact cells a n d / o r increases in PI3K activity recovered in antiphosphotyrosine, or anti-receptor, antibody-directed immunoprecipitates. Abbreviations: PDGF, platelet-derived growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; CSF-1/M-CSF, colony stimulating factor 1/macrophage colony stimulating factor; SL/SCF, steel ligand/stem cell factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; NGF, nerve growth factor. The p85a and p85/3 subunits of PI3K can bind to and be tyrosine-phosphorylated by the product of the neu oncogene (erb-B-2, recently identified as a receptor for heregulin) when these proteins are co-expressed in a baculo virus/insect cell expression system [70], though no data has yet been presented on the association of PI3K with this receptor in intact mammalian cells. Agent

Receptor-PTK

Increase in

PI3K activity associated with

PtdIns(3'4'5)P3 and/or Ptdlns(3'4)P2 in intact cells

antiphosphotyrosine antibody imm.ppts

anti-receptor antibody imm.ppts + +

PDGF (AA,AB,BB) EGF FGF CSF-1/M-CSF SL/SCF HGF/scatter factor Insulin

PDGF-R (a,fl) EGF-R FGF-R CSF-R (c-fins) SL/SCF-R (c-kit) HGF-R (met) Insulin-R

+ + + +

+ + + +

+

+

IGF-1 NGF

IGF-R-type 1 NGF-R-c-trk

+ +

+ +

+ + + + IRS-1 + + gpl40 trk (?)

Reference

[10,12,243] [20,119,244,245] [20] [26] [246,247] [248] [23,24,122] [20,249,261] [21,125,126]

33 lized rapidly in lysates of either 3T3 cells [30] or neutrophils [16] by a predominantly membrane-associated Ptdlns(3,4,5)P 3 5-phosphatase (that in neutrophils is EDTA-resistant and hence distinct from the membrane-associated Ptdlns(4,5)P a 5-phosphatase also present in these cells) and possibly by a soluble, vanadatesensitive, inositol phospholipid 3-phosphate phosphatase. Similar studies of the metabolism of PtdIns(3,4)P 2 in neutrophil lysates indicate it is metabolized by a highly active, soluble, inositol lipid 3-phosphate phosphatase to yield Ptdlns4P and a 4-phosphatase activity that generates Ptdlns3P(the relative fluxes through the 3- and 4-phosphatase activities are not yet clear [16]). Ptdlns3Pcan be degraded by a soluble inositol lipid 3-phosphate phosphatase which is similar to the activities against Ptdlns(3,4)P a and PtdIns(3,4,5)P 3 described above [31,32].

A.

Human Neutrophils

AOoel=

• Ptdlns(3,4,5)Pa O • Ptdlns(3,4)P= []

8 -I

--iN.--

g.

0 3T3 Cells 1.2

x

--

--

~

10

.

.

.

.

.

.

20

30

Time (sees) •

09.

•

#

A

•

"' '7~ " ~ &-

:

•

f.z/* 6-~--~-~-~

0

---i

. . . . . . .

~

I

I

i

0

1

2 Time (min)

-

-

~:~---

I

i

3

4

Fig. 4. FMLP- and PDGF-stimulated accumulation of 3-phosphorylated inositol lipids in human neutrophils and 3T3 cells. The rapid accumulation of Ptdlns(3,4)P2 and Ptdlns(3,4,5)P 3 in FMLP-stimulated human neutrophils (A) and in PDGF-stimulated 3T3 cells (B) (for further related data see Refs. 16,30). In neither case do the levels of Ptdlns3P change dramatically. These two examples have been chosen to illustrate the qualitatively identical pattern of changes in 3-phosphorylated inositol lipids which occur during the stimulation of very different cell types with agonists utilizing very different receptor-mechanisms.

AGONIST-STIMULATED ACCUMULATION

AGONIST-SENSITIVE DEGRADATION Ptdlns(3,4,5)P3

:§:k";, Ptdlns(3 4)P2 .....

,,

Ptdlns(4,5)p2 ~

=

Ptdlns3P

Ptdlns4P

Ins(1,4,5)Pa DAG

;Nc " , .....

'"

Ins(1,4)P2 DAG

Ptdlns Fig. 5. Hydrolysis of inositol phospholipids. Agonists can stimulate accumulation of Ptd]ns(3,4,5)P3 and Ptd]ns(3,4)P 2. These molecules, unlike Ptd]ns(4,5)P 2 and Ptd]ns4P, do not appear to be substrates of a phospholipase C [27,28] but are rapidly degraded by pbosphomonoesterase activities (X-Pase). The possible pathways of hydrolysis for which some evidence exists are depicted. The Ptdlns(3,4,5)P 3 5-phosphatases (1) are probably distinct from the Ptdlns(4,5)P 2 5phosphatase (5) previously characterised (in both 3T3 cells and neutrophils they are predominantly membrane-associated [16,30] but, multiple, soluble forms of Ptdlns(3,4,5)P 3 5-phosphatases can also be resolved, L.S., unpublished data). The relative amounts of PtdIns(3,4,5)P3 degraded via (1) and an inositol phospholipid 3-phosphate phosphatase (2) have not been quantified (it has yet to be shown whether (2) is the same as the activity(ies) (6) and (4) that hydrolyze Ptdlns3P a n d / o r Ptdlns(3,4)P2). The reasons for this uncertainty are due to the fact the substrate in these experiments was Ptdlns([32p]-3,4,5)P3, which means a putative Ptdlns(3,4,5)P 3 3-phosphatase could have produced unlabeUed Ptdlns(4,5)P 2 and hence could only be quantified by [32p]pi release; thus a PtdIns(3,4,5)P3 3-phosphatase could not be distinguished from the combined actions of Ptdlns(3,4,5)P 3 5-phosphatase and Ptdlns(3,4)P e 3-phosphatase activities that are also present in these lysates. Similarly, the relative amounts of Ptdlns(3,4)Pe degraded via a PtdIns(3,4)P2 4-phosphatase (3) [16] or (4) are not defined (the relationship of (3) to the previously characterised Ptdlns4P 4-phosphatase (7) [281] is unknown). Ptdlns3P is degraded by a Ptdlns3P 3-phosphatase activity (6) [31] that in rat brain appears to be a homodimer based on a 65-kDa protein that is also found in a 65-kDa: 78-kDa heterodimeric protein associated with Ins(1,3)P 2 3-phosphatase activity [32,282].

All of the 3-phosphorylated inositol lipid phosphomonoesterase activities detected by these strategies are apparently active under basal-state, physiological assay conditions. Further, to put their activities in perspective, they hydrolyze Ptdlns(3,4,5)P 3 under these conditions at a significantly higher relative rate than that at which Ptdlns4P and Ptdlns(4,5)P 2 are hydrolyzed by their respective phosphomonoesterases [16]. In vivo the Ptdlns(4,5)P 2 and Ptdlns4P phosphomonoesterases are known to be counter-balanced by constitutively active lipid kinases that serve to maintain substantial levels of Ptdlns(4,5)P2, even in unstimulated cells [18,33]. Without comparably balancing kinase activities, the basal levels of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 in vivo would be expected to be negligible and, furthermore, any unsustained increase in their production

34 would be automatically reversed. Both of these predictions are apparent in the changes in levels of PtdIns(3,4)P 2 and Ptdlns(3,4,5)P 3 that occur upon agonist activation of ceils [10,16,23], indicating that the in vitro assays which have been used to define these degradative pathways are supplying credible data and, furthermore, that the metabolism to which these lipids are subject is very similar to that experienced by other authenticated 'second-messenger' molecules. A further implication of these observations is that both PtdIns(3,4)P 2 and Ptdlns3P can be derived by dephosphorylation of Ptdlns(3,4,5)P 3. Not only does this have a prejudicial influence on postulated functions for these lipids, but it also causes practical problems when interpreting experiments attempting to define the origins of these compounds in agonist-activated cells (see later).

II-A.2. Synthesis of 3-phosphorylated inositol lipids One of the major difficulties in this area has already been alluded to: that of the substrate specificity of lipid kinases and how it can depend on the assay conditions employed. This problem means that work with intact cells must drive investigations of the properties of lipid metabolizing enzymes, not only to identify potentially significant reactions but also to act as a yard-stick by which to evaluate the validity of in vitro assay protocols that might subsequently be used to study them. Nevertheless, to establish the relationships, identities and regulation of the enzymes that underlie these events, cells must be broken. Hence a large variety of experimental strategies have been used to study the synthesis of 3-phosphorylated inositol lipids that have included both in vitro and in vivo based approaches. However, there is a great deal of confusion surrounding the connections between lipid metabolizing activities detected in these two situations.

11-,4.2.1. Synthesis of 3-phosphorylated inositol lipids in vitro. To date, assays based on the use of broken cells (or fractions derived from them) and added phosphoinositides as a source of substrate have revealed only four potentially distinct types of enzyme activity that can catalyze formation of 3-phosphorylated lipids. (1) The PI3K that has been purified to homogeneity from mammalian cells, and is known to be regulated by receptor-controlled PTKs, can catalyze formation of all three 3-phosphorylated inositol lipids (see above and Fig. 2) [34-36]. However, the substrate specificity it displays in vitro, where it readily phosphorylates PtdIns, does not 'fit' with that implied by changes in the levels of these lipids in agonist-stimulated cells (see Fig. 4 and below; the purification and properties of PI3K are discussed in detail in Section III-B.1). (2) A membrane-associated PtdIns 3OH-kinase activity has been reported in yeast lysates [37]. However, in contrast to mammalian PI3K, the yeast enzyme does not apparently phosphorylate either PtdIns4P or Ptd-

Ins(4,5)P2 in vitro [37,77], suggesting it may be an authentic Ptdlns 3OH-kinase. A gene in yeast that probably encodes this PI3K activity was initially characterized as the locus of a vacuolar protein sorting mutant and has substantial homologies with mammalian PI3K (see Section III-B.1) [38]. However, on the basis of its apparent substrate specificity, this yeast enzyme does not seem to be a good candidate for a significant role in the agonist-activated formation of Ptdlns(3,4)P 2 a n d / o r Ptdlns(3,4,5)P 3. Subsequently the product of another gene TOR2 has been found to display a similar degree of sequence homology to the mammalian p l l 0 and yeast Vps34 proteins and has therefore been proposed to encode another PI 3-kinase [344]. However, though it is a candidate signalling molecule in the regulation of mitotic progression, determination of its significance must await the characterization of its products in vivo. (3) An inositol lipid 5OH-kinase activity, presumed to be responsible for the 5-phosphorylation of PtdIns3P that has been reported [13], has also been purified to homogeneity [39,40]. However, this activity almost certainly serves as a Ptdlns4P 5OH-kinase in the intact cell, and the absence of any positive evidence for the existence of Ptdlns(3,5)P 2 in cells only serves to further warn of the problems associated with interpreting data from in vitro assays alone. (4) A soluble Ptdlns3P 4OH-kinase activity in erythrocytes has been clearly resolved from the ubiquitous Ptdlns 4OH-kinase activity [41], suggesting that it is not another example of an enzyme showing low substrate specificity under in vitro conditions. Although the activity could also be detected in platelets [41,42], it was not present in 3T3 cells, suggesting this enzyme can not have a universal role in the formation of Ptdlns(3,4)P 2. Furthermore, studies of intact cells raise doubts about the significance of this activity, hence: (a) detailed analyses of the lipids present in intact mammalian erythrocytes have failed to detect any Ptdlns(3,4)P 2 [33] and, (b) three independent studies of the origin of Ptdlns(3,4)P 2 in intact cells have unambiguously shown this activity could contribute, at most, only a small proportion of the Ptdlns(3,4)P 2 in agonist-stimulated platelets, neutrophils and 3T3 cells (see below). Thus these four distinct types of phosphoinositide kinase (at lease one of which is almost certainly an 'assay artefact') represent the currently defined potential sources of 3-phosphorylated inositol lipids in intact cells. The substrate specificities the enzymes display in vitro do not match those apparently required to explain observations in agonist-activated cells. Further, although there have been several reports of agonists stimulating various 'PI3K-type' activities in cell lysates, they all suffer from one or more ambiguities in interpretation, e.g., most could equally well be explained by inhibitions of 3-phosphorylated phosphoinositide-

35 directed phosphomonoesterase activities. Hence the opportunity to short-circuit the need for a complete characterization of the agonist-sensitive reactions in intact cells by defining an enzyme activity with uniquely appropriate properties to explain events in vivo does not appear to be available. II-A.2.2. Synthesis of 3-phosphorylated inositol lipids in intact and permeabilized cells. To get around the interpretational problems associated with the use of broken cells and exogenous substrates, some workers have used radioactive isotope tracing techniques that enable pathways based on sequential phosphorylation/dephosphorylation reactions to be studied in intact cells [16,17,30,33,43,44,283,284]. In essence, cells are labelled with [32p]pi for a sufficiently short period of time that they are still far from isotopic equilibrium (in most mammalian cell systems isotopic equilibrium of the relevant phosphoinositide phosphates takes 6070 min to become established) and then stimulated with agonists. The relevant lipids are extracted, purified and the distribution of [32p]p between constituent phosphate moieties determined. This data is then used to predict the sequence of phosphorylation reactions via which the lipid was synthesized. The phosphate moiety that labels most rapidly within a target compound will be that with the smallest pool size (i.e., one that is only found in the compound of interest and not in its precursors) and was hence the last phosphate to be incorporated; more slowly labelled phosphates will be ranked progressively in the order in which they were inserted in the pathway. These techniques have also been applied to streptolysin-O permeabilized cells, in which [3,32p]ATP can be introduced almost instantaneously into the 'cytosol' of the cells [45]; thus these assays avoid the problems associated with the slow uptake of [32p]pi into ceils (see Refs. 44,16,33), but can still utilize membrane localized endogenous phospholipids as substrates. The above techniques enable the routes by which lipids are synthesized, in either agonist-activated or control cells, to be investigated. They cannot in isolation, however, pin-point the agonist sensitive reactions in these pathways. This question has been usefully addressed by experiments studying the initial, agonistinduced changes in the rate of accumulation of the individual 3-phosphorylated inositol lipids (e.g., see Fig. 4) [16,30]. Further, the use of permeabilized cells (where the accessibility barrier due to slow [32P]Pi entry into the cell is circumvented; see above) has enabled direct measurement of the initial rates of synthesis of these lipids on agonist-stimulation and hence whether stimulation of kinase a n d / o r inhibition of phosphatase activities are implicated in the increased accumulation of the relevant lipids seen in intact cells. Combined interpretation of these forms of experi-

ment enables some conclusions to be drawn about the likely routes of synthesis of Ptdlns(3,4,5)P3, PtdIns(3,4)P 2 and Ptdlns3P in agonist-activated mammalian cells and also the probable identity of the agonist-sensitive reaction(s).

II-B. The origin of Ptdlns(3,4,5)P3 in agonist-activated cells Data from experiments utilizing both (a) [T32p]ATP labelled, FMLP-stimulated permeabilized neutrophils [45] and, (b) [32p]Pi-labelled, FMLP-stimulated neutrophils [16], PDGF-stimulated 3T3 cells [30] and thrombin-stimulated platelets (N. Carter, C.P. Downes and S. Rittenhouse, personal communication), have all indicated that the order of addition of phosphates in the PtdIns(3,4,5)P 3 which accumulates on agoniststimulation is 1 ~ 4 ---, 5 ~ 3. Thus, the route by which PtdIns(3,4,5)P 3 is probably synthesized in a range of agonist-activated cells is PtdIns ~ PtdIns4P ~ PtdIns(4,5)P 2-~ PtdIns(3,4,5)P 3, i.e., PtdIns(3,4,5)P 3 is probably derived by direct 3-phosphorylation of PtdIns(4,5)P 2 (see Fig. 6B; but see also Fig. 6A and Refs. 17,43 for a contrary view). Further, FMLP and PDGF stimulate an almost instantaneous 10-20-fold increase in the rate of synthesis of PtdIns(3,4,5)P 3 in permeabilized neutrophils and 3T3 cells [20,45], respectively, indicating the similarly rapid and linear rise in the concentration of PtdIns(3,4,5)P 3 that occurs in intact cells (e.g., see Fig. 4 and Refs. 16,20,30), was a consequence of an 'activation' of a PtdIns(4,5)P 2 3OH-kinase activity [10,16] (the reason for the inverted commas is explained later in Appendix IV and Section IV.

II-C. The origin of Ptdlns(3,4)P e in agonist-activated cells The majority of 32p within Ptdlns(3,4)P2, obtained from all of the experimental situations in which PtdIns(3,4,5)P 3 was analyzed (see above, but see also Fig. 6A), was in the 3-position. This suggests the final phosphorylation in the synthesis of PtdIns(3,4)P 2 was mediated by a 3OH-kinase activity. However, because the distribution of 32p within the PtdIns(3,4,5)P 3 in these same extracts was 3 > 5 > 4, a PtdIns(3,4,5)P 3 5-phosphatase could have produced PtdIns(3,4)P 2 with a [32p] distribution precisely like that observed [16]. Hence these data argue that PtdIns(3,4)P 2 could have been derived by either direct phosphorylation of PtdIns4P or dephosphorylation of PtdIns(3,4,5)P 3. In many ways this question is directly analogous to the still unresolved debate over the substrate specificity of PICs and the origin of Ins(1,4)P 2 in vivo, i.e., is it from PtdIns4P or Ins(1,4,5)P3? From the point of view of the calcium-signalling fraternity, the immediate signifi-

36 cance of this problem receded with the discovery that Ins(1,4,5)P 3 and not Ins(1,4)P 2 was a messenger. If, however, the problem over the origin of Ptdlns(3,4)P 2

Receptor

Ptdlns ~ Ptdlns

B

= Ptdlns3P

Ptdlns ~ . ~ P t d l n s 4 P

Ptdlns3P

C

P Ptdlns(3,4)P 2

~

m Pldlns(3,4,5)P 3

Ptdlns(4,5)P2,1b"

Ptdlns(3,4)P2

Ptdlns(3,4,5)P 3

Ptdlns @ , ~ d l n s 3 P

Fig. 6. Kinase-mediated synthesis of 3-phosphorylated inositol phospholipids. The pathways possibly responsible for the kinase (K)-dependent synthesis of 3-phosphorylated inositol lipids in intact cells are described (discussed in Sections II-B, I1-C and II-E). Scheme B is in essence similar to that originally proposed by Cantley and co-workers [10]. Steps which are unambiguously implicated are shown with heavy arrows, those for which only indirect or in vitro evidence exists are marked with light arrows. All of the reactions depicted have been detected in broken cell assays. This scheme implies Ptdlns(3,4,5)P3 is the product of a Ptdlns(4,5)P2 3OH-kinase and is hence synthesized from a precursor with a relatively substantial presence in unstimulated cells. Scheme A is based on the internally consistent data of Maierus et al., [17,43] (although they are inconsistent with data supporting scheme B; i.e., in apparent contradiction to the 'equivalent' studies reported in Refs. 16 and 30) which were reproduced in both PDGF-stimulated 3T3 cells and thrombin-stimulated platelets and recently also observed in duckweed [278]. PtdIns(3,4,5)P3 is envisaged to be synthesized by a completely independent pathway terminating in a Ptdlns(3,4)Pa 5OH-kinase (that has yet to be detected in vitro). There are several problems associated with acceptance of this scheme. Firstly, the levels of Ptdlns(3,4)P2 in controls cells are sufficiently low (10-20 g M in the inner leaflet of the plasma membrane, 150 nM in the whole cell; see Table I) to suggest that the rate of a putative Ptdlns(3,4)Pa 5OH-kinase catalyzed reaction would be a function of the prevailing Ptdlns(3,4)P2 concentration; this would be highly unusual for a possible messenger-generating enzyme. Secondly, it is difficult to envisage control mechanisms that could produce a rapid and transient rise in Ptdlns(3,4,5)P3 without a detectable change in the levels of its putative precursor, Ptdlns(3,4)P2 (which subsequently rise substantially, see Fig. 4). Finally, these data also suggest the role for a PI3K activity is limited to phosphorylation of Ptdlns and hence that the translocation of PI3K which occurs in PDGF-stimulated 3T3 cells [9,20] plays no part in the parallel changes in 3-phosphorylated inositol lipid metabolism (since Ptdlns3P levels do not change on stimulation [20,30]; or that PI3K is manifest as a Ptdlns3P 4OHkinase and/or a Ptdlns(3,4)Pa 5OH-kinase in the activated cell). The model does receive some independent support however, from the description of both Ptdlns3P 4OH-kinase [42,41] and Ptdlns 3OH-kinase activities in vitro (e.g., Refs. 8,35). The strongest evidence in favour of scheme C ('direct 3OH-phosphorylation' of PtdIns) is found in work with yeast, that has suggested that they possess a substantial complement of Ptdlns3P [37,242] and a membrane-associated, Ptdlns-directed PI3K activity [37,77]. The relatively high levels of Ptdlns3P found in unstimulated mammalian cells could also be the result of direct phosphorylation of Ptdlns; this could be the result of the agonist-sensitive PI3K activities depicted in scheme B being manifest as Ptdlns 3OH-kinase activities in quiescent cells and/or a distinct Ptdlns 3OH-kinase activity (like that present in yeast?).

Ptdlns4P ~

Ndlns(4,5)P2 ~

~dlns3P

Ptdlns(3,4)P 2

\\ \\ ',

/

Ptdlns(3,4,5)Pa

Fig. 7. Agonist-activated metabolism of 3-phosphorylated inositol lipids. Receptors (including those which interact with G proteins, possess intrinsic protein tyrosin kinase activity or associate with src-type protein tyrosine kinases) can increase the activity of a Ptdlns(4,5)P2 3OH-kinase (3-K) and this leads to increased accumulation of Ptdlns(3,4,5)P3 and hence Ptdlns(3,4)P2, by way of a Ptdlns(3,4,5)P3 5-phosphatase. Ptdlns(3,4)P2 is recycled to Ptdlns via both Ptdlns3P and Ptdlns4P. There is some evidence that a Ptdlns 3OH-kinase activity may exist which is agonist-insensitive and responsible for the accumulation of Ptdlns3P seen in many unstimulated cells (see Appendix III).

can be resolved, it will dramatically change the credibility of Ptdlns(3,4)P 2 a n d / o r Ptdlns(3,4,5)P 3 as potential signals (discussed in Section VI-A). There are a large number of issues which address this question (see Appendix II and Refs. 16,30 for a detailed discussion), the most critical of which are observations indicating that in numerous agonist-activated ceils Ptdlns(3,4)P 2, in contrast to Ptdlns(3,4,5)P 3, only begins to accumulate after a pronounced delay (or 'lag', relative to Ptdlns(3,4,5)P3; see Fig. 4 and Refs. 16,30,22). These observations are probably most simply explained by suggesting Ptdlns(3,4)P 2 is derived by dephosphorylation of Ptdlns(3,4,5)P 3.

