Interaction of tyrosine kinase oncoproteins with cellular membranes

307

Biochimica et BiophysicaActa, 1155 (1993) 307-322 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00

B B A C A N 87278

Interaction of tyrosine kinase oncoproteins with cellular membranes Marilyn D. Resh

*

Department of Cell Biology and Genetics, Memorial SIoan-Kettering Cancer Center, 1275 York Avenue, Box 143, New York, N Y 10021 (USA) (Received 17 September 1993)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

307

II.

Biosynthesis and targetting of m e m b r a n e - b o u n d tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . A.I. T r a n s m e m b r a n e proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2. Receptor internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. gag-onc fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Non-transmembrane, extrinsic tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.1. pp60 vsrc as a paradigm for myristylated oncoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . C.2. Additional acylation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C.3. T h e SH4 motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

308 308 308 309 309 309 311 311

III.

Activation of m e m b r a n e - b o u n d tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Receptor oligomerization activates t r a n s m e m b r a n e proteins . . . . . . . . . . . . . . . . . . . . . . . . . B. Activation of extrinsic tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

312 312 312

IV.

Mechanisms of transformation: role of the m e m b r a n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.1. Structure of the membrane-spanning domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2. Mutation in the t r a n s m e m b r a n e domain activates the neu proto-oncogene . . . . . . . . . . . A.3. Specific information can be encoded within t r a n s m e m b r a n e sequences . . . . . . . . . . . . . B. Is m e m b r a n e localization necessary for transformation? . . . . . . . . . . . . . . . . . . . . . . . . . . . .

313 313 313 314 314

V.

Interaction of src family m e m b e r s with m e m b r a n e - b o u n d proteins . . . . . . . . . . . . . . . . . . . . . . . A.1. Interaction of p56 Ick with CD4 and CD8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2. Other m e m b r a n e interactions of p56 lck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T h e T-cell antigen receptor interacts with tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . C. Supermolecular complexes form with GPl-linked proteins at the cell surface . . . . . . . . . . . . .

315 315 316 316 317

VI.

Signal transduction: from plasma m e m b r a n e to the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.1. Intermolecular communication at the plasma membrane: link to p21ras . . . . . . . . . . . . . A.2. Other signal transducers associate with the P D G F receptor . . . . . . . . . . . . . . . . . . . . . B. Signalling from m e m b r a n e to the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Unanswered questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

317 317 318 319 319

VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

320

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

320

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

320

I. Introduction

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* Corresponding author. Fax: + 1 (212) 7173317.

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308 Membrane bilayers also perform a structural role, by serving as a scaffold or structural framework which facilitates organization of enzymes into multi-protein complexes. Several features of biological membranes are particularly well suited for formation of these complexes. Although proteins are fluid within the plane of the bilayer, lateral diffusion of membrane proteins is limited compared to proteins in solution. Moreover, transmembrane 'flip-flop' is extremely slow, thereby preserving the unique identities of the inner and outer surfaces of the bilayer. Thus, kinetic parameters favor an increase in the local concentration of key proteins at the membrane interface. Cellular proteins which are targetted to the plasma membrane have the potential to regulate transmembrane signal transduction, and are poised to serve as organizing centers for signal transduction complexes. It is therefore not surprising that many oncoproteins are localized to the plasma membrane, the site at which perturbation of normal cellular processes is initiated. Membrane-bound protein tyrosine kinase oncoproteins can be classified into two general types: those which span the membrane and those which do not. The latter class, which lack a hydrophobic transmembrane sequence, bind to the inner face of the plasma membrane, as well as to other intracellular membranes. The tyrosine kinase domain in both classes of oncoproteins is oriented towards the cytoplasmic side of the membrane, where it has access to intracellular protein substrates. In addition, a growing number of soluble, intracellular tyrosine kinases have been described, including the Janus kinases (Jakl, Jak2), focal adhesion kinase (Fak), spleen tyrosine kinase (syk), Interleukin-2 inducible T-cell kinase (Itk), and c-src kinase (Csk) [13,134]. It is important to consider that the majority of known oncogenes are derived from 'normal' cellular proteins, and that mutation a n d / o r overexpression of a 'proto-oncogene' can result in oncogenic activation. For example, several tyrosine kinase oncoproteins are mutated versions of growth factors or growth factor receptors (Table I), and their expression results in constitutive activation of cell signalling pathways. Moreover, overexpression of the wild-type form of a growth factor receptor has been shown to lead to ligand-dependent transformation [46] (e.g., E G F receptor). Thus, a discussion of membrane-bound oncoproteins must obligatorily include the transmembrane growth factor receptors, many of which participate in both normal and oncogenic signal transduction. This review will focus on the structural and functional role of cellular membranes in mediating signal transduction by protein tyrosine kinase oncoproteins and growth factor receptors. Three particular questions will be addressed. First, how are protein tyrosine kinase oncoproteins targetted to membranes? Second,

TABLE I Membrane-bound tyrosine kinase oncoproteins Transmembrane Oncogene Proto-oncogene homologue/function v-erbB EGF receptor neu/c-erbB2 EGF-like receptor (neu ligand) v-fms CSF-I receptor v-kit c-kit receptor (mouse W locus) trk NGF receptor met Hepatocyte growth factor receptor ros sevenless (Drosophila) ret (c-ret)? v-sea 9 v-ryk ? Non-transmembrane/extrinsic src family src, yes, lyn, fyn, blk, hck, Ick, fgr, yrk abl family abl, arg Other fps, fes

what is the molecular nature of the interaction between oncoprotein and membrane? Third, how are signals generated at the membrane and transduced to the cell interior?

II. Biosynthesis and targetting of membrane-bound tyrosine kinases II-A. 1. Transmembrane proteins The mode of delivery of tyrosine kinases to cell membranes is dependent on protein domains outside of the kinase region. As illustrated in Fig. 1, transmembrane tyrosine kinases contain membrane-spanning sequences as well as extracellular ligand binding domains. The biosynthesis of transmembrane tyrosine kinases follows a pathway similar to that established for other transmembrane proteins destined for the plasma membrane. Nascent polypeptides are synthesized on membrane-bound polysomes, processed through the endoplasmic reticulum and Golgi apparatus, thereby acquiring N-linked glycosylation, and are delivered to the plasma membrane. For example, the v-erbB oncoprotein is synthesized as a 61 kDa protein precursor, and processed to 66 and 68 kDa forms by glycosylation in the endoplasmic reticulum. Some of the molecules are transferred to the Golgi and further processed to a larger 74 kDa species by glycosylation. Only a minor portion of the total verbB proteins are ultimately expressed at the cell surface [12]. I1-A.2. Receptor internalization Receptor tyrosine kinases are removed from the cell surface by a process known as down regulation. Subse-

309 quent to ligand binding, receptors aggregate into coated pits and are internalized via endocytosis [166]. Two different fates await the endocytosed receptors. Some receptors (e.g., insulin receptor) are recycled to the cell surface following ligand dissociation. Other receptors such as EGF are targetted to lysosomes, where they are degraded. Tyrosine kinase activity is required for the latter pathway; kinase-negative EGF receptors are internalized but rapidly recycled [69]. Down regulation thus provides a mechanism for turning 'off' the signals generated by cell-surface receptors. One would predict that receptors defective in internalization could potentiate ligand-induced signals to a greater extent than wild-type receptors. Such a prediction is verified by a C-terminal truncation mutant of the EGF receptor, which fails to internalize and exhibits super-sensitivity to low concentrations of EGF [177]. Mitogenic responses are achieved using EGF concentrations nearly 10-fold lower than those required to activate the wild-type receptor. Moreover, cells expressing the mutant receptor become morphologically transformed when exposed to low concentrations of EGF. Downregulation appears to have evolved as a mechanism to prevent transformation by physiological concentrations of ligand. H-B. gag-onc fusion proteins Many retroviral oncoproteins are synthesized as gagonc fusions, examples of which include v-abl (pl60gag'abl), v-fps (p130gag-fps), and v-yes (p90 gag'yes, p80gag-yes). The gag (group-specific antigen) gene is found at the 5' end of all retroviruses and encodes the internal structural proteins of the virion. Gag proteins are synthesized as polyprotein precursors, and subsequently cleaved into 4-6 products by a virus encoded protease. Gag-onc fusions generally contain 5' gag matrix and capsid sequences. Although the gag-onc