H-D. The pattern of agonist stimulated 3-phosphorylated inositol lipid metabolism The data we have addressed indicate that the answer to the critical question targeted at the beginning of this section, is that the primary agonist-sensitive reaction is catalyzed by a PtdIns(4,5)P 2 3OH-kinase activity. This appears to be true for a wide range of receptors, including examples that are thought to utilize G proteins (e.g., FMLP) and others that utilize various PTKs (e.g., PDGF) to relay their signals. It is also true for a variety of cell types, from those which are mitogenically competent, through to mitotically inactive sub-cells such as platelets. The fact that so many diverse receptors can mobilize 3-phosphorylated inositol lipid metabolism via a single type of lipid kinase activity, strongly supports the view that this might be a signalling reaction and hence that PtdIns(3,4,5)P 3, as possibly its only product, is the signal [2,16,30]. The scheme presented in Fig. 7 incorporates the above arguments and suggests a common scheme by which agonists can stimulate the observed changes in

37 3-phosphorylated inositol lipid metabolism. The strength of this scheme is based on the ‘unification’ it suggests between the PI3K activity that can translocate to receptor-PTKs and the PtdIns(4,5)P, 30H-kinase activity that is stimulated by many different agonists in intact cells, i.e., they can both catalyze the same reaction [lo]. If these activities are synonymous, however, then PI3K must be manifest as a PtdIns(4,5)P,-directed 30H-kinase in the context of an agonist-activated cell [16]. This clearly requires that the ability of PI3K to phosphorylate PtdIns and PtdIns4P when presented in the form of lipid micelles in vitro, is differentially lost in intact, agonist-activated cells. A precedent for just such a shift in apparent substrate specificity has been established by studies of the receptor-regulated PtdIns(4,5)P,-metabolizing family of PICs (see Appendix I). This work has suggested that although PICs can readily hydrolyze PtdIns under certain conditions in vitro, they preferentially hydrolyze PtdIns(4,5)P, in the context of an agonist-stimulated cell. This conclusion also leads us into potential confusion or ambiguity over the nomenclature of PI3Ks, since the reactions they catalyze in vitro may not be equivalent to those they catalyze in vivo. In a field where a number of the relevant issues have still to be resolved, we have adopted the following ‘makeshift’ positions: (1) PtdIns(4,5)P, 30H-kinase activities are responsible for agonist-stimulated accumulations of PtdIns(3,4,5)P, in agonist-stimulated cells; (2) PI3K activities are due to enzymes capable of phosphorylating any combination of PtdIns, PtdIns4P and PtdIns(4,5)P, in the 3-position in vitro; and, (3) the PI3Ks are the closely knit family of PISK activities which have been recently purified and the genes for which have been cloned (Section III-B.l). Whether the PI3K.s characterized so far encompass the major, or even all, possible PI3K activities in cells is still an open question (discussed in Section III-B.2). It is also unclear whether PI3K activities represent all the PtdIns(4,5)P, 30Hkinase activities responsible for agonist-stimulated accumulation of PtdIns(3,4,5)P,; it is certainly likely that a PI3K is the prototypic example of a PtdIns(4,5)P, 30H-kinase activity which is activated by PTKs (see above and Section III-B.l), but it is by no means clear that a currently defined PI3K will be involved in all forms of PtdIns(4,5)P, 30Hkinase activity (discussed in Section III-A.2). II-E. The origin of PtdIns3P in cells

The very small or non-existent changes in the concentration of PtdIns3P that occur in the presence of agonists that can simultaneously stimulate dramatic translocations of PI3K was initially very confusing, because it was anticipated that in the cell the enzyme would phosphorylate PtdIns just as it would in vitro.

The picture outlined above begins to rationalize these observations by suggesting that in vivo, PI3K predominantly uses PtdIns(4,5)P, as a substrate. However, this does not explain the origin of the PtdIns3P that is so relatively abundant in unstimulated cells. Many of the experimental strategies utilized to study the metabolism of PtdIns(3,4)P, and PtdIns(3,4,5)P, have been applied to PtdIns3P. The pattern of results that have been obtained lead to precisely the same interpretational problems that had to be faced over the origin of PtdIns(3,4)P,: it could be synthesized by either a Ptdlns 30H-kinase and/or a PtdIns(3,4)P, 4-phosphatase (see Appendix III for a similar consideration of the relevant issues). In essence, although it is possible to account for the apparent agonist-insensitivity of PtdIns3P within a model proposing it is solely derived from the dephosphorylation of PtdIns(3,4)P,, the likely existence of PtdIns 30H-kinase activity in cells (see Appendix III> suggests PtdInQP can be derived by both mechanisms. Cantley and co-workers [34] have suggested that this PtdIns 30H-kinase activity is the phenotypic manifestation of an agonist-sensitive PI3K in unstimulated cells (as a result of its cytosolic localization changing its substrate supply). It can be argued, however, that the synthesis of PtdIns3P in unstimulated cells is the result of a distinct PtdIns 30H-kinase activity (which may have completely different roles) and that agonist-sensitive PtdIns(4,5)P, 30H-kinase activities are essentially inactive in unstimulated cells (see Fig. 7). III. Receptor-coupling to increases in PtdIns(4,5)P, 30H-kinase activity The preceding section has argued that a variety of receptors with fundamentally different structures and associated transduction mechanisms can all increase the rate of a PtdIns(4,5)P, 30H-kinase reaction in their target cells. The ‘activations’ of PtdIns(4,5)P, 30H-kinase they produce must ultimately be achieved by increasing its access to substrate and/or by changing its catalytic parameters (discussed in Appendix IV). The question is, what events intercede? III-A. How closely coupled is the activation of PtdIns(4,5)P, 30H-kinase activity to the activation of receptors?

The critical issue in this section is the level at which PtdIns(4,5)P, 30H-kinase activity resides in the stratified structure of receptor-directed signalling cascades (see Appendix VI). If this activity is the primary, receptor-regulated step in a new signalling system, then precedent suggests that it must be capable of interacting directly with a receptor-transduction mechanism. The following discussion addresses whether Ptd-

38

III-A.1. Implication of PTK activities in the regulation of Ptdlns(3, 4,5)P3 accumulation Receptors that are known to utilize PTKs in their signal transduction pathways fall into three classes: (1) those with intrinsic PTK activity (receptor-PTKs) [5];

Ins(4,5)P 2 3OH-kinase is directly activated by the two common classes of transduction mechanisms which are utilized by receptors stimulating PtdIns(3,4,5)P 3 accumulation; the activation of PTKs and the activation of G proteins. TABLE III

'Activation' of PI3K a n d / o r Ptdlns(3,4,5)P 3 accumulation by src-type and other related PTKs

Reports of association of PI3K with cellular src-type PTKs (either receptor, or mT-activated) or transforming, virus-derived src-type or related PTKs, are tabulated along with the evidence of other relevant associations or increased accumulation of Ptdlns(3,4,5)P 3. A number of the receptors listed are described by their activating ligand (ligand-R). CD3 and CD4 are cell-surface molecules acting as receptors for MHC II antigen complexes, mlgM is a B-cell antigen receptor and CD2 is a receptor for LFA-3; in each case in vitro activation is usually produced by antibody cross-linking. 'Activation' of an src-type PTK is usually accepted to have occurred when immunoprecipitates (imm.ptts) prepared with antibodies directed against it show increased rates of tyrosine phosphorylation of either the src-type PTK itself (i.e., autophosphorylation) a n d / o r an exogenous non-physiological substrate (e.g., enolase). The receptors or ligands (1-3, 5-8) are thought to physically interact with, and primarily signal through, src-type PTKs [263-265]. (4) The IL-4 receptor is a member of the cytokine-receptor family and is known to stimulate tyrosine phosphorylation of target proteins, though the PTK(s) responsible have not yet been identified [224]. (9) and (10) are thought to regulate src-type PTKs via their primary, G-protein-mediated effects on calcium levels a n d / o r PKC activity [146,147]. (11,15-16,17,19): the levels of PtdIns(3,4,5)P 3 in cells transformed by expression of mT or the transforming oncogenes v-src, v-yes, v-fps or v-abl were found to be chronically increased [51-53,129]. Although GM-CSF activates both lyn and yes in neutrophils it only activates lyn in TF-1 cells and, therefore, in the latter case, mirrors the effects of IL-3 [25]; a result in keeping with evidence that these receptors use a common, signal-transducing, /3 subunit [266,267]. The GM-CSF-stimulated increase in p85 immunoreactive protein in anti-lyn antibody-directed imm.ptts suggests a physical translocation of PI3K to lyn has occurred, rather than activation of prebound PI3K [25]. The IL-2 receptor in T cells is found associated with, and can activate, lck [46] (but in B cell lines which do not express lck it associates with and can activate lyn [268]). Although PI3K activity is translocated to the activated IL-2 receptor [120], evidence could only be found for its association with fyn [250] (which was shown to be be activated by IL-2 in these cells Ref. 250). The situation is made even more confusing by observations in similar cells, that stimulation of the C D 3 / T c R ('antigen' receptor) complex, an event reported to lead to the activation of fyn exclusively [259,260], results in an increase in PI3K activity in lck (acting as a form of positive control for the IL-2 response described above) and CD4 (TCR accessory protein) imm.ptts (but not fyn imm.ptts) [257]. It would seem that some of these associations may result from activation of an as-yet unidentified receptor-associated tyrosine kinase(s). IL-4 stimulation of FDCP-2 cells promotes the association of PI3K with both an unidentified 170-kDa protein (which is heavily tyrosine phosphorylated on stimulation) and to a lesser extent with the IL-4 receptor itself [224]: IL-3 stimulation of the same cells results in a relatively much smaller association of PI3K with phosphotyrosine-containing proteins and these proteins are likely to be different to those implicated in IL-4 stimulation [224]. The FMLP and thrombin receptors are thought to transduce their signals via G proteins and both can cause increased recovery of PI3K activity in src-type PTK imm.ptts [57,65] (although thrombin has not been shown to 'activate' src-type PTKs, FMLP has been shown to 'activate' lyn via a pertussis toxin-sensitive mechanism [57]). Other, indirect evidence suggests these apparent G-protein-mediated translocations are driven by calcium or protein kinase C signals (see Section IV-C.4). Activating receptor (R)

(1) GM-CSF-R (2) IL-3-R (3) IL-2-R (4) IL-4-R (5) mlgM (6) CD4 (7) CD2 (8) C D 3 / T c R (9) FMLP-R (10) Thrombin-R (11)(12) (13) (14) (15) (16) (17) (18) (19)-

Cell type

Neutrophils TF-1 cells T cells FDCP-2 cells B cells T cells T cells T cells Neutrophils Platelets

Increased accumulation

Activated src-type or

Increased P13K activity ( + ), or p85 immunoreactivity/85-kDa protein (*), in imm.ptts of:

Ptdlns(3,4,5)P 3

other ~8) PTK

PTK

PY proteins

+

lyn, yes lyn lck, fyn (?)

+ lyn*, + yes + lyn + fyn (?)

+

lyn Ick

fyn (?) lyn

+

+ + + + + a

+ a + a + a No + a

c-src/mT c-yes/mT c-fyn/mT c-fyn b v-$rc v-yes v-fps ~8) v-ros ~s) v-abl ts)

Reference

Receptor

+ lyn* + lck

+ + * p170 + +

+ IL-2 + IL-4 + mlgM + CD4

+ / c k (?) + lyn + fyn, +c-src +*src/mT + *yes/mT + fyn/mT *fyn + *src + yes + fps + ros +abl

+ + + + + + * + + + + +

+ C D 4 / C D 3 (?)

[25] [25] [46,120,250] [224] [84,85,251,252] [253-256,259] [258] [257-260] [14,57] [22,65,204] [11,12,51,52,66,137,141] [262] [250,269] [70] [6,129,143,262] [129,262] [129,262] [7,129,262] [53]

PY: phosphotyrosine-containing proteins; (?): see legend; a sustained increase in cells transformed by the relevant virus-derived, src-type or other PTK, or by expression of polyoma virus mT antigen; b overexpressed with p85 in sf9 insect cells.

39 (2) those that closely associate, via their cytoplasmic domains, with various non-receptor PTKs, e.g., those of the src family [46] (some receptor-PTKs may also recruit and potentially utilize src-type PTKs [1,47]); and finally (3) those with other clearly defined primary signal transduction mechanisms (e.g., G proteins) that can stimulate PTK activity indirectly [48,49]. Receptors falling into all three of these categories can stimulate a Ptdlns(4,5)P 2 3OH-kinase activity (see Tables II and III; although because of the indirect manner in which the latter class operate they do not usefully contribute to this part of the discussion). Direct evidence that these receptor types use their regulated PTK activities to increase Ptdlns(3,4,5)P 3 accumulation is limited to (a), demonstrations that the responses are abolished by various PTK inhibitors but not by inhibitors selective for other signalling systems [25,50] and (b), that ceils expressing transforming versions of cellular PTKs contain elevated Ptdlns(3,4,5)P 3 levels, but that PTK-inactive, non-transforming versions of these proteins do not [51-53]. However, this relative paucity of experimental support (looking from the 'outside of the cell - in') for the involvement of PTKs in the regulation of a Ptdlns(4,5)P 2 3OH-kinase activity becomes trivial, as soon as the huge body of data derived from experiments studying this response from an 'inside of the cell - out' perspective is accepted to mean a PI3K activity is implicated in agonist-stimulated accumulation of Ptdlns(3,4,5)P 3. This work has characterized a PI3K activity that can physically associate with receptor-activated PTKs (both receptor and src-type PTK varieties) and possesses all of the structural hallmarks of PTK-regulated proteins (see Section IV-B for a more complete description of these events). Furthermore, the fact that PI3K interacts directly with PTKs and in parallel with other authenticated signalling proteins (such as PIC7, rasGAP and GRB2/SEM5) indicates that it is not a 'downstream' response driven by signalling reactions that are more tightly coupled to receptor activation.

III-A.2. Implication of G proteins in receptor-stimulated accumulation of Ptdlns(3,4,5)P3 A significant number of receptors that belong to the 7-transmembrane-domain class of receptors can stimulate accumulation of PtdIns(3,4,5)P3 (Table IV). These receptors do not contain intrinsic PTK activity or associate with non-receptor-PTKs, but have been found to universally transduce their signals by coupling to heterotrimeric G proteins, (discussed in the legend to Fig. 11; these are distinct from low-molecular-weight monomeric GTP-binding proteins, e.g., the rho and ras families) [54,55]. This implies that a PtdIns(4,5)P 2 3OH-kinase activity can be regulated by G-protein-dependent pathways.

This notion is supported by some experimental evidence. Mastoparan (a 14aa peptide that appears to mimic the regions of receptors that activate heterotrimeric G proteins) can stimulate rapid accumulation of Ptdlns(3,4,5)P 3 in neutrophils [56]. FMLP-, PAD and ATP-stimulated accumulations of PtdIns(3,4,5)P3 in neutrophils, and thrombin-stimulated accumulation of Ptdlns(3,4,5)P 3 in platelets, can be abolished or reduced, respectively, by pertussis toxin [15,45,57,58] (which causes ADP-ribosylation of, and hence inhibits, receptor coupling via ao, a i and ~'r containing members of the Gi family of heterotrimeric G proteins [59]). GTPyS , an irreversible activator of both monomeric and heterotrimeric GTP-binding proteins can enhance Ptdlns(3,4,5)P 3 accumulation in permeabilized platelets and neutrophils [22,45]. A critical question, however, is whether these Gprotein-dependent 'activations' of Ptdlns(4,5)P 2 3OHkinase are direct or mediated via other signalling systems that are driven by the same G proteins. Potential intermediaries between a G-protein and a PtdIns(4,5)P 2 3OH-kinase activity could include PIC and its associated primary signals diacylglycerol (and hence PKC activation and PtdOH formation) and Ins(1,4,5)P3 (and hence calcium mobilization) [3,4]. They could also include a huge collection of secondary effects of these primary signals, such as protein (tyrosine, threonine or serine) phosphorylation and phospholipase A 2 or D activation (which might also be stimulated directly by G proteins) [60,61]. Furthermore, some evidence already suggests several of these signals might regulate 3-phosphorylated inositol lipid metabolism. For example, in platelets PKC-activating phorbol esters can directly increase PtdIns(3,4)P 2 accumulation [62]. Further, calcium can augment [63], whereas PTK/PKC-inhibitors [62] and reduction in integrin receptor function [63,64] can diminish, thrombin-activated accumulation of 3-phosphorylated inositol lipids (indicating some of thrombin's effects may be via secondary pathways controlling or sensing cell adhesion, which in turn are possibly calcium a n d / o r PTK-mediated). Moreover, the observations that thrombin and FMLP can cause small but significant increases in the recovery of PI3K activity in antiphosphotyrosine and anti-src-type PTKantibody directed immunoprecipitates [57,65] (see Table IlI; these are diagnostic markers for the involvement of PTKs in a regulatory process) suggest that these ligands may regulate PI3K activities via PTK-coordinated mechanisms. In a recent study which has addressed this issue, however, the rapid and large increases in accumulation of PtdIns(3,4,5)P 3 elicited by FMLP in neutrophils did not appear to depend on PTK, PKC, phospholipase A2, C or D activities (discussed in the legend to Fig. 11). This work suggests that G proteins might indeed interact directly with a PtdIns(4,5)P2 3OH-kinase activity and, therefore, that

40

a range of extracellular mediators which do not directly activate PTKs can access a putative PtdIns(3,4,5)P, signal within the context of their other primary signals [45,57]. Furthermore, because G proteins classically mediate more rapid and transient activations of effectors than signals transduced by PTK-coordinated mechanisms, this additional option broadens the scope of the type of signal that PtdIns(3,4,5)P, might provide. Indeed, the fact that G-protein-linked receptors seem to elicit the most intense (as judged by the initial rate of formation of PtdIns(3,4,5)P,), rapid and transient activations of PtdIns(3,4,5)P, accumulation thus far described, further strengthens the notion that the interceding coupling reactions must be relatively direct and probably do not entail PTK-driven reactions 114,571. Despite the potential increases in scope for a PtdIns(4,5)P, 30H-kinase activity that the possibility of G-protein coupling has to offer, very little work with the ‘inside of the cell - out’ approach of studying a PtdIns(4,5)P, 30H-kinase activity that can physically interact with heterotrimeric G proteins is available for consideration (i.e., the analogous approach to that which has yielded such dividends for understanding the relationship between PI3K and receptor-regulated PTKs). The reasons for this include the technical problems resulting from the fact that G proteins activate their targets by direct, but relatively weak and readily dissociated interactions, that do not involve covalent modifications, and hence leave no evidence beyond their life-time of an impact on an effector. This means that the evidence that G proteins can interact directly with PtdIns(4,5)P, 30H-kinase is still indirect and the critical reconstitution experiments remain to be performed. At the beginning of this section we noted that one of the reasons for suspecting that apparently G-protein-dependent increases in PtdIns(4,5)P, 30H-kinase activity might be indirectly produced via activation of a downstream PTK, was that FMLP and thrombin can stimulate increases in PI3K activity recovered in antiphosphotyrosine and anti-src-type kinase antibody directed immunoprecipitates. It is now clear that FMLP can activate the non-receptor src-type PTK lyn via a G-protein-dependent pathway in neutrophils [571. The signalling complex formed by lyn in response to FMLP contains PI3K activity and hence might be expected to drive accumulation of PtdIns(3,4,5)P,. However, the differential scale, time courses and ‘pharmacological’ sensitivity of this response compared to that produced by other agonists on the same cells, strongly suggested it contributed, at most, 5-10% of the rapid FMLPactivated production of PtdIns(3,4,5)P, (this is discussed later in Fig. 11). The cellular significance of this G-protein and PTK-dependent pathway may be found in the context of the plethora of downstream events

occurring in the later stages of the cell’s responses to an agonist. There are currently no equivalent estimates of the possible contribution PTK-dependent processes might make to thrombin induced accumulation of PtdIns(3,4,5)P, in platelets (and could therefore range from O-100%); this information would be particularly valuable in assessing both the significance and cause of the large translocations of PI3K activity that occur during this cellular response and the origins of the cell-specific capacity to support G-protein-activated PtdIns(3,4,5)P, accumulation. III-B. Characterization of enzymes potentially responsible for PtdIns(4,5)P, 30H-kinase activity III-B.l. Characterization of a PI3K activity which can tramlocate to actiuated PTKS An early clue as to the identity of the PI3K activity

which can translocate to activated PTKs was the correlation between the presence of an 81-85-kDa phosphoprotein(s) and PI3K activity in various immunoprecipitation protocols (initially using antibodies against the mT: c-src complex [66] or the PDGF-receptor [12]). This information was reinforced by subsequent data describing the presence of 85-kDa proteins in purified preparations of PI3K from bovine brain [35] and led to efforts to isolate an 85-kDa protein and clone cDNA encoding it on the suspicion that it was PI3K. cDNAs encoding these 85-kDa proteins were cloned in parallel by three different approaches: (1) by utilizing sequence data derived from peptides generated from an 85-kDa protein present in ‘conventionally purified’ preparations of PI3K from bovine brain [67]; (2) by sequencing peptides generated from an 85-kDa protein purified from mouse fibroblasts by virtue of its ability to translocate to activated PDGF-receptors [68]; and (3) by a so-called ‘GRAB’ technique, where an expression library was screened to identify clones producing proteins able to bind tightly to a tyrosine phosphorylated proteolytic fragment of the EGF receptor (derived from the region of the EGF receptor thought to mediate interactions with signalling proteins) [69]. cDNAs encoding two p85 proteins were characterized in these studies and termed p85a and p85p [67]. Recombinant, purified p85a and pS5p did not possess PI3K activity but could translocate to, and be tyrosine phosphorylated by, activated PDGF, EGF, CSF-1 and C-erb B2 receptors, the mT: c-src complex and the src-type PTK c-fyn [70]. These two proteins could also compete with PI3K binding to PDGF and CSF-1 receptors and anti-p85cr and /? antibodies could immunoprecipitate PI3K from a variety of cells (although p85a antibodies always recovered more activity), suggesting they were potential authentic regulatory subunits of PI3K and could be responsible for its translocation to activated PTKs [70].

41 Subsequent purifications of PI3K from bovine brain [38,71,72], bovine thymus [36] and rat liver [34] clearly correlated the presence of PI3K activity with a protein of native molecular mass 190 kDa. Further, this protein was composed of tightly associated p l l 0 and p85 subunits (which could be dissociated under reducing conditions in the presence of SDS) suggesting that native PI3K was in fact a heterodimer [34]. The p l l 0 protein had largely escaped earlier attention as a putative PI3K because it was not as heavily phosphorylated as p85 in in vitro kinase assays of PTK-containing immunoprecipitates [12,66]. cDNA encoding the p l l 0 subunit has now been cloned and, critically, has been shown to encode a protein with PI3K catalytic activity when expressed in the absence of p85 [38]. Recombinant p l l 0 also binds tightly to p85 and the resulting complex can then translocate (and hence carry PI3K activity) to activated receptor-PTKs [38,71]. These results therefore define a major PI3K activity in ceils which can translocate to activated PTKs as comprising a heterodimer of 85-kDa regulatory and ll0-kDa catalytic subunits. The primary structures of these two subunits contain significant homologies to other proteins (Fig. 8). Of particular relevance to the mechanism of translocation into PTK-coordinated complexes are the two src-homology region 2 (SH2) domains contained within the 85-kDa subunit [6769,73]. These domains are known to form tight complexes with tyrosine phosphates (discussed in Appendix VII)) and convey the observed ability of p85 (and hence the p85/pl10 catalytically active complex) to bind to autophosphorylation sites on receptor-PTKs [5,70] (discussed in Section IV-B; this tight association was the reason for the success of two of the strategies for the purification and cloning of p85 noted above). p85 also contains one SH3 domain, one or more potential SH3-binding domains and a region of homology to the breakpoint cluster region gene (bcr, see Fig. 8) [67-69,73,74]. The function of SH3 domains in proteins is only beginning to be understood, but they appear to function as potential sites for specific and tight interaction with certain 'proline-rich domains' present in other proteins, e.g., in the binding of GRB2 to the ras G T P / G D P exchanger SOS [73,75,83,341-343]. Sequences containing, or closely related to, the core consensus for binding of SH3 domains from abl, src and GRB2 are found in both p85a and p85/3 [341,343,345]. There is preliminary evidence to suggest that one of these sites may provide a target for interaction of a p85 with the SH3 domain of the lck PTK (personal communication of Dr. Chris Rudd). Not all of these possible sites are present in both o~ and /3 isoforms of p85 suggesting that SH3 domain interactions could provide a route for differential regulation of PI3K complexes dependent on the identity of their p85 subunit. The bcr domain is thought to convey an

ability to bind to, and activate the rate of GTP hydrolysis by, some members of the rho family of small-molecular-weight GTP-binding proteins [74,76]. The implications of these potential interactions for the 85-kDa protein are discussed in Section VI-A. The 110-kDa subunit possesses a remarkable homology with a yeast vacuolar protein sorting mutant (Vps34; see Fig. 8) [38]. Vps34 has subsequently been shown to possess PtdIns 3OH-kinase activity [77] and hence this discovery has generated a great deal of excitement about possible functions of PI3K in protein sorting or vesicle trafficking [78-80] (discussed in Section VI-B). Very recently, the 110-kDa subunit has also been shown to possess significant sequence homology with another yeast gene product, TOR2 [344]. The function of TOR2 is unknown, other than that it is essential for growth and may be a target of rapamycin action. TOR2 shows more extensive homology with mammalian p l l 0 than with Vps34, though within each domain the overall degree of sequence conservation found is roughly identical. It has yet to be demonstrated that TOR2 is in fact an inositol lipid 3OH-kinase. We have also identified a region in the N-terminal half of p l l 0 (which is absent from Vps34) which is homologous to a region in a number of membrane-associating/phospholipidbinding proteins (called CaLB [81]; see Fig. 8); this region has obvious implications for both the mechanism of catalysis and activation of PI3K (discussed in Appendix IV and Section IV-B). PI3K can become phosphorylated on both serine and tyrosine residues in cells [12,66,70,72] (this is discussed in Section IV-B.1 in the context of how the enzyme might be activated). Substantially purified preparations of PI3K from rat liver, bovine brain, and insect ceils overexpressing mammalian PI3K, could all auto-phosphorylate serine residues in both p85 and p l l 0 subunits. This serine autophosphorylation correlates with a decrease in PI3K activity, suggesting it may be an important regulatory mechanism [72,82]. It remains to be established, however, whether this property is intrinsic to the p85/pl10 complex or is conveyed by the tight binding of an as yet unidentified kinase present in both insect and mammalian cells [82].