tyrosine kinases are primarily localized to the inner surface of the plasma membrane, the mechanism of targetting is not completely understood. N-terminal gag sequences contain intrinsic membrane targetting information [180,181], which for mammalian retroviruses comprises, in part, a myristylation site (see below). The gag sequences per se are not required for v-fps mediated transformation [54], and can be replaced by N-terminal sequences from v-src [17]. Other oncoproteins are initially produced as gag-onc fusions, but the gag sequences are subsequently removed. An example of the latter mechanism is the v-fms oncoprotein of feline leukemia virus, which arose by insertion of c-fms sequences into the gag open reading flame. The primary translation product is a glycosylated precursor, p180 gag-fms, that is proteolytically cleaved, resulting in a gag fragment (p55 gag) and the v-fms glycoprotein (pl20v4ms). Following further carbohydrate addition in the Golgi, the mature glycoprotein p140 vfms is translocated to the plasma membrane, where its presence correlates with transformation [129]. H-C. Non-transmembrane, extrinsic tyrosine kinases II-C.1. pp60 vsrc as a paradigm for myristylated oncoproteins The mode of membrane trafficking and attachment for the extrinsic tyrosine protein kinases is quite different and best exemplified by the v-src oncoprotein [121]. pp60 v'src is synthesized on flee ribosomes in soluble form [88], and post-translationally attaches to membranes. The nascent polypeptide does not contain an N-terminal signal sequence nor are any sites for N- or O-linked glycosylation present [158]. In addition, no obvious stretches of hydrophobic amino acids, typically found in the transmembrane protein domains, are evident. Despite the lack of a 'hydrophobic' protein do-

Transmembrane Extrinsic >

outside inside l insert Fig. 1. Structure and orientation of membrane-bound tyrosine kinases. Transmembrane proteins contain an extracellular ligand binding domain, a transmembrane region of hydrophobic amino acids, and an intracellular tyrosine kinase domain. In some instances, the kinase domain is interrupted by an insert region. In contrast, extrinsic tyrosine kinases lack extracellular and transmembrane domains, and are anchored to the bilayer via a combination of acylation (myristylation and/or palmitylation) modifications and ionic interactions (see text).

310 main, 80-90% of the pp60 vsrc molecules in a Rous sarcoma virus-transformed cell are membrane bound at steady state [36,122]. A key to understanding the nature of pp60 v-src membrane association came with the finding that the Nterminus of the mature src oncoprotein is modified by myristylation [22]. Covalent attachment of the 14 carbon saturated fatty acid, myristate, is catalyzed by the enzyme N-myristyl transferase (NMT), which exhibits strict substrate specificity for N-terminal glycine residues [161]. During translation, the initiating methionine residue of src is removed by methionine amino peptidase, thereby exposing glycine-2 as the N-terminal amino acid [56]. Myristylation occurs co-translationally [42] before the first 50-80 amino acids are polymerized on the ribosome. Following completion of chain elongation, the nascent myristylated polypeptide chain is released from the ribosome in soluble form. N-terminal myristylation is essential for directing membrane association of pp60 vSrc. Non-myristylated v-src mutants do not bind to membranes, in vivo or in vitro, and do not transform cells [39,40,73]. However, myristylation alone is neither sufficient for membrane binding of src oncoproteins, nor for proteins in general. More than 50 proteins are now known to be N-terminally myristylated, and these myristyl-proteins are found in multiple cellular locations, including the plasma membrane, Golgi, endoplasmic reticulum, nucleus, as well as the cytoplasm. Thus myristate per se is not a specific targetting signal. It is now established that within pp60 vsrc, myristate functions in conjunction with specific adjacent aminoacid residues to direct membrane attachment. Using a combination of site-directed mutagenesis and peptide competition experiments, Silverman and Resh [143] showed that the myristate moiety plus a motif of three

alternating lysine residues is both necessary and sufficient for membrane association of pp60 vsrc. The 'myristate + lysine' sequence is also found in members of the src family of tyrosine kinases (Fig. 2), and defines a new membrane targetting motif [145]. Identification of src binding components in the membrane has been facilitated by the development of in vitro membrane binding systems [120]. pp60 vsrc exhibits saturable and specific binding to membranes in vitro. Membrane binding is inhibited by myristylated, but not non-myristylated, src peptides. These results led to the hypothesis that a specific myristyl-src binding component or 'src receptor' exists in the membrane, that recognizes N-terminal myristyl-src sequences. Using myristylated src peptides as probes, followed by chemical crosslinking, two groups have identified 32 kDa and 50 kDa src binding proteins in platelet membranes, and a 32 kDa protein in fibroblast membranes [51123]. The purified 32 kDa protein has been identified as the A D P / A T P carrier (AAC), an integral membrane protein primarily localized to the inner mitochondrial membrane [142]. Myristylated src peptides can gain access to a high affinity binding site on the AAC, while the larger pp60 .... c polypeptide cannot. The AAC is therefore a 'receptor' for myristyl-src peptide but not for pp60 vSrc polypeptide. Although no definitive sequence information is available for the 50 kDa protein, it too is unlikely to be a bona fide pp60 ..... receptor based on its peptide binding specificity ([144]; Sigal and Resh, unpublished results). To date, no specific protein receptor for pp60 vsrc in the plasma membrane has been identified. It is therefore possible that the membrane 'receptor' for pp60 v.... may be a specific lipid, rather than a protein. There is precedence in the literature for high affinity binding of proteins containing basic amino acids to acidic phos-

N 2

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Fig. 2. Multi-domain structure of the src family of tyrosine kinases. The four src homology regions are diagrammed (with positions in the src sequence indicated). Conserved amino-acid residues within the SH4 motif are highlighted in bold type. The N-terminal myristate moiety is attached after removal of the initiating methionine residue. If no gap is introduced into the fgr sequence, residue 10 (GLU) would also align with the SH4 consensus.

311 pholipids in the membrane [79,103]. Thus, in addition to the myristate, the lysines within the src N-terminus may provide further binding energy, through interaction with negatively charged membrane phospholipids. Biophysical evidence supporting this type of membrane interaction is now available for myristyl-src peptides [21], and a model for pp60 vsrc membrane interaction can be proposed which takes into consideration the amphipathic nature of the src amino terminus [Sigal and Resh, manuscript in preparation]. The myristylated, basic N-terminus of src would confer a general membrane binding affinity which would allow pp60 src to be targetted to several different membranes within cells, depending on the presence of acidic phospholipid. Indeed, pp60 v-src is found in the plasma membrane and perinuclear membranes in fibroblasts [117,122], whereas the presence of pp60 csrc has been reported in endosomal membranes [76], in chromaffin granule membranes [60], as well as in the plasma membrane [53]. Moreover, the requirement for myristate is not absolute, as myristate can be replaced by slightly longer or shorter fatty acid analogs without adverse effect on membrane binding activity [113]. Specificity of binding could be achieved through subsequent protein-protein interaction, mediated by the downstream src homology (SH) domains ([75]; Okamura and Resh, manuscript submitted). The molecular details of the latter interactions remain to be established. II-C.2. Additional acylation reactions may enhance membrane binding of src family members It is clear from the above discussion that additional components beyond myristate are required to provide stable binding of pp60 Src to cellular membranes. The presence of the myristate + lysine motif in many of the src family members [143], and its contribution to membrane binding of lyn, fyn and yes [145] is now established. In contrast, one of the family members, p56 lck, is myristylated but lacks the lysines present in the N-terminii of the other family members. In T cells, trafficking of p56 j~k to the plasma membrane is assisted by interaction with the CD4 transmembrane glycoprotein (see Section V-A.1). However, p56 ~ck can be expressed in non-lymphoid cells, where it is still efficiently bound to membranes. It is therefore likely that an auxiliary modification serves to enhance p56 ~ck membrane binding. The biochemical identification of this modification was recently determined [107]. p56 l~k is acylated via ester linkage with the 16-carbon fatty acid palmitate. The chemistry and kinetics of palmitylation are distinct from that of N-terminal acylation via myristate. Although the exact site of p56 ~k palmitylation has not yet been determined, it is tempting to speculate that it is the cysteine residue at position 3. The latter statement

is based on the recent reports that cysteine-3 of G protein o~ subunits is palmitylated [91,108]. By analogy to p21 ras, palmitate addition should increase the avidity of membrane binding [64]. Moreover, palmitylation has been shown to be a reversible reaction, potentially allowing p56 Ick to contact multiple membrane-bound substrates (see below). Of the nine members of the src family, seven contain a cysteine residue at position-3 (Fig. 2). Further research will be required to determine if these other family members are palmitylated. In addition, several unanswered questions remain. First, is myristylation of the N-terminal glycine (gly-2) a prerequisite for palmitylation? One might expect myristate to facilitate accessiblity to the palmitoyl transferase, which has been reported to be membrane-bound [78]. Second, is palmitylation required for interaction of src family members with other membrane proteins (see Section V)? For example, protein-protein interactions can be influenced by palmitylation, as evidenced by the /3adrenergic receptor [104] and the nucleotide releasing protein GAP43 [156]. Finally, is palmitylation regulated, and if so, what is the effect on membrane binding and biological activity? The rapidly emerging field of protein acylation is likely to provide important answers to these exciting questions. II-C.3. The N-terminal sequences of src family members form a new src homology motif." SH4 Members of the src family of tyrosine kinases share several regions of sequence similarity, denoted src homology (SH) domains [81]. These regions comprise the C-terminal tyrosine kinase (SH1) and internal SH2 and SH3 domains. Based on the above discussion, it is clear that an additional conserved sequence motif is present within the N-terminii of the src family members, previously assumed to be unique. This motif contains the first nine amino acids of the mature protein, plus an N-terminal myristate: myr- G S/C I/V K S/C K X K D/E I propose that the above motif, in keeping with existing nomenclature, be designated SH4 (Fig. 2). At least two functions are encoded within this sequence. The SH4 motif contains amino acids necessary for N-terminal myristylation, including gly-2 (which becomes the Nterminal residue upon removal of the initiating met) and ser-6, both of which are conserved in nearly all known myristylated protein sequences [161] (Fig. 3). Lys-7 is also required for myristylation of pp60 v-Src [74], but is not found at position 7 in most other myristylated proteins. However, the other SH4 amino acids are apparently not part of a myristylation motif, since they are not highly conserved among non-src family myristyl-proteins (Fig. 3), and substitution of lys-5, lys-9,