III-B.2. Characterization of a Ptdlns(4,5)P2 30H-kinase activity that might be regulated by G proteins There appear to be two distinct types of pathway by which G-protein-linked receptors can regulate PI3K activities (see above, Section III-A.2). One, shown to be used by the FMLP receptor in neutrophils, ultimately results in activation of src-type PTK activity [57] and hence the PI3K this regulates is very likely to be of the p85a : p l l 0 format known to be regulated by other receptor-sensitive src-family PTKs [25,84,85]. The alternative pathway (which is the major pathway used by FMLP during short periods of stimulation), which may

42 not involve PTKs, is most simply explained by direct interaction between a G-protein and a PI3K activity. The critical reconstitution experiments, which will es-

tablish whether this connection is direct, remain to be done. However, if this mode of regulation does operate then the implications are substantial, not only for the

Regulatory subunit SH3 SH3B?

bcr

SH3B?

p110 binding site ?

pss~ SH3B?

SH3B?

p85 I~ pS0

Catalytic subunit peSUr~ng site ?

,-~ strong VPS 34 homology

p110 CaLB

~

site

w e a k V P S 34

homology

-']unique

VPS34 (p95) i

i

A'rP binding site

a

V///////////l

(p288)

!

i

A TP binding

CaLB

file

CaLB domain in p110 P65b P65a Pkcy Nfl Gap cPLA2 PIC6 PIC~I PIC72 PICyI p110 TOR2 CON

P65b P65a Pkcy Nfl Gap cPLA2 PIC6 PIC~I PIC72 PICyI p110 TOR2 CON

Lm...Qng.k rlkKkKTtIk L . . I P E .... k k k K F e T K V h L..iPDprnl ..tKqKTRtV ..qSvvyhee sppqYqTsyL L.nSvQv ....... aKTHar IstTPDsr ...... kRTRhf I.hGvtgDv. ..asrqTaVV Mf.GlpvDt. rrkaFKTKts I.cGAEyEs. ..nKFKTtVV V.aGAEyDsi ...KqKTefV IyhGGEp... icdnvnTqrV qiiAAEIEe .... iiKyKkL

kNT.LNPyYN rkT.LNPVFN kaT.LNPVWD QsfGfNGLWr E..GqNGVWs NND.INGVWD tQQGfNPwWD QgNAVNPVWE NDNGLsPVWa vDNGLNPVWp ..PcsNPrWN ...PqNSdkr

E...SFSFEV E...qFTFkV E...TFvFNL f...AgPFsk Ee...FvFDd E...TFeFiL te...LeFEV Ee..PIvFkk ptqekVTFEI ak..PFhFQI E...wLnYDI ..... L T m r r

.... P f e q i q k v q V v V T V i D .... P y s l g s . k t L . M A V Y D ..kpGD . . . . . v e r R L S V e V qtqiPDyAg. IiVKFLdAL l..pGDIn ..... RFeITL ...dPNqen .... vLeITL ..avPDLA.. .IVRFMVeD vv.iPsLAc. ..LRIAVYE yd..PNLAf. ..LRFVVYE ..snPEfAf. ..LRFVVYE yi..PDLP.. .raaRLcLSI ...eTwnT .... rLlgcqkN

eLPlm..D.r gISdPYVeVe L..G.E..d .... kfKTKvv

..NGINPVW.

e .... ftFEI

.... P D I a . . . . . L R f . V y e y D . . . f s k n d

NLkKM..Dvg ELPaL..Dmg IIP...mDPN ILSIc.qDPN k L P v ..... K afGdM...ID QLPKVnkNkN iISgqflsdK hLPKL...GR hLPK...NGR yVn...vNiR ELSaLvnESy

GLSDPYVKIh GtSDPYVKVf GLSDPYVKLk .LiNPihgIv hfTNPYcNIy T.PDPYVELf SIvDPkVtVE kVG.TYVEVD SIAcPFVEVE GIvcPFVEIE dIdkiYVRtg nrAynvVvra

dIdkvf.VS .......... y nStgAelrHw sDMLANp .................. hrpIa QwhtlQvvEe i I G e . f k V P M N T ........ v d f g h V t e E w r D L q S a e . . . . . . . . . . . . . . . keeQekLg DicfelryvP FMGa.msfGV selLKAp.vd gwyKiLnqE ........................ egEyfnV PvAdaDncN1 ...... y L P g i d e e t S e e s . . I L t P T s P y p p a L q S D l s i t a .... n l n l S N M t S l a T s q h S p G I D k e n v e ..... S k d P d i l f M R c q l s r . l q K G h a T D e w f L L S s h . i p ik . . . . . . . . . . . . . . . . . . . . SIEpgSIr t L G . t A t f P V sS . . . . . . . . . . M K v G e k K e V p f I f . . n q v te . . . . . . . . . . . . . . . m I L E m S L E v c S c P F I G . q S t I P w NS . . . . . . . . . . L K Q G Y r H . V h L L S k n . . . . . . . . . . . . . . . . . . . . . . . . . GdQhpSaT F I G . h r i L P V Q A . . . . . . . . . . IRPGYhy. I c L r n E r n q p i m l p a l f v y i E V k d y v P d t y A d v I E a l S N P laSllvfce .... MRPvl.. eSeeELYSsc rQLrrQeel ......... nn QLflyDThqn IrnaNrdAlv FLAqaTf.PV kG.LKTgyra vPLKNnYSEg LELaSllvki dvfpakqenG DLsPfgGasL rerscDaSgP a k e e h . . c P L a ......... w G n i N L F d y t d t L V . . . . . . . . . . . . . . sG k M . A I N I w p V P h G L E D I I N P r V r s l v i k P k E d a q . . . . . . . . . . . V r i K f a N L c r k . . . . . . . . . . . . sG r M . A l a k k V L N t l L E E t d D P f I g . . s . . P v ns . . . . . . . . . slk..yse,

evd tag Lqk LsP VrA dlr Lfv Iry kef Lfh Igv dhP

v d L l s . . . . . . . . . . . . . . g .m .... t.il p.g. E d . s n p l.p

415 1285 303 2800 707 145 750 816 1203 1242 461 1644

YDk..iGKND FDr..FSKhD WDwdrtSRND iDT ....... sNk...TKk. mDA.nYvmDE YDS..sSKND e g g ...... k eDy..sediE eDm..FSdQN csv..kGRkg iDv..WqRil

43

kinetic scope of the signals this pathway might generate but also, in the light of recent evidence suggesting that SH2 domain :tyrosine phosphate docking is responsible for activation of PI3K activities in these situations ([86,87] and see below) for the physical characteristics of the PI3K activity that is regulated in this manner. This is because the mechanisms by which G proteins activate effectors are very different to those by

which PTKs activate their targets. Hence in the case of PICs, the only signalling system that has been established to be regulated by both G proteins and PTK-coordinated pathways, highly structurally distinct families of PIC (fl and y, respectively) are dedicated to coupling with each of these classes of transduction reaction [4,88,89]. Indeed, the differences between/3 and y PICs are manifest at a level such that they only possess

Fig. 8. Structure of PI3K and homologies with other proteins: (A) structure of p85 and pllO subunits. A major PI3K activity in mammalian tissues has recently been purified, 'cloned' and shown to be a heterodimer of tightly associated 85-kDa and 110-kDa subunits [34,35,38,67-69]. Schematic bar diagrams representing the primary structures of the t~ and fl isoforms of the 85-kDa 'regulatory' and the ll0-kDa 'catalytic' subunits are shown, along with related p50, Vps34 and TOR2 proteins (see below); the amino terminal is shown to the left. Regions of significant homology to domains in other proteins are variously shaded and labelled appropriately. The regions of the two subunits which are thought to interact to form the tight complex are also shown (I. Hiles and M. Waterfield, personal communication; hut see also Ref. 337). At least 4 different isoforms of the 85-kDa protein (designated a,/3, 3' and 3; [67,70], M. Waterfield, personal communication) and a truncated 50-kDa form (likely to be derived from a by alternative splicing; L. Cantley, personal communication) have been characterized. These genes are co-linear with extensive regions of amino-acid sequence homology (highest in the SH2 domains and the region between them, i.e., in the putative P110 binding domain). Antibodies specific for the t~, 13 and 3' isoforms of p85 are all capable of immunoprecipitating PI3K activity from cell lysates, indicating that they can each form tight complexes with the 110-kDa catalytic subunit ([70], and I. Gout, personal communication), pS0 is found tightly bound to membranes in preparations from rat liver, but it is not yet known whether it is associated with PI3K activity (L. Cantley, personal communication). In the tissues and cell lines thus far examined (from human, mouse, rat and bovine sources), the a-form of p85 is by far the most abundant isoform (accounting for its discovery as the major p85 which is present in purified preparations of PI3K or which could translocate to PDGF receptors) [70]. The significance of the different isoforms of p85 for conveying different properties to PI3K activities is not yet known. The gene for p85a has been localized to human chromosome 5 whilst that for p85/3 is on chromosome 19 [285]. The p85 subunit contains two SH2 domains, one SH3 domain and a region with homology to rho-GAP, n-chimaerin, p190 and bcr (the bcr domain [74,76]) [67-69]. The significance of the bcr homologous domain for PI3K function is not known. However, with the exception of the extreme C-terminal, this region shows lowest sequence homology between t~ and /3 p85s. It may therefore provide a point at which different proteins may interact with each isoform to regulate, or be regulated by, the PI3K complex. SH2 domains are thought to bind directly to specific tyrosine phosphate residues on proteins (see Appendix VII) and therefore their presence in the 85-kDa protein has obvious implications for the mechanism by which it may bind to autophosphorylation sites on PTKs (see Section IV-B). It is now thought that SH3 domains direct protein-protein interactions by binding to proline rich sequences, however for p85s, neither the identity of their target nor the significance of their binding is known. Sequences showing similarity to a consensus for the binding of abl, src and GRB-2 SH3 domains [341,343,344] are found in each of the available p85 sequences (in p85a residues 83-93 S_PPTPKPRPPR and 303-313 PAPALPPKPPK; in p85/3 residues 203-213 G_PALEPPTLPL 294-304 A_PPALPPKPPK and 714-724 A_PGPGP_PPAAR) and these are indicated in the figure. Two different p110 proteins have been observed in purified preparations of PI3K from rat liver [34] and Southern blotting has revealed that two closely related genes for the p l l 0 subunit are present in rat and human DNA [38], but the relationships between these observations and their significance for PI3K activity are not yet known, p l l 0 shares significant sequence homology with two yeast gene products; Vps34, identified as a vacuolar protein sorting mutant and TOR2, a protein essential for cell-cycle progression and in which a mutation can convey rapamycin resistance. Vps34 shows a low level of homology to p l l 0 over almost its whole length with maximum homology (33% identity) over a stretch of approx. 450 amino acids containing a single unique insertion. Since Vps34 is a PtdIns 3OH-kinase, this domain is likely to convey some form of PI3K activity [77]. A corresponding region shows maximal homology between p l l 0 and TOR2, though it contains a Vps34-1ike insertion, leading to the suggestion that it encodes a lipid kinase domain. Weaker homology to the remainder of p110 extends over the entire C-terminal half of the TOR2 protein (its N-terminal shows no homology to other known proteins and is represented only as a broken bar). This includes a region which corresponds to the calcium- and phospholipid-hinding (CaLB) domain in p110. No such region occurs in Vps34. A putative ATP-binding domain which shares some homology with the ATP-binding domains of protein kinases is marked in each protein. (B) Identification of a 'CaLB' domain in the llO-kDa subunit of PI3K. This table shows an amino-acid alignment of homologous domains present in the p l l 0 subunit of PI3K, the yeast PI3K homolog TOR2 and in cPLA2, ras-GAP, the Neurofibromatosis gene product (NF-1), PKC3,, p65 (synaptotagmin duplicated domains A and B) and various phosphoinositidase Cs (PIC/31, ~, Yl and Y3). The CaLB domain was originally identified as part of the C2 region conserved in PKCs (t~,/3s and y) and also recognized in PICy [294]. A similar domain has since been recognized as a repeat in p65/synapotagmin [295], in ras-GAP and cytosolic PLA2 (where it is implicated in Ca2+-dependent binding to membranes) [81]. A CaLB domain sequence is also present in the NF-I gene product. Each of these proteins has been reported to bind to a n d / o r be influenced by lipids and in several cases to show a preference for phosphoinositides [289,290,295-298]. Our analysis suggests that a similar domain is also present in PICs/3 and 8 and that in each case the position of this sequence is similar, lying to the COOH terminal side of the second region of the proposed split catalytic domain [301,306]. The presence of the CaLB domain in p110 and TOR2, but not in Vps34, suggests that it is not an essential component of PI3K's catalytic site but may confer particular phospholipid binding properties upon these enzymes (e.g., allowing membrane localization following activation discussed in Appendix IV and Section IV-B). The amino-acid sequences of the CaLB domain from each of these proteins are presented in standard single letter code (these are given in Table V). Residues which are identical or represent conservative substitutions (within the Dayhoff groups: GPAST: FWY: ILMV; NQED and RKH) present in >/4 of the 11 sequences are indicated by upper case lettering, and those present in >~8 sequences, and which may aid in delineating a consensus (con) for such domains, are indicated below the sequences. Numbers above the alignment simply provide a guide to the length of the alignment, those at the right hand side indicate the position of the COOH terminal residue shown in its respective protein. The sequences represented are: rat p65 [295]; bovine PKCy [299]; bovine ras-GAP [300]; human cPLA2 [81]; bovine PICS; bovine PIC/31 [301]; bovine PICy 1 [302]; rat PIC3'2 [303]; bovine p 110 [38]; yeast TOR2 [344] and NF-1 [305].

44 significant sequence homology in their putative catalytic and CaLB domains (i.e., the G-protein-regulated /3 isoforms do not contain either the SH2 or SH3 domains found in the 3' family [4,90]) and early crosshybridization strategies failed to tie them together and only revealed further isozymes within these families [90]. The discovery of the 7 and/3 families was entirely due to the fact that the scale of their difference in structure was adequate to mean they could be readily resolved by standard chromatographic techniques [91]. The potential existence of structurally distinct isoforms of PI3K may be susgested by the disparate sequences of the Vps34 and TOR2 [38,344] products in yeast. However, without confirmation that the TOR2 product is indeed an inositol lipid 3-OH kinase the significance of this as a precedent of multiple PI3 kinases in other systems is uncertain. There is limited evidence of a distinct form of PI3K activity being implicated in situations in which PtdIns(3,4,5)P3 accumulation is activated by G-protein-linked receptors. Hence the G proteins which are implicated in these pathways in myeloid-derived cells are Gi 2 a n d / o r Gi 3, however, presumed dissociation of these same proteins in permeabilized 3T3 cells (by GTPTS [20]), platelets (by adrenaline [58]) and NGll5 401L cells (by met-enkephalin [92]) failed to activate Ptdlns(3,4,5)P 3 accumulation, despite positive evidence that all of these cells contain PI3K activities (this apparent specificity could also be supplied by tissuespecific expression of Gi/37 subunits see Section V-C). However, chromatographic analysis of PI3K activities in a number of cells including neutrophils has revealed very limited variation in the gross properties of PI3K activities, to date all appeared to possess a common p 8 5 : p l l 0 format [34-36,38,71,72]. The only exception is a l l0-kDa activity purified from thymus [36], however, a PI3K with this native molecular mass could not be identified in neutrophils (Stephens et al., unpublished observations) suggesting this activity is not a 'G-protein-linked' isoform. The implication of these results is if G-protein-linked isoforms of PI3K occur in ceils they must be sufficiently divergent to have been completely undetected in either the assays used to date or in the isoforms of PI3K detected by Southern blotting [38,67]. Recent evidence of strictly activation-dependent types of PIC [94] and the precedent set by the divergent sequences of PIC/3 and 7s [4] suggest this is plausible. Alternatively, G-protein-regulated PI3Ks activities may possess a basic structure like that or possibly identical to, the known PTK-regulated PI3K activities. Although this apparently breaks the pattern established by regulation of PICs, the regulatory:catalytic subunit based make up of PI3K might be used to enable this form of flexibility. Indeed, a region of homology between p85s and part of the bcr gene product that can activate the GTPase activities of

specific low-molecular-weight GTP-binding proteins (see Fig. 8) has been suggested to represent a potential input-port for signals from GTP-binding proteins [74], implying, contrary to the small amount of evidence available on this issue, that all known PI3Ks are potentially G-protein sensitive. This suggestion is also at odds with the pertussis toxin insensitivity and the currently defined structures and functions of these low molecular weight monomeric GTP-binding proteins [54,76]. However, potentially PIC-like, signal-transduction-mechanism-specific coupling could still be generated by the presence of appropriate G-protein-related adaptations only within a particular p l l 0 a n d / o r p85 isotype. IV. How do agonists 'activate' Ptdlns(4,5)P 2 3OHkinase activities

This section considers the process by which PtdIns(4,5)P2 3OH-kinase activities physically interact with transducing proteins and attempts to dissect out of this the factors that might enable the interaction to be manifest as an increase in the rate of accumulation of PtdIns(3,4,5)P 3. The fact that Ptdlns(4,5)P 2 3OH-kinase activities seem to connect with receptor signalling pathways at the level of classical transducing proteins (i.e., receptor-regulated PTKs or G proteins), the sole functions of which are to couple receptor stimulation to the activation of a variety of signalling pathways, means that examples of signalling proteins experiencing analogous activations are already described [4,5,95], and can be used as a framework within which to consider PtdIns(4,5)P 2 3OH-kinase activities (indeed, they may be induced by the same G-protein or PTK species [14,58,96,97]). In this regard the PIC family of signalling effectors are a particularly appropriate model, because not only do they utilize the same substrate as Ptdlns(4,5)P 2 3OH-kinase activities, but they have been definitively established to be regulated by both G proteins and receptor-regulated PTKs. Consequently, at a number of levels in the following discussions we will try to clarify problems emerging from work with 3-phosphorylated inositol lipids by reference to the regulation of the analogous PIC. How are these families of critical signalling enzymes regulated by the transducing proteins? In the relatively straightforward case of the membrane localized effector adenylate cyclase, an enzyme which uses cytosolic substrates, binding to free G-protein a subunits (released from activated Gs proteins) leads to a 'simple' increase in its Vmax [98]. In the case of soluble enzymes which use membrane-associated substrates, however (i.e., PIC and PI3K), there are two additional factors that must be considered in order to understand their regulation. These are derived from the potential need

45 for soluble e n z y m e s to i n t e r a c t with m e m b r a n e s as well as their substrates [99] (this can only b e avoided by true e n z y m e ' h o p p i n g ' m e c h a n i s m s ; see A p p e n d i x IV) a n d the fact that their substrate is effectively c o n c e n t r a t e d into only a small part of the space to which they have access. H e n c e ' a c t i v a t i o n ' of a n enzyme such as P I 3 K (i.e., an increase in the rate of synthesis of PtdIns(3,4,5)P 3) can b e provided by a c h a n g e in its catalytic activity (like that described for a d e n y l a t e cyclases above), b u t also by m o d u l a t i n g processes that t e n d to c o n c e n t r a t e , 'localize' or ' t r a n s l o c a t e ' a P t d I n s ( 4 , 5 ) P 2 3 O H - k i n a s e activity to a place where its substrate is c o n c e n t r a t e d , a n d / o r by c h a n g i n g its ability to i n t e r a c t with m e m b r a n e s (this is the r e a s o n for the inverted c o m m a s we o f t e n place a r o u n d activation in the context of P t d I n s ( 4 , 5 ) P 2 3 O H - k i n a s e activities). T h e fol-

®

lowing sections c o n s i d e r how o n e or m o r e of these p a r a m e t e r s might be affected by the i n t e r a c t i o n of P t d l n s ( 4 , 5 ) P 2 3 O H - k i n a s e activities with receptortransduction mechanisms.

IV-A. Interactions between Ptdlns(4,5)P2 30H-kinase activities and receptor-regulated PTKs T h e r e are several m e c h a n i s t i c a n d structural t h e m e s that recur in the activation of signalling p r o t e i n s by all k n o w n r e c e p t o r - t r a n s d u c i n g PTKs. It has b e e n argued, however, that t h e r e may be differences in the precise s e q u e n c e a n d significance of some of the p a r t i c i p a t i n g events, d e p e n d i n g u p o n w h e t h e r they have b e e n derived by a src-type-, or receptor-, PTK. H e n c e the following sections c o n s i d e r i n d e p e n d e n t l y , the regula-

.......- -"

"•

"

p110 ~

Ptdlns(4'5)P2

p85

Ptdlns(3,4,5)Pa

PI3K

pPT~K K ~

® SH2

p110 ~ PI3K

Ptdlns(4'5)P2

I "~.~Ptdlns(3,4,5)P3

Cytosol Fig. 9. Regulation of Ptdlns(3,4,5)P3 accumulation by receptor-PTKs. Schematic models are presented of possible SH2: tyrosine-phosphate interactions responsible for causing the 'translocation' of PI3K into receptor-PTK-coordinated complexes; symbols are used qualitatively and do not represent the numbers of entities present; sites for activated, PTK-mediated tyrosine phosphorylation ( ~ ) are shown as P; SH2 domains are portrayed as SH2; the potential interactions between SH2 domains and tyrosine phosphates are indicated (--.). In the absence of agonist the receptor subunits are in their PTK-inactive, monomeric configurations and PI3K is predominantly soluble and is not manifest as a PtdIns(4,5)P2 3OH-kinase. The effects of agonists on basal systems are shown in A and B. (A) Translocation of PI3K to activated receptor-PTKs. This is a generalized model which is likely to apply to a number of receptor-PTKs, e.g., PDGF-receptor, CSF-1 receptor and EGF-receptor (see Table II for a complete list). (B) Translocation of PI3K to insulin receptor substrate protein-1. This is a model which applies to insulin stimulated translocation of PI3K into phosphotyrosine-containing complexes. It appears that the major site of PI3K binding is a 185-kDa substrate of the activated insulin-receptor-PTK, the insulin receptor substrate protein (IRS-I) [86,122]. IRS-I was originally characterized as a soluble protein [286] but some evidence suggests that it may associate with specific intracellular organelles [241,287]. It is not known for certain whether a small amount of PI3K binds to the insulin receptor directly [24,124,261], or whether a ternary complex containing the insulin receptor, PI3K and IRS-I can exist [86]. The concept that PI3K binds or physically translocates to receptor-PTKs relies on surprisingly limited evidence that the mass of PI3K present in receptor nucleated signalling complexes rises on stimulation. The majority of reports of PI3K translocations in intact cells actually describe increases in activity in anti-receptor antibody directed immunoprecipitates and/or increases in the phosphotyrosine content of 85-kDa proteins (see Table II), and hence could be explained by an association and/or an activation or phosphorylation of a prebound enzyme. However, use of recently available antibodies to the p85 subunit of PI3K has indicated that, at least in a limited number of examples of cell stimulation, translocation of PI3K is likely to occur [86,127]. Further, the translocation of PI3K to activated receptor-PTKs has been well documented in several in vitro 'reconstitution systems' (e.g., Refs. 67,68,70,274,308).