312 N-Terminal Sequence Conservation I

I

9 8

4O

7

~

6

30

4

2O

3 2

10

rnyr GLY SER ILE LYS CYS VAL CYS aa#

2

3

4

5

6

X GLU 7

8

9

l0

Fig. 3. Nlterminal sequence comparison between src family members and other myristylated proteins. The first nine amino-acid residues of 44 known myristyl-protein sequences were compared to the SH4 consensus sequence, at each position. The data for position 6 includes both serine and threonine residues.

or asp-10 [74,143] does not affect myristylation of pp60 ....c. The presence of additional conserved residues suggests that a second function may be encoded by the N-terminal SH4 sequence. Several lines of evidence implicate a role for SH4 amino acids in membrane targetting a n d / o r interaction with membrane-bound proteins. Myristate plus three lysine residues form a critical motif within the N-terminus of pp60 vsrc which directs membrane association of this oncoprotein [143], several other members of the src family [145], as well as chimeric proteins containing this motif at the Nterminus [75,111]. In T cells, the first 10 amino acids of p59 fyn mediate interaction with the zeta chain of the T-cell receptor [58]. Finally, the SH4 motif contains conserved cysteine residues that are likely to be palmitylated, and thereby influence membrane binding. III. Activation of membrane-bound tyrosine kinases

IliA. Receptor oligomerization activates transmembrane receptors The plasma membrane forms a physical barrier between the ligand binding domain of a transmembrane receptor and its catalytic tyrosine kinase domain. A mechanism must therefore exist to transmit signalling information received by the receptor at the extracellular surface through the bilayer to the intracellular domain. There is now ample compelling evidence to implicate receptor dimerization as the mechanism of

receptor activation [166]. Binding of ligand has been shown to promote receptor dimerization and receptor activation for the EGF receptor [15,185], PDGF receptor [66], and CSF-1 receptor [87]. The ligands can be monovalent (EGF) or divalent (PDGF), and receptor oligomers are evident both in vitro and in vivo [166]. The intermolecular interactions promoted by receptor dimerization result in activation of receptor tyrosine kinase activity. The receptor dimerization model predicts that stabilization of receptors in dimeric form should result in constitutive activation. Such a case is evident for the neu/c-erbB protein (see below). Moreover, it should be possible to block signal transduction by promoting heterodimer formation between wild-type and mutant receptor forms. As predicted, kinase defective EGF receptors function as dominant negative inhibitors. Hybrid oligomers are formed with wild-type EGF receptor and EGF-mediated signal transduction and transforming activities are blocked [77,116]. Likewise, point mutations in the kinase domain of c-kit result in dominant negative loss of function in W mutant mice [118]. The region encoding dimerization information is apparently present in the extracellular, ligand binding domain. A soluble, purified fragment of the EGF binding domain of EGF receptor forms dimers and inhibits tyrosine kinase activity of wild-type EGF receptor in vitro through heterodimer formation [10,85]. However, in vivo the ability of the soluble domain to exert dominant negative effects on EGF receptor function is vastly reduced. Tethering of the ligand binding domain to the membrane apparently increases the efficiency of intermolecular interaction, allowing effective heterodimer formation.

III-B. Oncogenic activation of extrinsic tyrosine kinases Retroviral oncogenes were acquired by transduction of normal cellular proto-oncogenes concomitant with the introduction of a limited number of deletions and mutations. All members of the src family of tyrosine kinases share a conserved tyrosine in their C-terminal tail (Tyr-527 in c-src), which is negatively regulated by phosphorylation [37]. Dephosphorylation of this site, or removal by mutation, results in an activated form of c-src that is capable of mediating cellular transformation [29,80,114]. The kinase that phosphorylates the C-terminal tyrosine of the src family members, CSK (c-src kinase) [105,106], also contains SH1, SH2, and SH3, but not SH4 domains. The currently accepted model portrays an intramolecular interaction between the SH2 domain (and possibly SH3) and tyr-527, which serves to keep src kinases in their inactive state [130]. Mutations within SH2 and SH3 regions apparently disrupt this interaction, thereby unleashing the catalytic domain from repression [67,137].

313 The subcellular distribution of proto-oncogenes and oncogenes is often different. As noted in Section IIC.I., pp60 vsrc is localized to the plasma membrane and perinuclear regions in fibroblasts [117,122]. In contrast, several studies have demonstrated localization of pp60 csrc to juxtanuclear vesicles, which likely represent endosomal membranes, in chicken, rat and mouse fibroblasts [41a,76,117]. In chromaffin cells, most of the pp60 c-src molecules are concentrated in chromaffin granules [60], which are endosomally derived. However, Ferrell et al. [53] report a plasma-membrane localization for pp60 .... c in platelets. Oncogenic activation of pp60 c.... is correlated with a shift in its distribution to a 'detergent-insoluble matrix' fraction, loosely defined as the cytoskeleton [62,92]. Many of the targets of src's tyrosine kinase activity are cytoskeletal associated proteins [63,109], and it is likely that the cytoskeleton represents a biologically important site for transforming activity. Activation of the c-abl proto-oncogene is also associated with mutations that alter its subcellular localization. p150 c-abl(Iv) is a myristylated, nuclear protein [169] which is activated by an N-terminal deletion that changes its distribution from the nucleus to the cytoplasm. Acquisition of the abl gene by Abelson murine leukemia virus resulted in deletion of N-terminal abl sequences and replacement with myristylated gag sequences, such that p160 gag-v-ab! is mostly plasmamembrane bound [55]. A alternate method of activation is evident in chronic myelogenous leukemia, where fusion of the c-abl and bcr genes results in chimeric proteins (p185 bcr-abl, p210 bcr-abl) that are activated by intramolecular interaction between bcr sequences and the abl SH2 domain [112]. IV. Mechanisms of transformation: role of the membrane IV-A.1. Structure o f the transmembrane domain

Transmembrane-spanning regions of proteins generally consist of 20-25 hydrophobic amino acids. Thermodynamic considerations favor the insertion of this region into the hydrophobic membrane interior, the basis of which is known as the hydrophobic effect [159]. If a hydrophobic molecule is introduced into an aqueous environment, the hydrogen bonding network of the surrounding water molecules must be rearranged and re-ordered. This increase in order, i.e., decrease in entropy would lead to an unfavorable increase (AG > 0) in free energy: AG = A H - - T A S . Consequently, insertion of a hydrophobic transmembrane domain into the phospholipid bilayer is entropy-driven. The hydrophobic nature of the bilayer interior also dictates considerations for secondary structure of transmembrane regions. With the notable exception of bacterial porins, nearly all transmembrane-spanning

regions assume an alpha-helical structure in the membrane [149]. This structural preference is due to the ability of the polar main-chain atoms within the helix to hydrogen bond to each other, leaving only the hydrophobic side chains exposed to the lipid interior. Assuming an average bilayer width of 35-40 A, and a rise of 1.5 ,~ per amino acid in an alpha helix, one can calculate that a stretch of 23-26 amino acids would be required to span the membrane in alpha-helical form. This is exactly the number of residues found in most transmembrane domains. o

IV-A.2. Mutation in the transmembrane domain activates the neu proto-oncogene

The neu oncogene was isolated in 1981 by transfection of D N A from rat neuroblastoma cells [141]. Neu and its human homolog, c-erbB2 or HER2, encode transmembrane receptor-like tyrosine kinases with striking sequence similarity to the E G F receptor [7]. At least one putative ligand for neu has been identified and cloned [68,178], termed NDF or heregulin, which contains an EGF-like domain. The neu proto-oncogene is susceptible to oncogenic activation by several molecular mechanisms. Deletions within the extracellular or intracellular domains [8] or overexpression of the normal proto-oncogene [47] activate the transforming potential, c-erbB2 is often overexpressed in breast and ovarian cancer and is correlated with poor clinical outcome [150]. However, the primary activating mutation which converts the rat proto-oncogene to an oncogene maps to a single point mutation within the neu transmembrane domain, resulting in change of a valine at position 664 to a glutamic acid residue [7]. Activation of neu is position and amino-acid specific. Mutation of val-664 to either glu or gln is highly transforming, to asp is weakly transforming, and to gly, his lys or tyr is not transforming. Mutation of residues adjacent to position 664 to glu does not activate neu [8] but the amino-acid sequence context surrounding glu-664 is important [27]. Point mutation within the neu transmembrane domain activates its protein tyrosine kinase activity [8] and may therefore serve to influence the conformation or the oligomeric state of the receptor in the membrane. Theoretical predictions for preferred three-dimensional structure suggest that non-transforming neu sequences contain a sharp bend within the alpha helix of the transmembrane domain, whereas the transforming sequences are mostly alpha helical [16]. Direct determination of the tertiary structure of peptides containing the neu transmembrane domains confirmed the adoption of alpha helical structures, but failed to reveal any significant differences between normal and mutant sequence conformations [61]. One might therefore be tempted to conclude that altered receptor dimerization is the consequence of the