46 tion of Ptdlns(4,5)P 2 3OH-kinase activities by src-typeand receptor-PTKs. The arguments presented in Sections II and III indicate that the PI3K that has been purified and 'cloned' is in fact the prototypic (the only?) example of a PI3K activity that is regulated by PTKs, and that this enzyme is expressed as a PtdIns(4,5)P 2 3OH-kinase activity when 'activated' by them. Hence, throughout this section we will be referring to PI3K as the PTK-regulated Ptdlns(4,5)P 2 3OH-kinase activity. IV-B. Interactions between receptor-PTKs and PI3K Cell surface receptor-PTKs are composed of one or more single membrane-spanning polypeptide chain(s) with an extracellular domain which recognizes the appropriate ligand and an intracellular domain which possesses intrinsic PTK activity. Activati6n of these receptors by the appropriate ligand is usually thought to cause dimerization of homologous subunits and to activate the PTK contained within them (reviewed in Refs. 5,100). The activation of the PTK intrinsic to the receptor is known to be essential for the receptor to mediate its appropriate effects on the target cell. An early puzzle, however, was that the major intracellular target for this PTK was the receptor itself (i.e., receptor autophosphorylation, possibly mediated by crosssubunit phosphorylation in the active homodimer). This seemed at odds with the precedent set by the control of enzymes by serine/threonine protein kinases, where it is the target enzyme which is phosphorylated. The recent purification and cloning of proteins which bind directly to these receptors on stimulation (of which PI3K was one of the first) has, however, begun to reveal the rationale behind receptor-PTK autophosphorylation and to provide a new perspective on the use of PTKs and protein phosphorylation in general, to control enzyme activity. A mechanism for agonist-stimulated translocation of PI3K to receptor-PTKs is shown in Fig. 9A. It is based on recent observations that (1), specific tyrosine autophosphorylation sites on receptor-PTKs are required for the binding of particular proteins [96,101-107] (2), the primary sequences of proteins known to associate with receptor-PTKs in an agonist-dependent fashion contain one or more SH2 domains (e.g., PI3K [67-70], ras-GAP [73,103,105,108], PICy [90,107,109], c-src, fyn, c-yes [47,110], vau [111] and GRB2 [112]; although the binding of raf may be an exception [113,114]) and (3), SH2 domains display a high affinity for tyrosine phosphate residues on proteins [73,115-118] (see Appendix VII). Thus, agonist-induced activation of the receptorPTK results in the autophosphorylation of key tyrosine residues in the receptor cytoplasmic domain and these tyrosine phosphates are then envisaged to act as cues for the recruitment of SH2-bearing proteins to the receptor, including the 85-kDa/l10-kDa PI3K het-

erodimer (although these tyrosine phosphate cues are currently considered products of autophosphorylation reactions they could also be supplied by other receptor-PTKs, or non-receptor PTKs). Hence PI3K can be immunoprecipitated from stimulated cells by antibodies to either the receptor (e.g., PDGF [12], EGF [119], IL-2 [120], CSF-1 [26] receptors; because the SH2: tyrosine phosphate-mediated receptor:PI3K interaction is sufficiently strong to survive standard washing protocols used to dissociate 'non-specifically' bound protein from immunoprecipitates) or to phosphotyrosine (~.g., Refs. 12,20,23,24; presumably through 'free' tyrosine phosphates present either on PI3K itself or a tightly associated protein, such as the receptor). Although the insulin receptor contains an intrinsic PTK which has the potential to self-phosphorylate, the mechanism by which it stimulates translocation of PI3K appears to be distinct from the receptor-PTKs described above (Fig. 9B). The major substrate of the insulin receptor-PTK is a 185-kDa protein(s) (IRS-1) [121], that becomes phosphorylated on multiple tyrosine residues in response to insulin and appears to be the predominant site to which PI3K translocates [86,122-124]. Indeed, a recent study has shown that insulin treatment of rats leads to the association of PI3K with IRS-1 in both liver and muscle, two of the main metabolic targets of insulin, indicating that this association is relevant in living animals [339]. Some evidence suggests the NGF receptor may activate PI3K via a 100-110-kDa phosphoprotein [125] in a manner analogous to that by which the insulin receptor uses IRS-1; however, it appears PI3K can also interact with the NGF receptor directly [126]. Hence activation of receptors containing intrinsic PTK activities can culminate in the formation of membrane-localized signalling complexes containing collections of mainly cytosolic proteins, including PI3K, that have been apparently anchored in place by the binding of their SH2 domains to tyrosine phosphate residues on the host receptor (the mechanism by which receptors can actually select the proteins to be recruited is discussed in Section V-B.1) and these events correlate with the activation of a PtdIns(4,5)P2 3OH-kinase activity in the context of an intact cell. Furthermore, the process of recruitment of PI3K to activated receptors can be effectively accomplished without an apparent need for tyrosine phosphorylation of PI3K [38,101] nor do the levels of tyrosine phosphorylation of PI3K apparently change in response to stimulation [86,127] (this is discussed in Section IV-B.1). Clearly this suggests the process of association of PI3K with the signalling complex via SH2 domain:tyrosine phosphate docking can lead to an increase in Ptdlns(3,4,5)P 3 and, therefore, that PI3K is 'activated' by it. There are several levels at which PI3K activation could be provided (see above). The shift from cytosol

47 to the plasma membrane environment of the signalling complexes could, by concentrating the enzyme in the vicinity of its substrate, lead to an activation (although this does require that PI3K can still gain access to Ptdlns(4,5)P 2 whilst tethered in this way). The potential significance of this type of membrane localization can be discerned in the failure of non-transforming, myristoylation-defective mutants of v-src and v-abl to localize to the plasma membrane, or to increase basal accumulation of Ptdlns(3,4,5)P3, in the manner of their parental oncogenes [53,129] (this is discussed in Section IV-C.1). This somewhat starkly implies that translocation of what is probably a very small% of total soluble PI3K (at least in ceils that are expressing wild-type levels of receptor-PTKs) (e.g., Ref. 9) can lead to a very large and rapid increase in the rate of Ptdlns(3,4,5)P 3 production, purely on the basis of a rise in the local concentration of enzyme (without an accompanying change in any of the catalytic constants of the enzyme). This view is softened if the membrane-anchored signalling complex is envisaged to activate PI3K by enabling controlled, orientation-specific interactions between a bound PI3K and its substrate (see Appendix IV). Recent evidence, however, indicates that the SH2 domains of PI3K may play a more positive role in its activation, than that of being mere membrane-localizing anchors. Two groups have shown that soluble PI3K activities can be activated directly in in vitro assays by tyrosine phosphorylated IRS-1 or appropriate tyrosine phosphorylated peptides (these were based on sequences surrounding the tyrosine phosphate moieties to which PI3K is thought to bind in IRS-1, the PDGF receptor and mT) [86,87]. Although the scale of the activations reported were small, they were defined under assay conditions in which, in contrast to the cellular situation, 'unbound' PI3K would be active (i.e., using exogenous lipids; see Appendix I). Interestingly, the native, multiply tyrosine phosphorylated protein activated PI3K with greater potency than doubly tyrosine phosphorylated peptides and these were more potent than singly, and in turn unphosphorylated peptides, indicating a structure-activity series that was consistent with this representing an authentic effect that could be expressed in vivo. Furthermore, these results suggest that activation might depend on both of PI3K's SH2 domains becoming bound to tyrosine phosphate cues (this conclusion dovetails with some reports that two tyrosine phosphate residues in the PDGF receptor are required for it to express maximal affinity for PI3K [103]; discussed in Section V-B.1). Thus, these results suggest that the allosteric forces generated by the binding of the SH2 domains within the 85-kDa subunit of PI3K to the appropriate phosphotyrosine target can enhance the catalytic potential of PI3K in the absence of receptor-directed translocation or tyro-

sine phosphorylation of PI3K. Hence, in vivo, this mechanism might act in concert with the process of translocation to provide an effective activation of PI3K and an increase in Ptdlns(3,4,5)P 3 accumulation. The precise mechanism by which SH2 domain docking might contribute to 'activation' of PI3K is unclear but there are two distinct (but not mutually exclusive) alternatives. Firstly, by eliciting a change in the ability of PI3K to interact with the phospholipid structures present in the assays (mediated by a domain that could be structurally and functionally unrelated to the catalytic centre of pll0, see Appendix IV; this task might be performed by the CaLB domain, see Fig. 8) or secondly, by a direct, allosteric impact on the catalytic properties of Pll0, presumably transmitted via the site of p85 : p l l 0 interaction. The existence of a functional link or 'physical connectivity' between the SH2 domains of p85 and the p l l 0 subunit of PI3K required by this form of mechanism, is supported by circumstantial evidence that the tyrosine phosphate binding specificity of free p85 is lower than that of p85:pl10 complexes [67,71]. A plausible mechanism by which SH2 dom a i n : p l l 0 communication could increase PI3K activity is by liberation of p l l 0 with a resultant release from a p85-mediated inhibition (this might be inferred from reports of a high specific activity, ll0-kDa protein-associated activity in bovine thymus [36]). However, in one study of phosphotyrosine peptide-induced activation of PI3K, the phosphopeptide:PI3K complexes were immobilized via p85 and extensively washed prior to assay [86], suggesting this explanation is invalid. IV-B.1. The role of tyrosine phosphorylation in the regulation of PI3K Throughout the preceding consideration of receptor-PTK-mediated activation of PI3K, no part was identified for a PI3K-directed PTK activity. This view of events is only just crystallizing and goes against considerable expectation, and the strong precedent set by regulation of PICy~, that 'tyrosine phosphorylation' of PI3K would result in its activation. The critical role for tyrosine phosphorylation of PICy~ (at residues 783 and, to a lesser extent, 1254) in coupling-activated receptor-PTKs to increased inositol phosphate production, has been elegantly demonstrated by constructing various point mutations of PICy 1 and then analyzing their abilities to potentiate PDGF-stimulated inositol phosphate production [130]. Mutants of PICyl lacking the appropriate tyrosine sites for phosphorylation by the PDGF receptor-PTK failed to couple receptor activation to increased inositol phosphate production, despite the fact that they showed wild-type levels of translocation. Further, meticulous kinetic studies have revealed that tyrosine phosphorylation of PICy I can lead to its activation [131,132] (that was largely manifest as a change in the

48

ability of PICy, to effectively interact with phospholipid-containing micellar structures; could this be another example of a ‘CaLB domain’-mediated interaction?). Thus, these studies establish that tyrosine phosphorylation of PICy, is critical to the processes by

which receptor-PTKs activate this enzyme. However, complementary studies of the effects of substituting the sites in the FGF [133,134], and EGF receptor-PTKs 11351 to which PICy, binds, have also revealed that effective docking of PICy,‘s SH2 domains is essential

Fig. 10. Activation of PI3K by receptors utilising src-type PTKs. A diverse and expanding group of receptors are considered to interact directly with and utilize non-receptor PTKs to transduce their signals (see Table III), including those for IL-2, IL-7, IL-3, GM-CSF, growth hormone and erythropoietin, and also the mlgM, mlgE, CD3/TcR, CD4 and CD2 receptors (e.g., Refs. 263-265). A number of these have been shown to regulate PI3K activity (see Table III) in various assays. However, the detailed mechanisms by which this is achieved are not understood. The figure schematically portrays a currently plausible model for the GM-CSF receptor [25]. (A) Unstimulated cell in which PI3K is inactive. The ligand-binding (Y and signal-transducing /.? subunits of the GM-CSF receptor are in their unstimulated, basal conformation but are nevertheless mutually associated and a src-type PTK is bound to the cytoplasmic domain of the p subunit (in the case of the GM-CSF receptor in neutrophils this could be lyn or c-yes [25]; these interactions are not SH2: tyrosine-phosphate based). The src-type PTK is inactive, possibly as a result of an intramolecular SH2: tyrosine phosphate interaction (src-type PTKs have a single SH2 domain; the phosphate may be supplied by a PTK-kinase, PTK-K), but membrane localized as a result of its association with a receptor and/or myristoylation [25,46,84,85,143,128,260,309,310]. (B) Stimulated cell in which PI3K is active. Upon agonist binding the src-type PTK becomes activated. There is no clear, common picture of how this is achieved, but current evidence suggests the following are possible; (1) by direct allosteric interaction between the receptor and src-type PTK causing activation of the PTK (by releasing its intramolecular constraint?) or (2) by agonist-induced loss of either a receptor-imposed block, or an might be inhibitory phosphate ([PI) in the src-type kinase (by a phosphotyrosine protein phosphatase, PTPPase +; this dephosphotylation stimulated by direct receptor-activation of a PTPPase or by a receptor-src-type PTK interaction which increases the susceptibility of the src-type PTK to attack by the phosphatase) (see Refs. above). The active PTK phosphorylates itself and target proteins ( + 1 possibly including the p subunit of the host receptor and PI3K. PI3K is bound into a complex (--+), probably via its SH2(s) docking with target tyrosine phosphate sequences (there are no tyrosine residues in either the src-type kinases or the majority of receptors in this family that are found within an ideal PI3K-binding consensus sequence; discussed in Section V-B.l). Alternatively, PI3K may in principle bind via its tyrosine phosphates to the src-type PTK’s SH2 domain. The process of recruitment into this complex activates PI3K resulting in increased production of PtdIns(3,4,5)P,. V-src has been suggested to interact with PI3K via a mechanism entailing docking of its SH2 domains to tyrosine phosphates on PI3K [1,136], and hence it could be argued receptor-associated src-family PTKs might regulate PI3K in the same manner. However, the notion that PI3K interacts with the host receptor or a distinct substrate of the src-type PTK (depicted as (?) in the figure), rather than the src-family PTK itself, draws strength from: (1) the precedent set for this mode of regulation by the receptor-PTKs (particularly in the way the insulin receptor uses IRS-I as the major target for PI3K binding; see Fig. 9); (2) evidence that stimulation by this group of receptors can promote the association of PI3K with the receptor, receptor-associated, or receptor-directed, proteins (Table III); (3) a currently defined mechanism of activation is built into the process (discussed in Section IV-B). (4) an analogy with the surrogate receptor-style with which mT binds to PI3K (discussed in Section IV-C.2). (5) the observation that PI3K does not bind to phosphotyrosine peptides containing the tyrosine residues of c-src that are its major sites of tyrosine phosphorylation [71]; (6) the fact that this mechanism naturally confers a level of receptor specificity (such that the system’s output signals would not be governed solely by the identity of the associated src-type PTK), and hence also introduces a flexibility that avoids the problems associated with signalling through a relatively limited selection of src-type PTKs.

49 for PIC 'activation'. Further, in the case of the EGFreceptor mutants, EGF was still able to induce wildtype tyrosine phosphorylation of PIC3,1 (both in magnitude and in the nature of the sites phosphorylated) [135]. Taking all of this data together, it suggests that SH2-mediated docking of PIC3,1 to the receptor-PTK, and the receptor-PTK-mediated tyrosine phosphorylation of PIC'Y1, can occur as independent events and yet they must both co-operate in some way in the stimulated production of inositol phosphates [135]. No evidence for direct activation of PIC3, by the docking of its own SH2 domains to tyrosine phosphorylated target proteins or peptides has yet been reported. It will be interesting to know whether this occurs, to enable a clearer insight into the role of SH2 domain binding in the activation of inositol phosphate production and, more generally, to compare the principles on which activation of PIC3q and PI3K are based. The expectation that receptor-PTKs would activate PI3K by tyrosine phosphorylating it, has been based on a large amount of predominantly circumstantial data. Hence, reports have been published showing that (a) PI3K (both p85 and p l l 0 subunits), p85a and p85/3 can be isolated from cells in a tyrosine phosphorylated condition [12,70,72,126,136,137]; (b) the level to which they are tyrosine phosphorylated can be enhanced by co-expression of receptor-PTKs [70]; (c) purified complexes containing activated receptor-PTKs can phosphorylate associated PI3K, p85a and p85fl [70] and, finally, (d) that specific protein tyrosine phosphate phosphatases can significantly inactivate purified bovine brain derived PI3K [72]. Although these observations make the right connections they do not identify the tyrosine residues that are implicated in each situation and therefore do not identify whether the effects may be related. Furthermore, the critical experiments which indicate that agonist-stimulated tyrosine phosphorylation of PI3K may occur in intact cells are based on data using antiphosphotyrosine antibodies to either immunoprecipitate PI3K activity or 32P-labelled 85-kDa proteins (presumed to represent a subunit of PI3K) and could therefore have been detecting tyrosine phosphorylation of protein(s) associated with PI3K or translocation of a pre-phosphorylated PI3K. The most informative experiments to date are those which have utilized antiphosphotyrosine antibodies to quantify the level of tyrosine phosphorylation of 85-kDa proteins in Western blots of protein initially immunoprecipitated by specific anti-p85 antibodies. These experiments have shown either small [126] or undetectable [86,127,224] changes in tyrosine phosphorylation of p85 in response to PDGF and NGF, or PDGF and insulin, respectively, indicating that PI3K does not undergo stimulated tyrosine-phosphorylation on the same scale as PIC3q in analogous situations (see above). Although none of these experiments are definitive,

and we still await studies of point mutations in the critical tyrosine residues of PI3K and the relevant receptors (i.e., studies analogous to those on PIC3'I), they do suggest that tyrosine phosphorylation of PI3K probably does not contribute to the activation of this enzyme by these growth factor receptors. IV-C. Interactions between src-family PTKs and PI3K

PI3K was originally discovered as a protein which tightly associates with transforming, viral PTKs. Some of these viral PTKs are now considered to be constitutively activated forms of cellular src-type PTKs. These cellular src-type PTKs are envisaged as being normally quiescent, but can be stimulated by specific cell surface receptors for certain growth factors and cytokines. These receptors belong to a diverse class that do not interact with heterotrimeric G-proteins, or possess intrinsic PTK activity, but appear to associate, via their cytoplasmic domains, with various non-dedicated PTKs (predominantly from the src family [1] but also including more recently characterized species such as ZAP-70 [138] and perhaps FAK-125 [139]) and to use them to transduce their signals (see Fig. 10 and Table III). This pattern of observations can be readily extrapolated to predict that (i) cells transformed by expression of oncogenic PTKs or by oncoproteins that can activate normal cellular src-type PTKs (e.g., mT) might contain permanently elevated levels of Ptdlns(3,4)P 2 and PtdIns(3,4,5)P 3 and (ii) receptors utilizing normal cellular src-type PTKs to transduce their signals will also activate, although relatively transiently, PI3K and hence production of Ptdlns(3,4,5)P 3. Recent experiments suggest this logic is sound, hence ceils expressing mT (and hence containing activated c-src and possibly c-yes and c-fyn), v-src, v-abl or v-yes have now been shown to contain chronically elevated levels of Ptdlns(3,4,5)P 3 and Ptdlns(3,4)P 2 [51-53,129]. Moreover, some of the receptors thought to interact with src-type PTKs have been reported to stimulate accumulation of PtdIns(3,4,5)P 3 (see Table III). These data suggest that a Ptdlns(4,5)P 2 3OH-kinase activity, probably due to PI3K [25,84], can be activated by either receptor-dependent or independent pathways that contain src-type PTKs. How do these receptors which utilize src-family PTKs couple to PI3K activities? The answer is currently far from clear and there is a great deal of confusion in this literature, resulting from incomplete or apparently contradictory data. To date, of those ligands thought to primarily utilize src-type PTKs in their signalling pathways (see Fig. 10 and its legend), only GM-CSF [25] and IL-2 [120] have relatively clearly defined connections between activation of their receptors and increased accumulation of PtdIns(3,4,5)P 3. Stimulation of the receptor for GM-CSF can activate

the src-type PTKs lyn and c-yes, PUK activity translocates to complexes containing these PTKs and cellular levels of PtdIns(3,4,5)P, rise rapidly [25]. The molecular interactions that negotiate these steps and hence the manner in which PI3K is activated and converts this into an increase in PtdIns(3,4,5)P, production is discussed below. IV-C.1. Transforming src-type PTK-mediated accumulation of Ptdlns (3,4,5) P3 In the ‘simplified’ systems containing constitutively active (i.e., viral), src-family kinases or the mT: c-src complex, in which the need for receptor stimulation is short-circuited, the increase in basal levels of PtdIns(3,4)P, and PtdIns(3,4,5)P, depends on PTK activity and complexes containing tightly associated src-type PTKs and tyrosine phosphorylated PI3K can be isolated from the transformed cells [1,6,11,51-53,65,70, 129,136,137,143]. Furthermore, transforming, or mTactivated, src-type kinases can tyrosine phosphorylate PI3K and/or p85 in vitro [67,70,137]. These observa-

tions have led to the suggestion that P13K is tyrosine phosphorylated by the transforming src-type PTKs, and then bound via these tyrosine phosphates to the SH2 domain of the PTK (although there is no direct evidence that such an interaction exists in these complexes) [l]. However, this model does not enable the ‘activation by SH2 docking’ mechanism thought to operate in PI3K’s interactions with receptor-PTKs to explain an increase in accumulation of PtdIns(3,4,5)P, (because the orientation of the SH2:tyrosine phosphate interaction is reversed). Moreover, the location of tyrosine residues in PI3K that become phosphorylated and whether these are activating have yet to be established. The only positive evidence suggesting a possible mechanism by which PI3K is activated in these complexes has been provided by the finding that non-transforming, myristoylation-defective mutants of v-src and v-yes, which fail to localize to the plasma membrane (but complex with and apparently tyrosine phosphorylate PI3K to wild-type levels), also fail to increase

51 basal PtdIns(3,4,5)P, accumulation [53,129]. These results with myristoylation-defective mutants indicate that the capacity of transforming src-family PTKs to increase basal accumulation of PtdIns(3,4,5)P, might reside in their ability to translocate to the plasma membrane (this argument was used earlier as evidence that translocation may be a plausible mechanism for the activation of PI3K by receptor-PTKs; see Section 1V.B). IV-C.2. mT: c-src-PTK complex-mediated of PtdZns(3,4,5)P3

accumulation

In cells transformed by expression of polyoma middle T antigen (mT) an activated mT: c-src complex forms in which mT is phosphorylated on tyrosine 315 by c-src and this tyrosine phosphate then binds PI3K via PI3K’s SH2 domains [1,11,12,66,137,140-1421. Al-

though PI3K can be tyrosine phosphorylated by C-UC on both p85 and ~110 during formation of mT : c-src nucleated complexes, the role of these phosphorylations is unclear [137]. This mechanism can draw on the established effects of both membrane localization and docking of PI3K’s SH2s to potentially explain the activation of PtdIns(3,4,5)P, accumulation. Such a process has now been described for the recruitment of the murine SOS1 protein via its associated GRB-2 adaptor in v-src transformed cells, where v-src phosphorylates tyrosine residues on the SHC-1 protein (a normal cellular target of receptor and src tyrosine kinases; [342, 3461) which then act as recruitment sites for the single SH2 domain of GRB-2 [342]. In many ways this view of mT : c-src complex-mediated activation of PI3K may be a better precedent for how receptor-stimulated, srctype PTKs might increase PtdIns(3,4,5)P, accumula-

Fig. 11. G-protein-regulated PtdIns(3,4,5)P, accumulation in neutrophils. The mechanisms by which FMLP might activate PtdIns(3,4,5)Ps accumulation in neutrophils. Symbols and abbreviations are as in Figs. 9 and 10. The FMLP receptor like all other members of the family of ‘seven-transmembrane-domain’ receptors is thought to interact via regions in its 2nd and 3rd cytoplasmic loops with the carboxy-terminal portion of the GTP-binding (Y subunits of heterotrimeric G-proteins [55,311]. This interaction effectively accelerates exchange of GDP for GTP and disassociation of the complex, ultimately resulting in release of free, potentially effector-activating, 1yand Py subunits (it is usually the a subunits that are responsible for effector activation; however, recent evidence suggests a more widespread role for py subunits; see below). Hydrolysis of the a subunit-bound GTP to GDP, by an intrinsic GTPase, and reassociation with Py, inactivate the system [54,150,185]. G-protein-coupled signalling pathways typically convey rapid and intense signals that are also relatively transient compared to PTK-mediated receptor activations of isoforms of the same enzymes. This is a result of the interactions leading to activation being rapid; the effector is usually either associated with the membrane (e.g., adenylate cyclase, [98,152]) or the membrane-associated cytoskeleton (e.g., phospholipase C p [I5611 and hence does not experience a large-scale recruitment from cytosol to membrane and the modification to the effector is non-covalent. Because the primary inactivation mechanism is built into the activation pathway (and may indeed be stimulated by its activation), signalling can be stopped similarly quickly. In FMLP-stimulated neutrophils, Gi, and/or Gi, are dissociated 1971.Although any of the released subunits could, in principle, be responsible for activating pathways leading to a PtdIns(4,5)P, 30H-kinase, there is good evidence that it is free Py subunits that activate PIC and hence production of the second messengers Ins(1,4,5)Pj and diacylglycerol[191]. There are four known isoforms of PIC that are sensitive to dissociated G-protein subunits; p,, &, & (41 and an incompletely characterized, soluble ~86 PIC in brain [313]. p, and & are known to be widely distributed and pz is particularly abundant in neutrophils [185]. All three p-isoforms can be stimulated by free, activated (Ysubunits from the Gq (but not Gi) family of G proteins (pt is activated by (Ye, or1 > crr4, ‘Y,~; & is activated by cxr6, (which is particularly abundant in haemopoietic cells), > LYE,a,,, a14 [189,190]. However, PICP,, & and p, are also activated by By subunits from neutrophil- or brain-derived Go and Gi preparations (PICp, > PI@, > PICp,) [172,185,191], providing a pertussis toxin sensitive mechanism for the activation of PIC. It is not yet clear what factors determine the relatively restricted tissue distribution of pertussis toxin-sensitive activation of PIC: the relatively widespread occurrence of PICp, and Gi suggest that it may be due to a specific interaction between receptors and G, and/or the capacity to produce sufficient dissociated py (1721in the appropriate tissues. FMLP- and ATP-stimulated accumulations of PtdIns(3,4,5)P, in neutrophils and U937 cells are driven by pertussis-toxin-sensitive, (G-protein-mediated pathways. These pathways are not stimulated by increases in Ca’+, diacylglycerol, CAMP or PtdOH, nor are they dependent on a secreted extracellular mediator or on the stimulation of adenylate cyclase, phospholipase A,, D or PTK activities, or integrin receptor function [14,15,45,57]. Hence in the figure we use single arrows to connect free G-protein subunits to PtdIns(4,5)P, 30H-kinase activity. This can only be accepted however, once the interaction has been reconstituted in vitro. The PtdIns(4,5)P, 30H-kinase in the figure that we imply can be regulated by G-protein subunits is described in accord with reports of the properties of a major soluble activity in neutrophils (approx. 45-50% of total assayable activity in lysates). This neutrophil-derived PtdIns(4,5)P, SOH-kinase activity was clearly immunologically distinct from a p85a-epitope-bearing PI3K activity in the same cell (nor was it recognized by p85p or y directed antibodies) but had identical chromatographic properties (including an apparent native molecular mass of 200 kDa) and may therefore have a similar p85/pllO format (Stephens et al., unpublished observations). We consider this connection plausible because; (i) of indications that the G-protein-regulated P13K activity may be distinct to the PTK-regulated, p85a-associated PI3K (discussed in Section III-B.2); (2) it is a major activity in neutrophils (consistent with the large activations of PtdIns(3,4,5)P, accumulation that FMLP can activate via a G-protein-dependent mechanism) and; (3) G-protein-regulated PICs have previously been found to be apparently predominantly ‘soluble’ and been purified from cytosolic fractions [4,312-3141. Despite the apparent PTK independence of the large, dramatic accumulation of PtdIns(3,4,5)P, in FMLP-stimulated neutrophils, FMLP can increase PI3K activity in antiphosphotyrosine-antibody-directed immunoprecipitates in a G-protein-dependent manner (discussed in Sections III-A.2 and IV-C.4 and Table III; though this effect is sufficiently small that it is easily missed [315]). We suggest, on the basis of precedent, that this is driven by PIC-derived Ca2+ and/or PKC signals. These signals elicit formation of a signalling complex containing an activated form of the src-type PTK lyn [57]. lyn is then envisaged as phosphorylating target proteins, possibly including a membrane-localized protein (analogous in function to IRS-I) that might serve as a recruitment target for SH2-domain-bearing proteins, including PI3K However, this FMLP-stimulated, G-protein- and PTK-dependent pathway makes a very small ( < 10%) contribution to the increased accumulation of PtdIns(3,4,5)P, that occurs during the first 10 s of stimulation [57,315].

52 tion than that offered by transforming src-type PTKs like v-src (see below and the legend to Fig. 10). IV-C.3. Receptor-stimulated, src-type PTK-mediated accumulation of Ptdlns(3,4,5)P3 The cytoplasmic domains of the IL-2 receptor, the CD3/TcR complex, mIgM, CD4 and mIgE form close associations with various normal, cellular, src-type PTKs (including, lck, lyn, fyn, c-yes and blk, see Fig. 10 and Table III; these interactions are present without receptor stimulation and are not based on SH2: tyrosine phosphate interactions). These associations are thought to enable receptor-stimulation to activate the associated src-family PTK [1]. The receptor-activated src-type PTKs are then envisaged to tyrosine phosphorylate themselves, the host receptors a n d / o r other target proteins. PI3K has been shown to be translocated into, and presumably membrane localized by, these signalling complexes [25,84,120,224], and hence this could in itself contribute to increased formation of PtdIns(3,4,5)P 3. Receptor-associating src-type PTKs have been demonstrated to form complexes with tyrosine phosphorylated p85a and /3 in vivo and tyrosine phosphorylate them in vitro [70] and, furthermore, stimulation of src-type PTK-associating receptors can lead to an increase in PI3K activity recovered in antiphosphotyrosine antibody directed immunoprecipitates (see Table III). However, none of these results establish whether PI3K is tyrosine phosphorylated in response to this class of agonists nor do they define the orientation of any SH2:tyrosine phosphate interactions which might be formed. In this regard, IL-4 stimulation of FDCP-2 cells has recently been shown to promote a relatively small association of PI3K with the IL-4 receptor itself, but a much larger association of PI3K with an unknown 170-kDa protein, which becomes heavily tyrosine phosphorylated on stimulation [224]. Furthermore, the p85 subunit of PI3K does not itself become tyrosine phosphorylated on IL-4 stimulation (a p85 protein could not be recognized by an antiphosphotyrosine antibody in anti-p85 antibody directed immunoprecipitates, [224]), suggesting an obvious analogy with the mechanism by which insulin promotes the association of PI3K with IRS-1 i.e., the SH2 domains of PI3K may engage with tyrosine phosphates on p170. However, this interaction between PI3K and p170 has yet to be shown to be direct and the identity of the PTK responsible for the phosphorylation of p170 is unknown (the IL-4 receptor is a member of the cytokine receptor family and would thus be predicted to utilize a src-type PTK in its signalling mechanism, but this has yet to be demonstrated). A further possible mechanism of regulation of PI3K by src type tyrosine kinases is suggested by the observation that the Ick PTK may bind via its SH3 domain

to a proline rich sequence present in p85 (Chris Rudd, personal communication; see Fig. 8). However, this complex is detected in unstimulated cells and as yet there is no correlation between this form of intermolecular interaction and PI3K activation. In the absence of any definitive data concerning the mechanism by which receptors use src-type PTKs to activate PI3K, the pattern of interactions between receptors, src-type PTKs and PI3K shown in Fig. 10 is constructed to resemble those found in receptor-PTKand mT:c-src--PI3K complexes, where 'activation' might be driven by the consequences of PI3K's SH2 domains docking to membrane-localized tyrosine phosphates. IV-C.4. Receptor-stimulated, G-protein and src-type PTK-mediated accumulation of Ptdlns(3,4,5)P3 It has been apparent for several years that in addition to receptors that use intrinsic or physically associated, non-receptor PTKs to transduce their signals, some G-protein-coupled receptors can regulate PTKs via their primary signalling mechanisms. For example, bombesin, endothelin, FMLP, thrombin and vasopressin can all elicit increased tyrosine phosphorylation of proteins via both calcium- and PKC-dependent mechanisms [48,49,57,65,144-147]. The identity of the PTKs responsible for coupling these reactions have not been established but there are indications that src-type PTKs [57,65,148] and p125 FAK [49] may play a role in coupling G-protein-linked receptors to formation of signalling complexes capable of tyrosine phosphorylating proteins, and furthermore, that PI3K may be recruited and hence activated by these complexes [57,65] (Table III). However the 'Ptdlns(3,4,5)P 3 responses' elicited by these pathways may be relatively small (with the possible exception of thrombin-stimulated platelets [62,149]?) and, moreover, the location of these complexes and the mechanisms by which the src-type PTK is activated are unclear (discussed in Section III-A.2 and Fig. 11). IV-D. Interaction between Ptdlns(4,5)P2 30H-kinase actiuities and G proteins Precisely the same set of potential mechanisms that we considered could lead to increased production of Ptdlns(3,4,5)P 3 via PTK-dependent pathways apply to a putative directly G-protein-mediated pathway. The identity a n d / o r characteristics of a G-proteinregulated Ptdlns(4,5)P 2 3OH-kinase activity and hence the precise molecular interactions that underlie an associated increase in the production of PtdIns(3,4,5)P 3 have yet to be defined. However, the properties of known G-protein-regulated signalling systems actually place significant restraints on all of these parameters (see Fig. 11 for a brief discussion and Refs.

53 59,95,98,150,151 for a complete analysis). The most obvious demand placed on an enzyme by the requirements of a G-protein-transducing system is for it to possess the structural domains necessary to bind to the subunits released from activated G proteins. It must also coordinate (i) the presentation of this binding site at the precise location where the potentially activating, G-protein-derived signal will be expressed, with (ii) the need to gain access to its substrate. Further, in order to achieve the observed rapid response times of G-protein regulation, it must accomplish these processes rapidly. One of the best understood examples of G-protein regulation is that of adenylate cyclase [98]. Adenylate cyclase is a membrane-associated protein that possesses both hydrophobic and hydrophillic domains [152] which probably confer on it a defined orientation within the membrane/cytosol interface. This closely defined orientation can be exploited by using it to place a putative G-protein-binding site (sensitive to active a S subunits) in an appropriate position for effective interaction with a membrane localized ligand and to simultaneously hold its catalytic domain in an orientation such that it gains effectively unlimited access to its cytosolic substrate. In this way adenylate cyclase uses its location to aid the process of speedily conveying a signal from the membrane into the cytosol. The problems faced by apparently soluble enzymes, such as PIC and the bulk of PtdIns(4,5)P 2 3OH-kinase activities thus far studied, in using G-protein signals as a source of control are focused on this issue of being ready and appropriately placed to take advantage of the benefits of coupling to G proteins. The fl-isoforms of PIC, which are dedicated to G-protein-dependent coupling, show substantial structural differences to the y-isoforms of PICs, which are coupled to PTKs [4,90,91]. For example, they lack the SH2 and SH3 domains present in PICT and the ability to serve as substrates for activated PTKs which can phosphorylate PICys [4]. Hence, the forces that result in activation of PICT1 (i.e., its tyrosine phosphorylation, docking of SH2 domains, and membrane localization; all of which are 'time-consuming' processes essentially involved in solving the problems of 'mobilizing' a soluble enzyme and giving it effective access to its substrate) would not necessarily apply to the fl isoforms of PIC. Indeed, PIC/31 is stimulated in reconstitution assays by activated all and Otq subunits from Gq-family proteins by a mechanism that apparently leads purely to an increase in its 'intrinsic' catalytic activity (its 'Vmax' increases) without apparent need for improved access to substrate [88,153] (in contrast to activation of PICT1 , where tyrosine phosphorylation leads to improved substrate access [131]). The implication of this is that in these reconstitution systems 'soluble' PIC/3 may be acting as a membrane-localized enzyme in the fashion of adenylate cyclase (the alterna-

tive is that PIC/3 does not interact with the lipid domain in any way other than via its substrate, i.e., it 'hops' [99,154]; however, this would presumably require the activating aq to 'hop' with it). There may be a number of 'unphysiological' reasons why PICfll gets unhindered access to PtdIns(4,5)P 2 in these reconstitution assays, e.g., the presence of several hundred nanomolar calcium and sodium cholate [88]. However, if PIC/31 is activated by a similar mechanism in vivo then it must be able to gain equivalent access to its substrate. In this regard, there is evidence that G-protein-regulated PIC/3s are associated with the membrane cytoskeleton [155,156] in a manner which may not be exhibited by PICys and, furthermore, that this association may be critical for allowing effective G-protein coupling [156]. This binding to the cytoskeleton is via specific, reversible but possibly relatively labile (non-SH2- or SH3-domain-mediated) interactions. Perhaps these cytoskeletal associations enable a 'soluble' PICfl to be engineered into a location that enables it to interact with both activated G-protein a subunits and its substrate (i.e., alleviating the potential problem of accessibility) and hence to fully exploit the potential speed of G-protein-transduction reactions. How these 'specialized' features of PICflls , that appear to adapt them to life as G-protein targets, might be translated into the structure a n d / o r regulatory features of a putative G-protein-dependent PtdIns(4,5)P 2 3OH-kinase cannot yet be defined. If, however, the apparent demand for instant access to PtdIns(4,5)P 2 is to be met, this implies that none of the mechanisms that lead to 'activation' of PI3K by PTKs can be relevant. Approx. 15% of 'PtdIns' 3OH-kinase activity in unstimulated platelets is associated with cytoskeletal proteins and this can be increased by stimulation of the G-protein-linked thrombin receptor [157,158]. Although this could represent evidence of associating with cytoskeletal elements and hence offer a mechanism that might enable its efficient activation by G proteins (indeed PIC activity showed a similar distribution), a number of other features of the response indicate it could equally well be ascribed to a PTKdriven translocation event. Thus, although the receptor for thrombin is G-protein-linked, its activation leads to an increased recovery of PI3K activity in antiphosphotyrosine and anti-src-type PTK antibody-directed immunoprecipitates [65]. Thrombin also stimulates a translocation of c-src to the cytoskeleton that parallels the analogous response with PI3K activity [157,158]. In addition, all of the PI3K activity in platelets has been reported to be associated with an 85-kDa protein possessing an epitope recognized by a monoclonal antibody raised against the p85 subunit of PI3K, suggesting the PI3K activity being studied was similar to the PI3K known to be regulated by PTKs [65]. Hence, although

54 this does not discount the possibility that the function of the cytoskeletal association is to enable G-protein interactions, it may have been initiated by a translocation to a cytoskeletal PTK.

V. How do agonists integrate Ptdlns(3,4,5)P3-encoded messages into their signalling repertoires?

V-A. Receptor and tissue specificity Two critical features in cellular communication are the capacities of receptors to generate intracellular signals that are not only distinct from those elicited by other receptors in the same situation, but that can also be adapted to fit the requirements of very different cells or circumstances. If all receptors and the pathways via which they regulate targets in cellular metabolism were widely different, then achieving receptor and tissue-specific signalling would not represent a problem beyond that of regulating the expression of the relevant receptors a n d / o r pathways (huge though this problem would be). However, the systems of intracellular communication we are faced with have evolved progressively into a state containing a huge diversity of receptors and associated signalling pathways and, as a result, varying degrees of homology exist between the components at all levels in these pathways (as a function of their varyingly distant common ancestors). Consequently, the organization of these systems is naturally manifest as being based on families. Thus, there are a relatively limited number of substantially different types of receptor-transducing reaction (G proteins [59], receptor-PTKs [100,159], receptor-serine kinases [160], receptor-tyrosine phosphatases [161], receptor-guanylate cyclases [162], receptor-ion-channels [163]) and of the signalling pathways they control (initiated by various critical regulatory proteins, e.g., PICs [4], adenylate cyclases [152], voltage-sensitive calcium channels [164], Ptdlns(4,5)P2 3OH-kinase activities [1], GRB2/SEM5 [165], ras GAP [76], guanylate cyclases [162], etc.; see Appendix V). Furthermore, the communication links between these broad classes of transduction and signalling protein show a parallel specificity [4,5,95,151]. Hence, those critical regulatory proteins which are controlled by a particular class of transducing reaction appear to be specifically designed for this interaction and contain many homologies with otherwise unrelated proteins that are also regulated by the same transduction elements. Signalling pathways that can be controlled by more than one class of transductional event therefore possess different types of critical regulatory protein which carry, in addition to those features associated with their output signals, the specific adaptions needed for receiving their respective inputs. Further, despite the limited number of types of transducing proteins and signalling pathways and the

way information is channelled between them, they must relay, but simultaneously maintain the individuality of, inputs from a far greater variety of receptors. Although the above view perhaps contains a historically derived bias, as a consequence of the way in which an appreciation of the diversity of receptor types preceded that of the diversity found in transducing reactions and signalling pathways (which is only really now becoming apparent, see below), it is still the current paradigm for addressing this subject and the problems it appears to present are still highly relevant. These problems are those of defining; (a) how often closely related receptors, that are dedicated to the use of a common transduction mechanism and therefore use a structurally related pool of critical regulatory signalling proteins, generate unique messages in a given cell, and (b) how one such receptor's messages can be moulded to suit different cellular contexts. Both receptor and tissue-specific signalling could be generated by the intervention of accessory regulatory factors, that were themselves receptor a n d / o r tissue specific. However, the best defined reactions of this kind that are potentially relevant to a Ptdlns(3,4,5)P 3 signal are apparently directed at the inactivation of signalling, e.g., receptor-specific kinases [166] and protein tyrosine phosphate phosphatases [167] and, although this can be important, it is a secondary consideration in a field still focused on understanding the mechanisms that generate signals. Therefore, the data that addresses the issues of receptor and tissue specificity in Ptdlns(3,4,5)P3-associated signalling is predominantly focused on the activation mechanisms and consequently this data will dominate the following discussion.

V-B. Receptor-specific and tissue-specific engagement of signalling pathways by PTK-coordinated mechanisms As described above, the problems to be faced are (i) how different receptors on a given cell produce characteristic intracellular messages when they all utilize PTKs to transduce their signals and, furthermore, when these PTKs have to operate via a family of critical regulatory signalling proteins that are all 'purpose-built' for activation by PTK-transduction processes and, (ii) how these types of transduction processes, that appear to be dominated by the specific structure of the activating receptor, can still generate different patterns of signals in response to activation of the same receptor but in different cellular situations. Section IV described work that has shown receptors can activate PTKs which drive the construction of a number of types of signalling complex (Figs. 9A,B and 10). The activated PTKs phosphorylate key tyrosine residues on proteins in the signalling complex and some of these phosphotyrosine moieties then act as

55 signals for binding and activation of SH2 domainbearing effector proteins. It is the events associated with the formation of these signalling complexes which dictate the major factors that can potentially confer individuality to a receptor's intracellular messages. Firstly, and probably most importantly, the selectivity of the recruitment process establishes the teams of critical regulatory or effector proteins that a particular signalling complex gathers and hence defines the codes in which its intracellular message will be delivered. Secondly, the process of translocating target proteins into signalling complexes is probably the most significant factor determining their kinetics of 'activation' (however, these tend to be fairly homogenous). What then are the features of this tyrosine phosphate: SH2 domain based interaction that enable PTK-coordinated signalling complexes to differentially recruit effectors like PI3K? V-B. 1. Receptor-specific activation of PI3K by PTK-coordinated mechanisms: the concept of a 'consensus sequence' for PI3K binding The high affinity of SH2 domains for tyrosine phosphates in proteins (KdS have been estimated to be in the nanomolar range [101,168,169]) that enables the efficient translocation of PI3K to recruitment stations in signalling complexes must be accompanied by a very high level of specificity. Without specificity in SH2 domain:tyrosine phosphate interaction, PI3K could translocate to any accessible tyrosine phosphate residues (not even necessarily membrane localized) and would also be in effective competition with every other SH2 domain bearing protein for all such sites. It is evident that different receptors that utilize tyrosine phosphates to recruit proteins into signalling complexes can do so with great specificity. Hence, the CSF-1 receptor can recruit and activate PI3K [26] but not PICy [170,171], whereas the PDGF receptor can recruit and activate both [105,130,173] (see Table V). Further, IGF-1 and insulin can activate PtdIns(3,4,5)P3 but not Ins(1,4,5)P3 accumulation in 3T3 [20] and U937 cells [25], respectively, indicating receptors utilizing associated recruitment stations (i.e., IRS-1, Fig. 9B) can also selectively activate effectors [169]. This specificity is supplied by interactions between the proteins harbouring the SH2 domains and the tyrosine phosphates to which they bind. The points of interaction are largely close to the tyrosine phosphate target (within roughly 10 amino acids) and in the surfaces immediately adjacent to the tyrosine phosphate-binding pocket in the SH2 domain [168,174,334336,338] (see Appendix VII). This sequence specificity means that a particular SH2 domain-bearing protein will bind preferentially to a tyrosine phosphate that is displayed within the context of a preferred local sequence [1,338]. This principle is clearly demonstrated

by recent work with the PDGF receptor; this has shown that phosphotyrosines at residues 751/740, 771, 1021, 579/581 and 1009 of the receptor convey a capacity for tight, agonist-dependent association of PI3K, ras-GAP, PLCy, src and p64, respectively

TABLE IV G-protein-linked receptors and other receptors that do or do not activate Ptdlns(3,4,5)P 3 accumulation in intact cells

Part 1 lists ligands with receptors that are known to belong to the 7-transmembrane-domain family- of receptors (i.e., G-protein-coupled) that have been tested to see if they can stimulate accumulation of Ptdlns(3,4)P 2 or PtdIns(3,4,5)P 3, along with other agents that are thought to interact with G-protein-mediated signalling pathways * Aluminium/fluoride is a non-specific activator of G proteins [271], that is effective on intact cells, and is commonly used as an indicator of G-protein involvement in a pathway. Although aluminium/fluoride ( 1 0 - 5 0 / z M / 1 0 - 5 0 mM, respectively) can activate PIC in neutrophils [270], it failed to activate PtdIns(3,4,5)P 3 accumulation [45]. However, these concentrations of aluminium/fluoride inhibited both FMLP-stimulated accumulation of Ptdlns(3,4,5)P 3 in vivo and PtdIns(4,5)P z 3OH-kinase in vitro, suggesting aluminium/fluoride has a direct inhibitory effect on PtdIns(4,5)P 2 3OH-kinase, which could mask any effect on a stimulatory G protein [45]. Abbreviations: FMLP, formylated met-leu-phe; Lt, leukotriene; Tx, thromboxane; PG, prostaglandin; PAF, platelet activating factor. Part 2 lists a number of other agents with various other mechanisms of receptor coupling that have been tested for their ability to stimulate 3-phosphorylated lipid production. Abbreviations: TNF, tumour necrosis factor; TGF, tumour growth factor; IL, interleukin; TPA, tetradecanoyl phorbol myristoyl acetate. A. G-protein-coupled receptors Ligand or agent

Increase in PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 in intact cells

Reference

FMLP LtB 4 LtD 4 LtE 4 ATP Thrombin TxA 2/U46619 PAF C5a

+ + + + + + + + +

[14-16] [15] [193] [193] [45,57] [22,204] [22] [45] [211]

PGE 1 Bombesin Angiotensin II Met-Enkephalin Bradykinin Vasopressin Adrenaline

-

[20] [20] [92] [92] [92] [20] [58]

Aluminium/Fluoride Mastoparan

- * +*

[45] [56]

-

[25] [92]

-

[92]

-/+ ?