314 transmembrane mutation. Indeed, activated forms of neu are preferentially stabilized in oligomeric form [175]. Dimerization could thus allow potential formation of intermolecular hydrogen bonds between residues on two different alpha helices [154]. However, experimental verification of alpha helical dimers for neu transmembrane domains has not been obtained [61] and may require the use of a phospholipid bilayer rather than analysis in solution. IV-A.3. Specific information can be encoded within transmembrane sequences The identification of a functional role for a transmembrane sequence is not unprecendented. Transmembrane domains have been shown to influence protein retention within the Golgi or endoplasmic reticulum [96]. Moreover, the presence of charged residues has been documented in proteins with multiple membrane-spanning regions, such as bacteriorhodopsin, as well as those with a single transmembrane domain [149]. Studies by Klausner's laboratory have established that positively charged within the transmembrane domain of the alpha subunit of the T-cell receptor stabilize assembly of the TCR complex, by interacting with acidic residues in the transmembrane region of the CD3 delta chain [34]. In the absence of pairing subunits with the appropriate reciprocal charge, the alpha subunit is targetted for degradation [14]. The molecular mechanism for charge stabilization has not yet been determined, and could involve formation of intermolecular hydrogen bonds or ion pairs. It is tempting to speculate that the transmembrane regions of other proto-oncogenes and growth factor receptors may also contribute to signaling function. Mutations analogous to Glu664 in neu activate the human c-erbB2 gene [136], the Drosophila EGF receptor homolog [179], and the insulin receptor [93], but have no observable effect on the activity of the EGF receptor [28]. Moreover, transmembrane domains of the EGF, PDGF, and insulin receptors can be mixed and matched without overtly affecting signalling properties [166]. Thus activation by unique mutation within the transmembrane domain may be restricted to neu and closely related family members, and may not be generally applicable to all transmembrane receptor-like tyrosine kinases. IV-B. Is membrane localization necessary for transformation? Paracrine regulation by exogenous ligands clearly requires that the extracellular portion of a growth factor receptor be accessible at the cell surface. Growth factor receptors that are impaired in cell surface expression are generally inactive, and not responsive to ligand. However, many transmembrane oncoproteins

are constitutively activated and therefore do not require ligand interaction for their stimulation. It is therefore possible that a trans-plasma-membrane disposition may not be absolutely essential. Several studies have addressed this issue. Early experiments with the v-erbB oncoprotein revealed that the majority of the protein was associated with intracellular membranes [115], but that transforming activity seemed to correlate with expression of a small amount of v-erbB on the cell surface [12]. The mode of membrane attachment was not specific, as the N-terminal 14 amino acids of v-src, containing a myristylated membranetargetting motif, could anchor the tyrosine kinase domain of v-erbB to membranes and permit verbB-specific transformation [99]. What happens when a tyrosine kinase oncoprotein is mistargetted? Deletion of the transmembrane domain of c-erbB2 results in a truncated oncoprotein which is retained in the endoplasmic reticulum membrane, but is still active as a transforming agent when over-expressed [72]. Cytosolically localized v-erbB mutants are capable of inducing a transforming phenotype, although with reduced efficiency [9,86]. Plasma-membrane localization apparently serves a concentrating function for the transmembrane tyrosine kinase oncoproteins, but can be bypassed by overexpression of mislocalized mutants. The requirement for membrane targetting of the extrinsic tyrosine kinase oncoproteins is generally more stringent, and has been extensively studied for the pp60 ..... oncoprotein. Deletion of the site of N-terminal myristylation [73] or of key N-terminal residues within the v-src sequence results in production of an oncoprotein which is cytosolic and inactive as a transforming agent, even when overexpressed [39,40,56]. Likewise, myristylation and membrane association are a prerequisite for transformation of chicken embryo fibroblasts by activated pp60 c.... [125], and for mitotic activation of pp60 c-Src[6]. Mutation of the myristylation site of activated p56 j¢k prevents membrane binding and transformation of rodent fibroblasts, and decreases lck tyrosine protein kinase activity [2]. Likewise, myristylation and membrane association are required for v-abl transformation of NIH 3T3 fibroblasts [41]. However, membrane binding is not always tightly coupled to other functions of oncoproteins. For example, soluble non-myristylated pp60 v-Src mutants exhibit reduced, but detectable mitogenic activity when expressed in embryonic chicken ceils [24]. Moreover, in some cases membrane binding can be dissociated from myristylation. Non-myristylated pp60 ¢Sr¢ still retains the ability to interact with polyoma virus middle T antigen [183], and complex formation results in membrane association and c-src activation. Similarly, interaction of non-myristylated p56 ]ck with the cytoplasmic tail of membrane-bound CD4 is impaired, but not