[20,57,62]

B. Other agents TNFa TGF/3 IL-I TPA

56 [93,103-105,173,182,273,340]. Consideration of the local amino acid sequences surrounding tyrosine phosphates known to act as PI3K-binding sites in a number of different proteins has led to the suggestion that tyrosine phosphates within the YXXMX sequence (see Table V) are favoured by the SH2 domain(s) of PI3K [1,103,105] (this is the origin of the notion of a 'consensus' binding site for PI3K; this sequence may not define the only PI3K-binding sites, however; see Table V). Thus it can be envisaged that, by autophosphorylating tyrosine residues in specific locations, receptors can gather a collection of effector proteins of their own 'choice'. Although in principle the selective mechanisms that enable specific collections of effector pro-

teins to become bound and activated by PTK-coordinated signalling complexes could exclude PI3K, the vast majority and probably all of the receptor-PTK complexes that have thus far been studied, do include it (see Table II). Although this view of PI3K:receptor interactions being dictated by very local modular parts of these proteins is remarkably predictive, it seems, in some cases at least, that additional factors may be involved. A number of SH2 domain-bearing proteins possess two different SH2 domains (e.g., p85, ras-GAP and PICy) and recent evidence suggests that both may co-operate in determining the strength of binding to target proteins. This suggestion was initially based on data which

TABLE V

Consensus sequence for tyrosine phosphates which bind PI3K There is strong evidence that the translocation of PI3K to activated receptor-PTKs (or analogous proteins) is mediated by the tight binding of SH2 domains in the 85-kDa subunit of PI3K to specific tyrosine phosphates in the cytoplasmic tails of receptor-PTKs. The short stretch of primary sequence surrounding the phosphorylated tyrosine residues and the particular structure of the SH2 domains in PI3K convey sufficient specificity to this interaction that the tyrosine phosphates recognized by PI3K are not recognized by the other signalling molecules thus far investigated (e.g., ras-GAP and PLC-y and vice-versa [1,101-105,173,176,182,272,273,275,337,338,340]. Thus, the possession of specific tyrosinecontaining sequences in a protein may be largely responsible for dictating the potential signalling systems with which it can interact (see Section V-B.1.). The sequences listed are thought to represent the major sites of tyrosine phosphorylation in the indicated proteins to which PI3K binds. These sites have been identified on the basis of site-directed mutagenesis and/or other evidence of direct binding to PI3K (e.g., competition of PI3K binding to the PDGF receptor by appropriately phosphorylated peptides) [1,86,101,103,105,137,141,176,274]. Also listed are the optimal peptide sequences selected from a random peptide library on the basis of tight binding to GST-fusion proteins containing either the N-terminal or C-terminal SH2 domains of p85a [338]: the library contained peptides which consisted of random sequences (excluding W and C) in the 3 positions C-terminal to a Y at position 4 in an otherwise defined 12-amino acid sequence. Tight binding of PI3K to the PDGF receptor may require the binding of both SH2 domains of PI3K to both tyrosine phosphates (751 and 740); it has yet to be established whether an analogous mechanism exists in other receptors (though this would often have to involve a 'non-consensus' site) but the question of whether one or two SH2 domains 'engage' on PI3K translocation has important implications for its possible mechanism of activation (see Section IV-B) and its specificity for binding to different receptors (see Section V-B.1). The EGF receptor (Table II) and many receptors which activate src-type PTKs (Table III and Fig. 10), are able to activate PI3K but do not contain appropriate YXXMX motifs (such a motif at Y919 in the EGF-R is outside of the region implicated in p85 binding [69]); thus, either this motif is insufficient to describe all possible PI3K binding sites, or as yet unidentified proteins (which do contain the YXXMX motif) are responsible for PI3K binding under these circumstances (i.e., proteins analogous in action to IRS-1). Protein

Position

Human PDGF-R-/3

740 (731 - human a; 708 - mouse fl) 751 (742 - human a; 719 - mouse/3) 721 315 608, 628, 658, 727, 939, 987 460, 546, 1010 728 1334 1331 721

Mouse c-fms/CSF-1-R Polyoma Middle T Rat IRS-1 Human Human Human Human

FGF-R insulin-R

rnet/HGF-R C-kit/SCF-R

Sequence Y M D M S/K YV P M L YV E M R YM P M E YM X M X YX X M X Y M MM R YT H M N YE V M L YM D M K Consensus Y X X M X

GST-fusion protein

Optimal sequence selected from a random peptide library

N-SH2 domain of p85 C-SH2 domain of p85

Y Y

(M/I/V/E) X

X X

M M

Y, tyrosine; M, methionine; E, glutamate; D, aspartate; P, proline; V, Valine; R, arginine; T, threonine; H, histidine; N, asparagine; L, leucine; K, lysine; G, glycine; A, alanine; I, isoleucine; Q, glutamine; C C'ysteine; N, asparagine; Q, glutamine; F, phenylalanine; S, serine; W, tryptophan. X, no defined amino acid. Abbreviations for proteins: see Table II.

57 showed that although the PDGF receptor can bind PI3K at either tyrosine 751 or 740 when present alone (both of these are 'consensus' sites), there was an apparently synergistic increase in the affinity with which PI3K was bound when both sites were available for interaction (most readily explained by postulating the SH2 domains through which PI3K interacted were physically connected) [103]. This concept receives independent support from experiments indicating, (a) that the two SH2 domains of PICy may act synergistically to promote binding to multiple tyrosine phosphates in both the PDGF and EGF receptors [107,109,118,173], (b) that doubly tyrosine phosphorylated mT/PDGF-receptor based peptides can activate PI3K with higher potency than singly phosphorylated peptides [87], and (c) that overexpressed SH2-domain fusion proteins constructed from the individual SH2 domains of p85 and/or ras-GAP require two SH2 domains to achieve measurable agonist-dependent binding to the PDGF receptor in intact cells [337]. The possible effects of this form of dual SH2 engagement could include altered selectivity, affinity and kinetics of binding and, in addition, an enhanced potential for catalytic activation (see Section IV-B). In this regard, the site-directed mutagenesis studies investigating the SH2 domain docking of PI3K to specific phosphotyrosines in receptors have not yet measured the consequences of these manipulations on the catalytic activation of PI3K (i.e., the agonist-stimulated formation of Ptdlns(3,4,5)P3). If dual SH2 engagement is accepted as a common feature of receptor:PI3K interactions then, in most cases, it must involve a currently unrecognized 'consensus site' for PI3K binding [337] (since two 'consensus sites' are not present in most of the receptors listed in Table V) or, binding of PI3K to a separate, receptor-directed protein (i.e., analogous to IRS-1). Evidence suggesting that the N-SH2 of p85 shows a reduced 'requirement' for the currently accepted PI3K 'consensus-sequence' is found in data showing that, (a) this SH2 domain can promote binding of a chimeric construct containing an SH2 domain of ras-GAP to PDGF receptors lacking phosphotyrosines 751/740 [337] and (b) this SH2 domain shows less 'selectivity' than the C-SH2 of p85 for the optimal 'consensus-sequence' in in vitro phosphopeptide binding studies [338]. The possibility that secondary SH2:tyrosine phosphate interactions may contribute to the fidelity and strength of binding between tyrosine phosphate bearing proteins and SH2 domain-bearing proteins indicates the potential importance of higher-order structure in the formation of these associations and suggests that their binding is not purely dictated by highly 'modular' (location-independent) SH2:target phosphate docking. This is supported by evidence that the association of the p l l 0 subunit can confer greater

specificity to p85's interactions with tyrosine phosphorylated receptors in vitro (e.g., although p85c~ will bind to tyrosine phosphorylated EGF receptors in vitro, PI3K will not [67,71]). Further, reports that tyrosine phosphorylated IRS-1 protein activates PI3K with greater potency than singly or doubly tyrosine phosphorylated peptides (that were based on its sequence and still contained the full 'consensus sequence' for PI3K binding) [86] suggest that more of the tyrosine phosphate-bearing protein is implicated in these interactions than just the local sequences around its target phosphate moieties. Perhaps it is all these additional factors that enable the relatively large 'wobble' in the stringency with which the 'consensus sequence' is often adhered to. Although the points of interaction between the PDGF receptor-PTK and the effectors PIC and PI3K are different, whether both can bind 'in parallel' to the same receptor molecule or whether they effectively compete for binding has not been definitively established. Evidence suggesting that antibodies to individual signalling components can each immunoprecipitate each other in agonist-stimulated cells overexpressing the PDGF receptor, indicates that signalling complexes can indeed form which contain multiple components [340]. Further, data in other cells suggests only a small proportion of receptors become bound to PI3K, even when maximally stimulated, indicating that in isolation this should not be limiting [178]. The notion that recruitment of PI3K into PTK-coordinated signalling complexes is receptor-specific can become confused when dealing with possible variations on the receptor-PTK theme, such as those seen in receptors utilizing src-type PTKs to transduce their signals or with receptor-associating proteins like IRS-1 (see Fig. 10 and 9B). In the former situation, a number of receptors can clearly activate the same src-type PTKs (see Table III) and hence the sites phosphorylated might not be a function of the activated receptor; a simple way to incorporate receptor specificity into this system, however, is to suggest that PI3K is recruited onto tyrosine phosphate moieties in the host receptor or a receptor-directed protein (see Fig. 10). Although many of the receptors associated with srctype PTKs do not contain tyrosine residues within PI3K 'consensus sequences', the precedent set by the EGF receptor and the discussion above indicates this may not be essential (Table V) and, furthermore, a large number contain tyrosines within a conserved sequence that is closely related to the currently defined consensus for PI3K binding. Use of proteins, like IRS-1, as surrogate recruitment stations for SH2 domainbearing proteins can only become a potential problem for the notion of receptor specificity if they serve as substrates for other PTKs. This however, has yet to be reported (in this regard IL-4 and IL-3 stimulation of

58 FDCP-2 cells appears to promote the association of PI3K with separate tyrosine phosporylated proteins [224]). We noted above that receptor specificity could be supplied by receptor-specific 'accessory factors'. EGFreceptor-directed protein tyrosine phosphate phosphatases have been described that could conceivably contribute to the process of making signalling via this receptor different to other related receptors [107,179181]. Further, by being specific for particular tyrosine phosphate cues [107], they might modulate the activation/deactivation of particular effectors, e.g., PI3K. However, it is currently difficult to envisage how such a phosphatase might be responsible for inactivating signalling (particularly in the case of PI3K, where activation seems dependent on maintained SH2 docking), because it would require the bound SH2:tyrosine phosphate proteins to be dissociated and current evidence suggests such interactions actually protect the relevant tyrosine phosphates against protein tyrosine phosphate phosphatase mediated hydrolysis [107]. A further and completely different type of mechanism by which 'accessory events' might lead to receptor-specific PtdIns(3,4,5)P 3 accumulation is via specific recruitment, tyrosine phosphorylation and 'activation' of other PTKs [182] (in a manner that is distinct to the activation of src-type PTKs by receptors without PTK activity). These PTKs might then go on and 'activate' PI3K, perhaps serving to amplify a direct but more limited recruitment of PI3K to the receptor itself. Despite the subjective focus on the role of polymorphism in signalling proteins being confined to tissuespecific adaptation of intracellular signalling mechanisms (discussed immediately below), multiple isoforms of effector proteins are commonly co-expressed in a single cell (e.g., PICs [183]). These isoforms presumably serve to enable differential coupling of a signalling pathway to the variety of different receptors which use it in single cell, i.e., different isoforms of PI3K (e.g., containing p85a, /3, y or 6) may exhibit receptorspecific characteristics of association into PTK-coordinated signalling complexes in a single cell (see figures in Ref. 70).

V-B.2. Tissue specificity in the activation of PI3K by PTK-coordinated pathways There are a number of post-receptor levels in the signalling systems which can regulate PI3K that are open to tissue-specific variation and which might therefore contribute to the ability of a PtdIns(3,4,5)P 3 signal to be adapted to the cell in which it appears. In the minimalist picture presented by a single receptor-PTK, only the effector protein is open to tissue-specific variation (see Fig. 9A). The closest available precedent for this situation is found in the interaction of PICys with receptor-PTKs. Two isoforms of PICy have been de-

fined (PICyl and PICy 2) that show clear differences in tissue distribution (PICy 1 being widely distributed whereas Y2 is particularly concentrated in lymphocytes) [4,184]. The relative coupling specificities of PICy 1 and Y2 are only just becoming defined but it appears T2, along with Yl, can be activated by receptors that are abundant in lymphocytes and use src-type PTKs to transmit their signals, whereas PIC71 appears to be the isoform that is activated by receptor-PTKs [4,184]. Perhaps this is evidence of a functional relationship where PICy 2 interacts more efficiently with tyrosine phosphate cues abundant in receptors that use src-type PTKs to elicit their signals. Furthermore, equivalent effector-type proteins that are dedicated to coupling with G proteins (e.g., PICfls and adenylate cyclases), have been shown to be made up of families of related isoenzymes which show distinct coupling properties and differential tissue distributions, which by interchangeable expression could supply tissue specificity to a receptor's signals [4,151,152,185] (see below). The existence of multiple isoforms of the p85 and possibly of the p l l 0 subunits of PI3K offers the opportunity for a receptor-PTK or a receptor utilizing a src-type PTK to show a tissue-specific capacity to gather PI3K (or alternatively viewed, a capacity for the p110 subunit of PI3K to interact with a different collection of receptor nucleated signalling complexes in a tissuespecific manner). The currently defined potential p85 subunits of PI3K contain non-identical SH2 domains [67,70] and hence tissue-specific expression of p85s might offer mechanisms by which to alter the pattern of signals a given receptor can generate. Although current evidence suggests that there are no large differences between p85a and /3 in terms of their capacity to interact with receptor-PTKs or src-type PTKs (in vitro or when hyperexpressed in vivo) [70], the capacity of p l l 0 to confer greater selectivity to associated p85s (than they show in isolation) [67,70,71] may well enable such a mechanism to operate. Furthermore, the early signs that in the rat p85/3 is preferentially expressed in the brain (unpublished data quoted in Ref. 70), suggest that the other essential requirement for this form of tissue-specific coupling may also be in place. Finally, the combinational use of different p85 and p110 isomers may allow a degree of adaptability in PI3K coupling that is a significant manifestation of the underlying advantages afforded by the heterodimeric subunit structure of PI3K. To achieve the same final tissuespecific effects in PIC-coupled systems would entail expression of a larger number of completely different species of PIC. Just as receptor-specific 'accessory factors' can supply receptor specificity, putative tissue-specific 'accessory factors' could supply tissue specificity to receptorPTK's signalling pathways. Such factors may include

59 protein tyrosine phosphatases, or agents that modulate the interaction of specific effectors and receptors. In the p85s of PI3K a number of possibilities for such regulation by protein-protein interactions are provided by the SH2, SH3, SH3-binding and bcr domains. It may be worth noting that it is in the structure of the bcr domain and the location and number of SH3-binding sites that individual p85s differ most, suggesting that these sites could mediate differential regulation of PI3K isoforms. The same range of options that are supplied by PI3K polymorphisms that could allow tissue-specific signalling (or broaden signalling options within a single cell) for receptor-PTKs could also achieve this for receptors that use receptor-regulated PTKs (often srctype) or receptor-substrate proteins (like IRS-1) in their pathways (Figs. 9B and 10). However, these pathways possess the additional options offered by the possibility of utilizing different forms of substrate proteins or receptor-associated PTKs.

V-C Specificity of signalling via G proteins Some of the interactions implicated in G-proteinmediated signalling and the advantages and/or consequences associated with their use in receptor pathways have been noted (see Fig. 11 and Section IV-D). Evidence discussed in Sections III-A.2 and III-B.2 suggests Ptdlns(4,5)P2 3OH-kinase activities can be activated by G-protein-linked receptors, but that the number of circumstances where an appropriate combination of the necessary factors (receptors, G proteins and effectors?) are co-incident is relatively limited (thus far to only haemopoietically derived cells). At present, there is simply insufficient data to allow a detailed consideration of how the connections are made which enable recepfor-regulated G proteins to activate Ptdlns(3,4,5)P 3 accumulation. In particular, the identities of the components implicated in several of the potential steps in receptor-stimulated, G-protein-mediated Ptdlns(3,4,5)P 3 accumulation are still unknown (see Section III-B.2 and below). However, recent studies of the specificity displayed by the components that make up other G-protein-regulated effector systems are beginning to define a general set of 'rules of engagement' that appear to govern the construction of these signalling pathways. Viewed from the outside, G-protein-mediated signalling pathways can usually be accessed by collections of receptors that are related via their 7-transmembrane-domain bearing structures. The specificity of the receptor: G-protein interaction which is critical to determining which G protein is activated by a particular receptor, appears to be a function of the receptorstructure itself [186] (see Fig. 11) and the a and /3 components of the G protein [95,187]. Typically, a

receptor only activates a single type or family of G protein (a G-protein family is defined here by its a-subunits), although there are exceptions [55,59,188]; therefore, because receptors are only transiently associated with G proteins and hence rapidly lose their potential grip on the shape of signalling events from that point on, receptor-specific signalling through a single G protein is predominantly dependent on the intensity with which different receptors can activate that G protein (as the speed of response is apparently largely governed by events downstream of receptor G-protein dissociation). This is compatible with observations in neutrophils, where all receptors that link through pertussis toxin sensitive G proteins (probably of the G i family, see below) activate Ptdlns(3,4,5)P 3 accumulation with similarly rapid kinetics, but with a wide variety of intensities [15,16,45,57]. Following activation, families of closely homologous G proteins exclusively interact with a particular class of G-protein-sensitive effector (e.g., Gq to PICs, Gs to adenylate cyclases) and, furthermore, individual members of these families couple with differing selectivities to the various isoforms of that class of effectors [4,59,189,190]. The G i family of proteins, however, appear to be unusual in this respect, because they do apparently have the capacity to interact with more than one type of effector [59,95] (perhaps this is associated with the capacity of their/3,/subunits to activate effectors independent of their a~ subunits [172,191], see below, and the fact that their o~i subunits might only indirectly regulate effectors [152]). The structural determinants which govern these interactions, and which therefore dictate why a specific G protein can modulate one effector but not another, are only just starting to be defined (e.g., Ref. 192). The specificity in G-protein signalling can thus be viewed as being defined by two major points of interaction: the specific interaction between a receptor and a particular family of G proteins, and the specific interaction between this family of G proteins and a particular class of effectors. The equivalent points of interaction in the discussion above, concerning PTK-coordinated signalling, would be the specificity of the sites phosphorylated by PTKs and the specificity of the resulting tyrosine phosphates for families of SH2bearing effectors, respectively. These interactions can, in turn, be seen as being controlled by the cell-specific expression of the appropriate components. There are numerous possible ways in which the interactions described above could theoretically produce tissue-specific, G-protein-regulated PtdIns(3,4,5)P 3 accumulation. Thus far, it seems that the ability of G proteins to stimulate Ptdlns(3,4,5)p 3 accumulation is confined to HL60 cells (LS, TJ, PTH, unpublished data), U937 cells (a promyeloid cell line) [25,57,193], neutrophils [15,45,56,57] and possibly

60 platelets [22] (see Table IV). In contrast, a collection of different receptors known to activate various G proteins were unable to stimulate this response in 3T3 cells [20], N G l l 5 - 4 0 1 L cells (a neuroblastoma cell line) [92], PC12 ceils (AN Carter, personal communication) and a smooth muscle cell line (L.C. Cantley, personal communication), see Table IV. Further, in the case of permeabilized 3T3 cells, this response could not be activated by GTPyS [20] (suggesting that the lack of G-protein-sensitive Ptdlns(3,4,5)P 3 accumulation observed in these cells was not due to use of inappropriate receptors but to a lack of appropriate G-protein : effector coupling). All of the above examples of G-protein-regulated Ptdlns(3,4,5)P 3 accumulation occur in cells of haemopoietic lineage and they are all sensitive to inhibition by pertussis toxin (indicating the involvement of a Gi-family member in the response). Further, all of these responses correlate with the occurrence of pertussis toxin-sensitive regulation of PIC (see the legend to Fig. 11 and Refs. 172,191,194 for a more detailed discussion of the G-protein regulation of PIC). Thus, although the number of relevant studies is still small, there does appear to be a pattern emerging which could plausibly result from cell-lineage-specific expression of certain relevant signalling components. In principle, the tissue specificity of G-protein-regulated PtdIns(3,4,5)P3 accumulation could be due to tissue-specific expression of either the receptor (although see above) a n d / o r the effector (a PtdIns(4,5)P 2 3OH-kinase activity?, see Section III-B.2) a n d / o r an appropriate G protein. If the G protein involved is a Gi-family member, then the ubiquitous expression of ai-subunits [59] suggests that either, (1) there is tissuespecific expression of the receptor a n d / o r the effector (in these models either the a a n d / o r /37 subunits could be responsible for activating the effector) and/or, (2) there is tissue-specific expression of a unique combination o f / 3 / 7 subunits (four distinct types of/3 and 7 subunits are currently defined [195]), which (a) effectively convey a tissue-specific expression to the G i protein and (b) are responsible, at least in part, for activating the effector. Further, if /33' subunits are responsible for activating PtdIns(4,5)P2 3OH-kinase, then this effect must be selective for the nature of the G protein from which they were released, since dissociation of G s in neutrophils does not stimulate PtdIns(3,4,5)P3 accumulation (indeed, it is inhibitory [15]): this property could be conveyed by either a specific combination of/3 and 3' subunits, by sensitivity to the co-incidental release of /37 plus a specific a (some recent evidence suggests released /37 and otS subunits can act synergistically to activate certain isoforms of adenylate cyclase [151,152]), or by some quantitative phenomenon, e.g., sufficient/33' to activate the effector can only be provided by dissociation of an abundant G

protein such as G i [172]. These issues can only be fully resolved by reconstitution experiments that determine which, or even if, G-protein subunits directly activate a defined Ptdlns(4,5)P 2 3OH-kinase activity. The answers to these questions are likely to be very important for our understanding of how different G-protein-coupled signalling systems have evolved and also how more than one such system can be simultaneously, but specifically, accessed by the same receptor in the same cell.

VI. Physiological function A strong theme of this article is the idea that agonist-stimulated changes in the levels of 3-phosphorylated inositol lipids represent a new and important signalling system (rather than a more indirect or 'downstream' response to stimulation by other signalling systems). This idea cannot be accepted until a target for the putative message generated by this pathway has been identified (i.e., the analogue of cAMPdependent protein kinase [196], protein kinase C [197] or a Ins(1,4,5)P3-regulated calcium channel [198]; some possible candidates are discussed in Section VI-B). Several lines of evidence, however, have persuaded many workers that it will in fact turn out to be correct. This evidence is, in essence, based on analogy with other such systems that have been established to fulfil this role and is founded on two main considerations: (1), that a class of enzymes, which can display PtdIns(4,5)P 2 3OH kinase activity, and catalyze the apparently initial, agonist-sensitive step in this pathway, can interact directly with receptor-coupling mechanisms in a manner which is very analogous to enzymes at the heart of other signalling systems (e.g., by coupling directly to receptor-PTKs or to G proteins in a manner independent of other signalling systems; discussed in Section III-A) and (2), the products of the putative effector enzyme(s) are low-molecular-weight diffusible molecules with no inherent catalytic activity and hence possess many of the credentials expected of classical intracellular second-messengers, i.e., molecules that relay signals originating from cell surface receptors to specific, potentially distant target proteins (see Appendix VI); these arguments do not, however, rule out the possibility that the enzymes responsible for PtdIns(3,4,5)P2 formation might also regulate signalling cascades based purely on protein:protein interactions, see below). VIA. Ptdlns(3,4,5)P3: an intracellular messenger? The most critical characteristic of a signal is that it can be clearly recognized in the context of the background in which it is acting. The precedent set by other acute signalling systems (e.g., those based on increases

61 in cAMP, cGMP, DG/Ins(1,4,5)P3 or decreases in cGMP) and, indeed, the nature of the control of metabolic pathways in general, suggests that the primary point of control exerted on a pathway (e.g., adenylate cyclase [152], guanylate cyclase [162,199], PIC [3] or cGMP phosphodiesterase [200,201]) is linked directly (i.e., by a product or substrate relationship) to a rapid and large change in the levels of a metabolite which then acts as a signal generated by that pathway. Thus, the clearest candidates for signals generated by activation of a Ptdlns(4,5)P2 3OH-kinase activity are its products, Ptdlns(3,4,5)P 3 (and Ptdlns(3,4)P2?), which are found at very low concentrations under basal conditions but the levels of which rise dramatically on activation by agonists. In contrast, the potential signal carried by the flip-side consequences of activating a Ptdlns(4,5)P2 3OH-kinase activity, a fall in PtdIns(4,5)P 2 levels, is relatively very weak (indeed, there is evidence that cells attempt to reduce the impact of Ptdlns(4,5)P2 3OH-kinase and P1C stimulation on PtdIns(4,5)P 2 levels [202,281]). This line of reasoning would suggest that the rapid increase in the cellular concentration of PtdIns(3,4,5)P 3 is an acute 'output signal' generated by agonist activation of this pathway. The credibility of this idea is further supported by a number of additional facts, these are; (1), Ptdlns(3,4,5)P3 is not known to be part of any other metabolic pathway (witnessed by its very low levels in unstimulated cells; and thus does not have a function which could be compromised or confused with a signal generated by the pathway under consideration here); (2), the pool of PtdIns(3,4,5)P3 generated on stimulation is likely to turnover rapidly (i.e., the putative signal can be switched off quickly) [15,16,20,21,45,57]; and (3), the structure of Ptdlns(3,4,5)P 3 is that of a very distinctive and uniquely polar phospholipid; it therefore has the credentials of being a small-molecular-weight messenger and, indeed, its headgroup has very strong similarities to the structures of Ins(1,4,5)P 3 and Ins(1,3,4,5)P4 [16] which are already known to carry 'second-messenger' information for another signalling system [3,203]. Taking all of these arguments into consideration, they point very strongly to the conclusion that the 'raison d'etre' of this pathway is to provide agonists with the opportunity to produce a potentially dramatic and transient rise in Ptdlns(3,4,5)P 3 concentration that is both extremely spatially localized (because the hydrophobic domain of Ptdlns(3,4,5)P 3 will tether it to the membrane leaflet in which it was produced) and intense (because of the very small effective volume it is restricted to) and therefore represents a highly plausible signal with a variety of possible roles. A consideration of some of the points made above in deducing that Ptdlns(3,4,5)P 3 is a likely signal molecule generated by agonist activation of this path-

way, can be used similarly to argue a case for PtdIns(3,4)P z acting as a signal. However, as discussed in Section II, the agonist-induced rise in Ptdlns(3,4)P 2 lags significantly behind that of Ptdlns(3,4,5)P3 accumulation (depending upon the agonist, a significant rise in Ptdlns(3,4)P 2 levels was not detected, in comparison to easily measurable increases in PtdIns(3,4,5)P3, for periods of time after stimulation ranging from 2-3 s to > 7 min) [16,22,25,30,63,92,204], suggesting that for substantial periods of time after stimulation it is difficult to make a case for an increase in Ptdlns(3,4)P 2 acting as a signal. Furthermore, it was argued that this rise in Ptdlns(3,4)P 2 may be derived, in part, from the hydrolysis of Ptdlns(3,4,5)P 3 [16,30], and thus at least one possible reason for the existence of Ptdlns(3,4)P 2 is simply to act as a route of degradation of Ptdlns(3,4,5)P 3 (i.e., in this role it would be analogous to Ins(1,4)P2 or AMP in the degradation of Ins(1,4,5)P 3 and cAMP, respectively [3,205]). These facts, in conjunction with the arguments in favour of Ptdlns(3,4,5)P 3 acting as a signal, suggest very strongly that Ptdlns(3,4)P 2 is not the sole information-carrying signal generated by this pathway, but do not exclude the possibility that it may act as some form of 'accessory signal' acting at later times of stimulation. In this respect, a similar situation is thought to occur in the PIC-based signalling system, where the primary signal, Ins(1,4,5)P 3, is metabolized via two routes (5-dephosphorylation and 3-phosphorylation), one of which (3phosphorylation) leads to the production of a further signal (Ins(1,3,4,5)P4), which acts in concert with Ins(1,4,5)P3 to control cytosol calcium concentrations [203]. Thus it might be imagined that dephosphorylation of a proportion of Ptdlns(3,4,5)P 3 via PtdIns(3,4)P2 allows the generation of an additional signal. However, the lack of definitive information on the proportion of Ptdlns(3,4,5)P3 metabolized via 5-dephosphorylation versus 3-dephosphorylation (Section II) and the plausibility of the notion that the degradation of Ptdlns(3,4,5)P 3 is required to be via a route which does not immediately regenerate its precursor (and the substrate for the PIC-based signalling system which often operates in parallel) does not allow any useful 'weight' to be placed on this argument. It should also be mentioned in the context of considering PtdIns(3,4)P 2 as a signal, that a Ptdlns3P 4OH-kinase activity has been discovered as an in vitro activity present in some cell extracts (e.g., platelets and erythrocytes), but not apparently in other cells (e.g., 3T3 cells) [41,42]. The analysis of the 32p distribution in [32p]Ptdlns(3,4)P2 synthesized in thrombin-stimulated platelets (A.N. Carter, C.P. Downes and S. Rittenhouse, personal communication) as well as FMLPstimulated neutrophils [16] or PDGF-stimulated 3T3 cells [30] (discussed in Section II), suggests that it cannot be made by a Ptdlns3P 4OH-kinase under

62 these circumstances; however, the idea that under different circumstances Ptdlns(3,4)P 2 might be specifically made by a different route, makes a small contribution in favour of the argument that it may represent an additional signal. The arguments in favour of Ptdlns3P acting as an acute signal molecule generated by this pathway on agonist stimulation are extremely weak. The lack of any significant changes in the already substantial control levels of Ptdlns3P (see Section II and Fig. 4) over prolonged periods of stimulation (although generally < 5 min) [10,16,20,22,23,26,45,57,92,120] mean that for it to act as a signal during this time it must be 'modified' in some way that has eluded detection (e.g., translocation to a different place). However, the fact that the characterized yeast PI3K predominantly, if not exclusively, synthesizes Ptdlns3P [37,77] suggests that it may have an independent role perhaps quite unrelated to those played by the receptor-sensitive lipids. The evidence discussed above is overwhelmingly convincing that the major function of the primary enzyme activity controlled by agonists in this pathway (which, at least in the case of PTK-mediated activation, we have argued is PI3K) is to synthesize at least one 3-phosphorylated inositol lipid with the credentials of a 'second-messenger'. Within this perspective, however, it is possible that PI3K possesses additional signalling functions. PI3K is translocated into PTK-coordinated signalling complexes in a manner analogous to a number of other 'signalling enzymes' (e.g., PLCT, ras GAP, GRB2/SEM5, c-src [1,5]). A number of these enzymes are thought to transmit their signals via direct proteinprotein interactions: c-src is a tyrosine kinase [206], r a s - G A P activates ras GTPase activity [207] (and may also recruit p190 [208] and p62 [209]) and, recently, GRB2/SEM5 has been shown to associate with and apparently activate the ras G D P / G T P exchanger SOS via an SH3 domain interaction [317,342]. Thus, in principle, it seems a similar function for PI3K should be considered. PI3K contains a number of homology domains which are recognized to mediate a number of these types of interactions (e.g., SH2, SH3 and bcr domains, see Section III-B.1) and purified preparations of PI3K appear to exhibit a serine kinase activity (measured as an ability to autophosphorylate, see Section III-B.1). Thus far, however, there is no clear indication of whether these points of interaction with PI3K should be viewed as points of 'input control' (i.e., regulation of lipid kinase activity) or 'output control' (i.e., regulation of the properties of associating proteins). For example, there are a number of circumstances where the activation of PI3K is correlated with the activation of small-molecular-weight GTP-binding proteins (e.g., ras, rac, and rho [76], see Section VI-B), and thus the predicted ability of PI3K to interact via its bcr domain with the rho family (see Section III-B.1) is

of considerable interest; at present, however, it is hard to guess whether the GTP-binding protein might be regulating lipid 3-phosphorylation (e.g., via regulation of the assembly of a signalling complex) or if PI3K is regulating the activity of the GTP-binding protein. VI-B. Potent&l targets o f Ptdlns(3,4,5)P 3 action

The preceding section has argued that hormonestimulated synthesis of 3-phosphorylated inositol lipids will turn out to be a new signalling system and that at least one of the messengers produced by it will be Ptdlns(3,4,5)P 3. The precedent set by the elucidation of the function of other signalling mechanisms suggests that, of the large collection of facts that together provide a convincing case for the function of the pathway, the most persuasive are those derived from in vitro reconstitution experiments where the direct effect of the messenger is exposed. Recently, evidence has been presented that polyphosphoinositides (i.e., PtdIns4P, PtdIns(4,5)P2, Ptdlns3P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3) can activate isozymes of PKC in vitro [288-292]. Further, Ptdlns(3,4,5)P 3 is effective at very low molar ratios compared to other lipids present in the assay and shows significantly greater relative efficacy than other polyphosphoinositides in activating the ~" isoform of the enzyme, suggesting that PtdIns(3,4,5)P 3 may be a physiologically relevant activator of this enzyme [292] (PKC r is an ubiquitous isoform of PKC which is not thought to be activated by phorbol esters or diacylglycerol and whose physiological targets are presently unknown [293]). However, the potency and selectivity of this effect of Ptdlns(3,4,5)P a are not sufficiently great in themselves to convincingly support this interesting idea and we must await the results of further study before it can be more clearly evaluated. At present, no other direct evidence for the function of Ptdlns(3,4,5)P 3 (or any other 3-phosphorylated inositol lipid) is apparent and hence the quest for this type of information is now a major preoccupation of this field. As a consequence, this section largely deals with a collection of more indirect and incomplete facts with which to derive clues as to the possible function of the pathway. One of the most natural ways of beginning to use the available information to search for possible functions of this pathway is to correlate its occurrence (i.e., the types of agonist and cell type involved) with that of 'eligible' cell responses, i.e., very much the approach which first linked Ca 2÷ responses to activation of a PIC [210]. On the basis of the arguments outlined in the preceding section, the primary requirements of such an 'eligible' response are that there is a clear gap in our understanding of how it is regulated and that it has properties consistent with its putative function as a component in the higher echelons of a signalling sys-

63 tem, e.g., whilst it may be regulated by other signalling systems, it is not totally dependent upon them and thus represents an essentially individual contribution to the signalling repertoire of different agonists. Any further characteristics of candidate responses can then be matched against relevant information for agoniststimulated Ptdlns(3,4,5)P 3 formation and some value placed on the ensuing fit; e.g., this might include a consideration of time-courses or the plausibility of the response being allosterically regulated by a diffusible, membrane-captive messenger. At present a number of agonists are known to stimulate PI3K a n d / o r the accumulation of PtdI n s ( 3 , 4 ) P 2 / P t d l n s ( 3 , 4 , 5 ) P 3 (Tables II-IV) and, although there is still relatively little information regarding 'negatives' (which must inevitably reduce the value of correlations based on 'positives'), a characteristic pattern for agonist-stimulation of this pathway is beginning to emerge (the individuality of this pattern compared to other signalling systems has been used earlier to argue that Ptdlns(3,4,5)P 3 formation is not a consequence of the activation of other known signalling systems). In particular, the rapid and intense stimulations of Ptdlns(3,4,5)P 3 formation mediated by G proteins appear to be confined to relatively few cell types, thus far only to cells of haemopoietic origin such as neutrophils and perhaps platelets (compare with the much more widespread occurrence of G-proteinstimulated PICs or adenylate cyclase). In contrast, slower and less intense stimulations of Ptdlns(3,4,5)P 3 formation are relatively ubiquitous responses to cell stimulation by receptor-PTKs (compare with the much more restricted or absent activations of PIC and adenylate cyclase, respectively). Thus a perspective is created in which the function of this pathway is required in a particularly rapid and powerful way by activators of platelets and neutrophils (e.g., FMLP, PAF and thrombin), but not during stimulation of other cells by G-protein-coupled receptors; yet it is also more generally required for growth-factor stimulation of many different cell types. This line of argument has led many workers to suggest that the function of this pathway lies in areas such as actin assembly, cystoskeletal rearrangements, shape change, membrane-ruffling, and membrane/cytoskeletal attachments [211,212]. These ideas are to varying extents overlapping but all suffer from our ignorance of the detailed processes involved and are therefore cripplingly vague, containing few specific, testable, hypotheses for the direct targets of Ptdlns(3,4,5)P 3. Recent evidence has suggested that some of these processes are controlled by members of the family of small-molecular-weight G proteins (e.g., ras, rac and r h o ) [213-216] and therefore these proteins might, in principle, be considered plausible targets for Ptdlns(3,4,5)P 3 action. Some evidence in favour of this idea is the reduction in both receptor-associated

PI3K activity and the GTP content of ras in agoniststimulated cells expressing PDGF-receptor mutants lacking Tyr 740/751 [340]. A close link between PI3K activity and ras activation does, however, seem unlikely, since there is now good evidence that ras activity (and hence perhaps other small-molecular-weight G proteins) may be directly controlled by the combined actions of other proteins, which are themselves directly or indirectly recruited to receptor-PTKs via their SH2 domains ( r a s - G A P , r a s - G A P associated p190 and, the SHC/GRB2/SOS connections [5,112,165,217,208,341343]), i.e., there appears to be no role for a primary effect of a diffusible messenger. Further, activation of rac and rho and their consequent morphological responses may be stimulated by hormones which do not activate Ptdlns(3,4,5)P 3 accumulation (e.g., bombesin acting on 3T3 cells) [20,215,216] and there is at least one situation where activation of PI3K appears to occur in the absence of ras activation (IL-4 stimulation of haemapoietic cell lines) [224-226]. More limited considerations of the panel of agonists which activate this pathway have also generated some ideas as to its possible function. In particular, the common association of PI3K activity with activated growth factor receptors and oncogenes has suggested, since the very early days of this field, a relationship between the metabolism of 3-phosphorylated inositol lipids and the control of mitogenesis [13]. This idea has received experimental support from studies showing that mutants of mT, c-src or v-src which are unable to support a translocation of PI3K are unable to sustain transformation in their host cells [1,11,12,51-53,66, 140,141,143,218]. Furthermore, mutants of the PDGFfl-receptor which lack an obvious PI3K translocation site (e.g., by point mutations of the critical tyrosine residues recognized by the SH2 domains of PI3K) are unable to support ligand induced thymidine incorporation in certain circumstances [1,105,175,219,340], though apparently similar studies with the PDGF-a-receptor [176,] and CSF-1 receptor [220-223,307] have yielded conflicting results. This idea is fairly limited in its scope however, since our present level of ignorance of the web of signalling events controlling mitogenesis severely limits an appreciation of any obvious points at which Ptdlns(3,4,5)P 3 may act. Further, it is clear that if Ptdlns(3,4,5)P 3 has a relatively common role in different contexts of cell stimulation (as might be expected of a widely used signalling system), then this role is neither universally necessary for, nor does it universally lead to, the stimulation of mitogenesis (many mitogenic stimuli which act through G proteins do not stimulate Ptdlns(3,4,5)P 3 accumulation [20] and many agonists which do stimulate Ptdlns(3,4,5)P 3 accumulation do so in non-mitogenic contexts, e.g., NGF- and EGF-stimulation of PC12 cells [21], or FMLP and thrombin-stimulation of neutrophils [14,16] and

64 platelets [22,204], respectively). One plausible point of Ptdlns(3,4,5)P 3 intervention in mitogenic signalling pathways, however, is in the activation of protein kinase cascades leading to the activation of the MAP family of serine/threonine protein kinases (variously known as mitogen-activated protein kinases; microtubule associated protein kinases; or ERKs, extracellular receptor-activated protein kinases) [227]. These kinases are thought to lead to the activation of the $6 ribosomal protein kinase p90 rsk in the context of mitogen activation, but are also activated in non-mitogenic contexts (e.g., in NGF- and EGF-stimulation of PC12 cells [228,229], insulin stimulation of adipocytes [230,231], and GM-CSF and FMLP stimulation of neutrophils [232]), where they may be responsible for the phosphorylation and regulation of transcription factors, cytoskeletal proteins and enzymes of intermediary metabolism. It is easy to imagine therefore, that the activation of these kinase cascades may underpin a number of cell responses which correlate with agoniststimulated accumulation of PtdIns(3,4,5)P3 [2]. Critical to this idea is the fact that the link between receptor activation and the top of these protein kinase cascades (currently a MAP kinase kinase kinase [233]) is ill defined. Recent evidence suggests this link may be the activation of ras-and/or raf- (a serine/threonine protein kinase with homology to the lipid-binding domain of PKC [234]) [227,235-239]. If this is correct, then it reduces the likelihood of a major role for PtdIns(3,4,5)P3 in these events, since there is evidence that neither ras (see above) nor raf [105,113,114] require PI3K activity for their activation. However, the construction and regulation of these protein kinase signalling pathways is clearly very complex, and, further, current evidence suggests more than one of these pathways may be activated in parallel by receptors, e.g., the pathway leading to activation of p70 $6 kinase is apparently independent of ERKs [240]; thus a role for Ptdlns(3,4,5)P 3 in the initial activation of one or more of these kinase cascades is still possible. In mammalian cells the p70 $6 kinase pathway is known to be sensitive to the immunosuppressant rapamycin [240, 347,348]. Thus the finding that a yeast PI3K homolog, TOR2, may also be a target of rapamycin [344] serves to fuel speculation that a lipid kinase product may be required for the activation of this signalling pathway. Further clues as to the function of this pathway may be derived from a consideration of what is known about the structure of the primary, agonist-stimulated enzyme in this pathway, PI3K. The ll0-kDa catalytic subunit of PI3K shares significant homology in its carboxyl terminal-half with a yeast vacuolar protein sorting mutant (Vps34) [38,78]. This homology was subsequently shown to convey a common ability to phosphorylate inositol lipids in the 3-position [77]. This discovery immediately suggests that PI3K may be in-

volved in a process such as 'vesicle management' or 'protein sorting', which might also be imagined to be critical to events in mammalian cells which correlate with Ptdlns(3,4,5)P 3 accumulation [79,80], e.g., the organization of the phagocytic vacuole in FMLP-stimulated neutrophils or, more generally, the targeting of endosomes containing endocytosed receptors to various cell destinations (some evidence in support of this is the appearance of PI3K in a 'light microsome' fraction after insulin stimulation of adipocytes [241]). However, it is not yet possible to assess whether these analogies are merely superficial or have a more profound basis. Thus, there are over 40 complementation groups which primarily lead to vacuolar protein-sorting defects in yeast with strong similarities to Vps34 and hence the lesion in Vps34 may be quite distally related to the mutant phenotype [80]. Further, assays of yeast PI3K activity have consistently reported a failure to phosphorylate Ptdlns(4,5)P 2 in the 3-position [37,77], and Ptdlns(3,4,5)P 3 has not yet been found in yeast [77,242], suggesting that the relationship between 3phosphorylated inositol lipid metabolism in yeast and in agonist-stimulated mammalian cells has yet to be established. In this regard, the Vps34 mutant does not contain any Ptdlns3P, indicating that its 'Ptdlns 3OH kinase' activity is entirely responsible for its synthesis [77]. This, when viewed in the perspective of the unusually large proportion of PtdlnsP which exists as this isomer in yeast (approx. 60% of total PtdlnsP is the Ptdlns3Pisomer in S. cerevisae grown in glucose-rich medium) [37,242], suggests that yeast, and Vps34 in particular, may be a better 'model' for the role of direct Ptdlns 3-phosphorylation than that of agoniststimulated synthesis of Ptdlns(3,4,5)P 3 (see Appendix liD. Whether the TOR2 gene product will provide a better model of mammalian PI3 kinase function must await the characterization of its catalytic action and the identification of its in vivo products. It is clear from the foregoing discussion that limited progress has been made thus far in attempting to find a function for this pathway. This is both because there is a dearth of good ideas to test and because it is difficult at present to test these ideas in a convincing fashion. In particular, it is difficult to prepare large enough quantities of both Ptdlns(3,4,5)P 3 (Ptdlns(3,4,5)P 3 has not yet been chemically synthesized and must therefore be prepared from biological sources) and appropriate 'controls' (e.g., similarly phosphorylated lipids or, ideally, the non-biologically occurring enantiomer of Ptdlns(3,4,5)P 3) to undertake a detailed investigation of its effects in vitro. It is hoped that the tools provided by the recent purification and cloning of PI3K (e.g., antibodies to, and genes encoding, PI3K) will allow the construction of model systems in which agonist-stimulated Ptdlns(3,4,5)P3-production may be enhanced a n d / o r suppressed in a controlled fashion and which

65 could therefore be used to effectively test various ideas for its function. It must also be considered that the present lack of any obvious 'gap' in our knowledge in which to 'fit' Ptdlns(3,4,5)P 3 may be because very little is presently known about the process which PtdIns(3,4,5)P 3 putatively controls, and hence 'PtdIns(3,4,5)P3-based' approaches (e.g., searching for PtdIns(3,4,5)P3-sensitive binding proteins or protein kinases, or searching in genetically tractable organisms for suppressors of PI3K-defects) may ultimately prove to be the most effective way forward.

Acknowledgements PTH is a Lister Institute Fellow; TRJ is a Mr. and Mrs. J. Jaff6 fellow of the Royal Society. We thank Catherine Brooksbank and Tony Corps for their valuable comments on reading the manuscript.

Appendices Appendix L The assay of PI3K in vitro with exogenous lipid substrates There are particular difficulties in deriving certain types of information from in vitro assays of phospholipid-metabolizing enzymes if the substrates are required to be presented as manipulable mixtures of purified phospholipids (often termed 'exogenous lipids'). Whilst it would seem that the fundamental, physiologically relevant reactions catalysed by these enzymes are faithfully represented in this type of assay, the regulatory properties and precise substrate specificities of these enzymes can be remarkably distorted by the assay conditions used. These discrepancies arise out of the quite different physico-chemical environments of the 'natural' membrane (i.e., a protein/lipid bilayer of extremely complex composition) and the available presentations of exogenous lipids, e.g., unilamellar or multilamellar liposomes of varying composition, mixed detergent/lipid micelles of varying composition, monolayers, etc. [99]. These differences in environment can be critical to the way in which the lipid-phase is recognised by the enzyme [99,154,316,317,319] (which is often the step in a lipid-metabolising reaction with the highest 'controlstrength', i.e., it often has the biggest effect on the rate of a process) and the way in which the substrate is presented to the enzyme within the lipid-phase (this is especially so with lipids possessing extreme physical characteristics, e.g., the very polar polyphosphoinositides [40]). Thus, in general, assays of this type should be used as qualitative indications that certain reactions may occur in vivo, but not quantitative indications of the extent that they do.

These difficulties in interpreting in vitro assays have been particularly frustrating in the study of soluble, agonist-regulated enzymes such as PIC and PI3K, where their mechanisms of regulation (i.e., agonist sensitivity) and substrate specificities (i.e., which polyphosphoinositides are substrates) are such key issues in understanding their role as signalling systems. In the case of PICs, a large body of work has described the relative rates of formation of its putative products in intact cells and one of these products, Ins(1,4,5)P3, has been established as an authentic 'second-messenger' [3,320]. Thus, through the course of this work, a consensus has developed which views PICs in vivo as being inactive in unstimulated cells, but active and PtdIns(4,5)Pz-selective on appropriate agonist stimulation [320,321]. Assays of PIC in vitro however, have yielded results which conflict to varying extents with this view. When assayed with high (mM range) Ca z+ and lipid/cholate micelles, PICs can be described as highly active, agonist-insensitive enzymes (e.g., Ref. 314), which are often PtdIns-selective (indeed, these were the conditions under which PICs were first described and led to the erroneous belief that they were PtdIns-selective in vivo) (see Ref. 320) for a review of much of the work that generated this view of events). If however, PICs are assayed under more 'physiological' ionic conditions [322], with the lipid substrates presented as unilamellar liposomes or endogenous membranes [314,323,324] they can retain a certain degree of sensitivity to appropriate stimulation and are most active against PtdIns(4,5)P 2 and PtdIns4P [323,324] (i.e., more nearly approach the anticipated situation in vivo). Further, the importance of considering the precise assay environment is particularly aptly demonstrated by studies of the regulation of PICvl, where the discovery of a potentially very important regulatory mechanism (tyrosine phosphorylation) was entirely dependent on assaying the enzyme under very specific conditions of lipid presentation [131] (mixed triton micelles with a low molar ratio of Ptdlns(4,5)P2; this is discussed in more detail in Appendix (IV) and Section IV-B.1). Initial studies of PI3K have presented problems which can be seen as analogous in many ways to those described above for PICs. The arguments presented in Section II suggest that PI3K is inactive in unstimulated cells but active and PtdIns(4,5)P2-selective on agonist stimulation (this view is supported by recent evidence indicating that the levels of 3-phosphorylated inositol lipids are unaltered in unstimulated cells over-expressing catalytically competent PI3K [38]). Assays of PI3K in vitro however, even when conducted with a carefully cell-environment-matched mix of phospholipids in the form of unilamellar vesicles, indicate that it is highly active when obtained from unstimulated cells and that it can readily phosphorylate PtdIns,

66 PtdIns4P and Ptdlns(4,5)P 2 in the 3-position [34-36]. The reasons for these apparent discrepancies could be that the interpretation of the intact cell data is simply wrong (and that PI3K is constitutively active against all three lipids in the intact cell) or that some feature of the in vitro assay environment is creating a misleading picture of the true activity/substrate-specificity of this enzyme in vivo. This latter form of explanation encompasses a huge number of possibilities, e.g., the cellular location of PI3K in the intact cell [38] may only allow it access to Ptdlns(4,5)P 2 amongst its potential substrates, or the effect of some component responsible for controlling the specificity of this enzyme is lost in the in vitro assays (indeed an uncharacterized component has been postulated to explain the apparent increase in Ptdlns(4,5)P 2 selectivity during PI3K purification from rat liver [34]). However, on the basis of the arguments outlined above, and the precedent set by PICs and other lipid-metabolizing enzymes, it must be considered highly plausible that the activity and substrate specificity of PI3K are simply distorted in assays using exogenous lipids, because of the forms of lipid presentation currently employed.

Appendix II. The origin of Ptdlns(3,4)P2 in agonistactivated cells As discussed in Section II-C, data obtained from 32P-labelling experiments using both intact and permeabilized cells suggests two potential routes by which Ptdlns(3,4)P 2 might be derived in agonist-activated cells; either (A), by a Ptdlns4P 3OH-kinase activity or (B), via a Ptdlns(3,4,5)P 3 5-phosphatase activity (the substrate for which is synthesized by a Ptdlns(4,5)P2 3OH-kinase). If Ptdlns(3,4)P z is derived solely by the dephosphorylation of Ptdlns(3,4,5)P 3 this suggests that: (1) Ptdlns4P 3OH-kinase activity (that can be expressed by PI3K, see Fig. 2, [10]) is an 'assay-artifact' and is not expressed in intact, agonist-activated cells (see Appendix I for a perspective with which to value this argument). (2) The lagged accumulation of Ptdlns(3,4)P 2 that is detected in both permeabilized and intact, agonistactivated cells (see Fig. 4) [16,22,25,30,45,63,92,204] can be naturally accommodated by the additional step required in its synthesis. A lagged accumulation of Ptdlns(3,4,5)P 3 relative to Ptdlns(3,4)P 2 has been reported in CD3- and CD2-stimulated T cells, but the Ptdlns(3,4,5)P 3 apparently measured in this study was not positively identified [258]. (3) The examples of agonist-stimulated cells that display increased accumulation of Ptdlns(3,4)P 2 but in which increased accumulation of Ptdlns(3,4,5)P 3 has not yet been detected ([64,149], see also Ref. 249), are

a result of either, (a) its misidentification, (b) its low levels compared to the detection limits of the assays (perhaps a result of particularly active Ptdlns(3,4,5)P 3 compared to Ptdlns(3,4)P 2 phosphatases), (c) a failure to efficiently recover Ptdlns(3,4,5)P 3 or, (d) an inappropriate selection of the time of stimulation (however, we also note the numerous examples of PIC activation where agonist-induced accumulation of Ins(1,3,4)P3, but not its precursor Ins(1,3,4,5)P4, have been reported). Situations in which agonists have been observed to activate relatively large accumulations of Ptdlns(3,4,5)P 3 but very little Ptdlns(3,4)P 2 (e.g., (GM-CSF-stimulated U937 cells [25]) may be explained by a differential expression of Ptdlns(3,4,5)P 3 3-phosphatase compared to Ptdlns(3,4,5)P 3 5-phosphatase activity. (4) The metabolism of Ptdlns(3,4,5)P 3 can be accounted for without the problems associated with proposing Ptdlns(3,4,5)P 3 5-phosphatase activity is an 'artifact' (see below). (5) That a single PI3K activity (that is Ptdlns(4,5)P 2directed) could be responsible for all of the changes observed in 3-phosphorylated inositol lipid metabolism in intact, agonist-activated cells. This potentially unifies the huge body of work implicating a PI3K activity defined in vitro in the signalling pathways utilized by tyrosine kinase-activating receptors (discussed in Section II-D), with the observed changes in the metabolism of 3-phosphorylated inositol lipids occurring in cells (not only stimulated by these receptors but also by receptors that utilise G-protein-mediated signalling pathways). Although this position can be achieved by assuming direct phosphorylation of Ptdlns4P, it requires far more complex arguments to do so (see below). (6) By proposing production of Ptdlns(3,4)P 2 is essentially governed by the metabolism of Ptdlns(3,4,5)P 3, this aligns the pathway with other signalling systems, like those centred around a PIC or adenylate cyclase, which encode their output signals in the form of the concentration of a single messenger molecule. It also makes the prospect that Ptdlns(3,4)P 2 is a potentially independent signalling molecule much less likely and this serves to simplify the process of investigating possible functions of the pathway by focusing attention on Ptdlns(3,4,5)P 3. If the Ptdlns(3,4)P 2 that accumulates in intact cells is solely derived by direct phosphorylation of Ptdlns4P (this does not necessarily mean a distinct PI3K activity is responsible for Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 production, but see below), then this suggests that: (A) Ptdlns(3,4,5)P 3 5-phosphatase is an assay 'artifact' or is inactive in stimulated ceils and, furthermore, that Ptdlns(3,4,5)P 3 must be metabolised via a pathway that generates a currently uncharacterized intermediate (Ptdlns(3,5)P 2) or a route (an inositol lipid 3-phos-

67 phate phosphatase) that is less active in vitro than the mechanism that we are proposing [16,30]. (B) To account for the lagged appearance of PtdIns(3,4)P2, we must propose that the substrate specificity a n d / o r supply of the agonist-sensitive PI3K must change during the early stages of its activation (in such a manner that Ptdlns(3,4,5)P 3 production is maintained through this period of change, despite the fact Ptdlns4P phosphorylation must rise from practically zero to rates of the same order as that of PtdIns(4,5)P2). The only alternative to this explanation, is to propose that the Ptdlns4P 3OH-kinase and PtdIns(4,5)P 2 3OH-kinase activities are independent. This, however, demands they are activated by the same receptor mechanisms with substantially different kinetics, adds unnecessary complexity and loses the clear focus on a single type of PI3K activity that is provided by the notion that a single form of the enzyme is activated in intact cells (and that concurs with current findings that there is only a single, or at most a limited and extremely closely related, set of PI3K activities in mammalian ceils; discussed in Section Ill-B). (C) If the added complexity of contemplating an independent Ptdlns4P 3OH-kinase is accepted, then the differences in the precise balance of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 accumulation detected under different circumstances (e.g., Refs. 16,25,63,64,149,249,258) are simply explained. (D) If an independent Ptdlns4P 3OH-kinase is envisaged to be responsible for Ptdlns(3,4)P 2 production, this endows the lipid with a degree of metabolic independence that might be argued to make it more likely to possess its own specific roles. Taking these arguments in balance, we consider the notion that Ptdlns(3,4)P 2 is derived from dephosphorylation of Ptdlns(3,4,5)P 3 offers the most simple explanation of the data currently available. Although operation of Ptdlns4P 3OH-kinase-, or Ptdlns(3,4,5)P 3 5phosphatase-based mechanisms of production of PtdIns(3,4)P2 are not mutually exclusive, to suggest both may occur (as, in many ways, it is tempting to do) is actually to propose a considerably more complex and far less experimentally testable explanation for the data than is currently necessary.

Appendix IlL The source of Ptdlns3P in cells Ptdlns3P is found in unstimulated mammalian cells at concentrations in the range of 1-2 /zM (generally 3-5% of Ptdlns4P levels in the same cells), whereas Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P3 are present at levels of 0.05-0.2 ~M (e.g., see Table I and Refs. 10,16,19). Upon appropriate stimulation, however, cellular levels of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 rise to become similar to those of Ptdlns3P, which, in contrast, display relatively small or undetectable changes in concentra-

tion (see Fig. 4 and Table I). These observations suggest that the majority of the Ptdlns3P in cells might be metabolically unrelated to the rapid agonist-stimulated changes in Ptdlns(3,4)P z and Ptdlns(3,4,5)P3. This raises the question of how Ptdlns3P is synthesized. The notion that direct 3-phosphorylation of Ptdlns occurs in cells is supported by the following observations. (1) Homogeneous preparations of PI3K can catalyze this reaction in vitro (however, see Appendix I for a discussion of the difficulties in interpreting the substrate specificity of PI3K in vitro) [34,35]. (2) Yeasts do not contain detectable quantities of Ptdlns(3,4)P 2 and Ptdlns(3,4,5)P 3 but do contain relatively large concentrations of Ptdlns3P (however, see the legend to Fig. 1 for the caveats associated with this statement) [37,77,242]. Moreover, 'standard' in vitro assays of total PI3K activity in yeast fail to observe 3-phosphorylation of either Ptdlns4P or Ptdlns(4,5)P 2 (in contrast to mammalian PI3K) [37,77]. (3) Majerus and co-workers have presented evidence that is compatible with direct 3OH-phosphorylation of Ptdlns in intact mammalian cells [17,43]. However, their data operate as an internally consistent package and parts of it should not be used in argument whilst others are rejected (this data is discussed in Fig. 6A). (4) Addition of 6 mM unlabelled MgATP to permeabilized neutrophils prelabelled with [3,-32p]ATP in the presence of maximal activating stimuli leads to an immediate 'chase' of radioactivity from PtdIns3P (the levels of label in its putative immediate precursor PtdIns(3,4)P 2 did not fall significantly in the same time frame) ([45] and L.S., unpublished observations). Although these data can not quantify the relative contributions of routes (A) and (B) they suggest a proportion of PtdIns3P in permeabilized neutrophils is derived by direct phosphorylation of PtdIns. The observations that indicate PtdIns3P is derived by dephosphorylation of PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 are as follows. (i) PtdIns[32p]3P can be detected as a breakdown product of PtdIns([a2P]3,4,5)P 3 in vitro [16]. However, because it was also apparent that a significant proportion of PtdIns([a2p]3,4)P 2 metabolism was possibly mediated by an inositol lipid 3-phosphatase activity [16], the turnover of PtdIns3P driven by this route might be low and hence the changes in PtdIns3P pool size that would be anticipated to be fuelled via this route may be relatively small. (ii) Despite (4) above, the synthesis of [32p]PtdIns3P from [T-a2p]ATP in permeabilised neutrophils proceeds with a significant delay relative to PtdIns4P [45].

Appendix ~ enzymes

'Activation' of soluble lipid metabolising

An agonist-induced 'activation' of a soluble enzyme utilizing a membrane localised substrate must ulti-

68 mately be produced by an increase in the rate of the relevant reaction in the membrane domain and the soluble enzyme must therefore, interact with the membrane. Hence, a concerted analysis of these events has to consider the interaction of the enzyme with both the membrane and its substrate. Bearing this dual consideration in mind, there are a number of distinct ways that an apparent activation of PI3K could be produced (only some of which are more generally applicable to soluble enzymes dealing with soluble substrates). (1) A simple increase in the mass of the enzyme in the system could supply the required increase in rate of reaction. However, precedent and the speed and scale of the 'activations' we are considering with PI3K make this unlikely and, furthermore, most workers would not consider the increased production of an enzyme constituted its 'activation' (however, the difference between this and some of the examples of activation considered below that are often deemed 'activations' become somewhat blurred). (2) A change in the catalytic constants of PI3K with respect to its substrates (i.e., apparent K m a n d / o r Vmax for Ptdlns(4,5)P z a n d / o r ATP) produced by either an allosteric or covalent modification of the enzyme. In this regard, the activation of PIC/31 in vitro by Gaq or G a l l was due, under the lipid-presentation conditions used, to an increase in apparent Vmax [88]. For both examples (1) and (2) to be effective, PI3K would have to have access to PtdIns(4,5)P 2. (3) A change in a membrane-association domain (e.g., the CalB domain? see Fig. 8) that increases the effective interaction of a soluble PI3K with a bilayer (for reasons of ease of analysis this is usually considered as being functionally independent of substrate recognition processes). This process is characterized by the K S association parameter in the analysis used by Denis and co-workers [317]. On the basis of a careful kinetic study of PtdIns(4,5)P 2 hydrolysis by tyrosine-phosphorylated vs. unphosphorylated PIC-T 1 based on this theory, it now appears that the 'activation' PIC-y 1 experiences as a result of tyrosine phosphorylation, under the assay conditions used (mixed triton micelles containing low molar fractions of PtdIns(4,5)P2), is largely a consequence of a change in this function [131]. Although in some senses this phenomenon could be regarded as a 'translocation' of the soluble enzyme to the membrane, it contrasts to more conventional 'translocational' mechanisms detailed below, in that a soluble enzyme 'activated' in this way would still be free to move throughout the entire aqueous region of the cell. (4) Translocation of PI3K to a target that results in its localising to a substrate-containing membrane could also supply an apparent activation of PI3K (however this would require that the localization mechanism does not block the capacity of the enzyme to interact

with its substrate). In its 'pure' form (i.e., isolated from a contribution of mechanisms of the type 1, 2 or 3 above) this would serve to concentrate PI3K into a space in which its substrate was also concentrated. On a much smaller, molecular, scale this type of mechanism has the potential to produce 'orientation-specific' interactions of PI3K with the membrane which could enable a far more efficient means of bringing PtdIns(4,5)P 2 and PI3K together (this might involve interaction with other localized facilitatory factors like PtdIns(4,5)P2-associating proteins or Ptdlns(4,5)P 2 itself, see Appendix V). Thus, these sorts of events could be envisaged to produce extremely subtle but effective activations of PI3K. Although in some ways this notion of translocation could be considered to have similarities to (3) above, it is sufficiently mechanistically distinct and has such clear analogies with events such as SH2-domain directed docking onto signalling complexes, cytoskeletal association, putative bcr-domain directed associations, or heterotrimeric G-proteintargeted associations, that it is most usefully considered in isolation. The significance of membrane localisation to PI3K activation is clearly evidenced by the failure of myristolylation-defective, non-membranelocalizing mutants of v-abl and v-src, that can apparently still recruit and tyrosine phosphorylate PI3K (or an associated protein), to transform cells or drive sustained increases in Ptdlns(3,4,5)P 3 like their parental oncogenes [53,129]. Appendix V. Ptdlns(4,5)Pe as a substrate

Although most phospholipids are most easily envisaged as freely diffusing within a planar membrane structure, for a molecule like PtdIns(4,5)P 2 this may be a very misleading picture. Considerable though largely indirect evidence indicates PtdIns(4,5)P 2 is largely found in the inner leaflet of the plasma membrane or membranes derived from it, however it can form tight and specific associations with proteins both within these membranes and in adjacent cytoskeletal structures [325-328]. Some of these PtdIns(4,5)P2-associated proteins are abundant, and may harbour substantial quantities of PtdIns(4,5)P 2 [329]. If the pool of PtdIns(4,5)P 2 utilized by PI3K (or PICs) is complexed with membrane-associated proteins (i.e., 'extrinsic' proteins) this could mean these enzymes would not have to truly interact with the membrane phase, and hence could avoid the kinetic barriers presented by the need to negotiate processes such as membrane 'penetration', 'adsorption' or 'scooting' that soluble enzymes (e.g., phospholipase A2s) which interact with authentic membrane-soluble substrates have to solve [99,154, 317,319]. Alternatively, if the pool of PtdIns(4,5)P 2 used by PI3K (or PICs) was viewed as associated with intrinsic membrane proteins, then, in the light of evi-

69 dence showing that the presence of structurally deforming features in phospholipid bilayers, such as proteins, can substantially aid the access of soluble phospholipid metabolizing enzymes into the membrane domain (and hence increase their effective activity) [154], this could nevertheless have major implications for the mechanism of operation of these enzymes. Even more radically, in view of the soluble/cytoskeletal distribution of Ptdlns4P 5OH-kinase and Ptdlns 4OH-kinase [13,330-332] and the existence of soluble Ptdlnstransfer proteins [318,333], even those pools of PtdIns(4,5)P 2 associated with apparently aqueous proteins might be actively metabolized and represent sources of substrate for PICs or PI3K. Appendix VI. Nomenclature of signalling cascades The 'meaning' of many of the phrases commonly associated with descriptions of the series of interactions that relay messages from extracellular mediators to target enzymes inside cells, have become more confused as each new layer of regulatory mechanism is peeled away. Despite this, we have regularly slipped into use of words and/or phrases like: transducing protein or step, signalling pathway or system, effector, second-messenger, signalling cascade, etc. We use these terms in the following ways. Transducing proteins or steps are those that convert changes in receptor conformation into another form of signal, this often occurs in the receptor (e.g., a change in an intrinsic PTK activity or the permeability of an intrinsic channel), but may be detected and transmitted by proteins able to closely interact with receptors (e.g., G proteins, receptor-associated src-type PTKs or 'ionophonic' channels). Signalling pathways are cascades of reactions that usually 'start' at their interactions with transducing proteins (e.g., pathways headed by activation of receptoroperated calcium channels, PICs or adenylate cyclases) or, in some circumstances, can be seen as starting with the transduction step itself (e.g., activation of a receptor-guanylate cyclase). In the case of signalling pathways that are headed by enzymes that have low-molecular-weight, non-catalytic, diffusible products, which serve to regulate the activity or function of other proteins via allosteric interactions; then these products are termed 'second-messengers' (e.g., cAMP, Ins(1,4,5)P 3, cGMP or Ptdlns(3,4,5)P 3 (?)) and the enzymes responsible for generating them are termed "effectors" (e.g., adenylate cyclase, PICs, guanylate cyclase, PI3K (?)). Appendix VII. SH2 domain : tyrosine phosphate interactions SH2 domains bind directly to specific tyrosine phosphate residues on proteins. This feature allows pro-

teins which contain SH2 domains, including PI3K (its 85-kDa subunit contains two, see Fig. 8), to bind to specific tyrosine phosphate cues in PTK-coordinated signalling complexes. The structure of a fusion protein containing only the amino-terminal SH2 domain of p85o~ [168] has recently been shown to possess many of the structural features present in the conformation of crystalline SH2 domain:phosphotyrosine peptide complexes that are thought to have functional significance for the host proteins [174,334,335]. Hence the huge diversity of protein types (and fragments obtained from them) within which SH2 domains can function, appears to be a result of their highly self-contained modular structure [174,334,336]. Although the overall structure of SH2s appears to be remarkably similar, despite substantial sequence differences, the residues implicated by site-directed mutagenesis in peptide binding are found in a relatively flat open surface (when in the uncomplexed state) that shows a degree of structural plasticity between the SH2s thus far analyzed which might account for the distinct recognition sequences that different SH2 domains demand be present around the relevant phosphotyrosine residue in their targets [174,336] (see Section V-B.1. and below). In contrast, the structure of the tyrosine phosphate binding site is probably highly conserved amongst SH2s and is formed by a rigidly defined pocket (containing several functionally critical positively charged residues) that is located in the peptide-binding surface (and is probably relatively dilated in the uncomplexed state as a result of the electrostatic interactions occurring between the positive residues) [174,336]. Binding to an appropriate tyrosine-phosphorylated target is presumably mediated by both of these regions and appears to be an example of induced fit; the negative charge on, and aromatic qualities of, the critical phosphotyrosine residue are probably the major interactive features that enable the positive residues in the SH2 pocket to come closer together and this might correlate with partial closure of the peptide-binding surface [174]. These changes in SH2 structure on target binding may drive other events in the host protein, including in the case of PI3K a trans-subunit activation of its catalytic domain [86,87] and suggest the role of the SH2 domains in PI3K may go beyond that of being simple site-selective anchors to one of specific tyrosine phosphate sensors. A very recent study has begun to systematically address the binding specificity of SH2 domains for different phosphopeptide sequences [338]. A semi-random peptide library was used to determine the optimal sequences for tight binding to individual SH2-domainGST-fusion proteins. The SH2 domains studied could be grouped into families on the basis of the general motifs that they recognized, e.g., the SH2 domains of p85a, PICT2 and SHPTP2 all preferred sequences

70

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