315 totally eliminated [140]. Thus, the efficiency of protein-protein interactions is apparently enhanced when tyrosine kinase oncoproteins are myristylated. One might therefore predict that expression of non-myristylated oncoproteins could result in cellular transformation under the appropriate set of circumstances. Indeed, non-myristylated v-abl proteins are still capable of transforming hematopoietic cells [41], and weak transformation of rodent fibroblasts has been reported with non-myristylated, activated c-src [6]. There are now several known families of tyrosine kinases encoding cytoplasmically localized proteins which lack myristylation, SH4 or other membrane binding motifs [13,163,173]. V. Interaction of src family members with membranebound signalling molecules

Although much attention has been focused on transmembrane tyrosine kinase receptors, it is important to emphasize that not all plasma-membrane bound receptors contain an intrinsic tyrosine kinase domain. However, in many instances, stimulation of the latter type of receptors has been shown to result in increased tyrosine phosphorylation of cellular proteins. It is therefore likely that coupling to intracellular kinases occurs, and indeed there is growing evidence in the literature [13] for direct physical interactions between transmembrane receptors and extrinsic tyrosine protein kinases (Table II; Fig. 4). The molecular bases for these interactions are considered below. V-A.1. Interaction of p561ck with CD4 and CD8 transmembrane proteins One of the best studied examples of a stable interaction between a transmembrane protein and a tyrosine protein kinase is the association of p56 ~ck with the T-cell proteins CD4 and CD8. The lymphoma cell kinase p56 Ick was originally identified in LSTRA, a lymphoma cell line derived from mice infected with Moloney murine leukemia virus [174]. Normal tissue

T A B L E II

Other membrane proteins Oncogene

Membrane protein

Cell/tissue

Reference

lck lck lck, fyn

CD4/CD8 IL-2 receptor CD48, CD55, CD59 Thy- 1 T-cell receptor Fc receptor CD36 Membrane Ig CD2 PDGF-receptor Polyoma middle-T B cell receptor IgE receptor EBV LMP2A

T cells T cells T cells

[138,164,172] [65] [152]

T B cells Platelets [71] B cells T cells fibroblasts fibroblasts B cells B cells B cells

[57,132] [157]

fyn fyn fyn, lyn, yes fyn, lyn, lck fyn, lck src, fyn, yes c-src lyn, blk, fyn lyn (src) lyn, fyn

[25] [11] [84] [183] [90,183] [49] [20]

expression of p56 lCk is primarily restricted to cells of the lymphoid lineage, where the protein is found complexed to the CD4 and CD8 transmembrane antigens [139,172]. CD4 and CD8 glycoproteins are classified as 'co-receptors' for the TCR, because they also interact with the MHC, a TCR ligand [44]. In T ceils, nearly 50% of the total p56 tck is associated with CD4 [172], based on co-immunoprecipitation experiments. Moreover, the association between lck and CD4 can be recapitulated in non-lymphoid tissue, based on the ability to detect complex formation by co-expression of p56 lck and CD4 in 3T3 fibroblasts [148]. The molecular basis for the interaction between these two proteins is now partly understood. The cytoplasmic domains of CD4 and CD8a contain two cysteine residues which interact with cysteine residues in the N-terminal domain of p56 lck (Fig. 4) [140,164]. Two pairs of cysteines are present in the lck N-terminus, at positions 3 and 5, and 20 and 23; mutation of either pair disrupts interaction with CD4. The interaction does not seem to require disulfide bond formation, but may involve coordination with a metal ion [164]. Moreover, complex formation is apparently tight and effi-

GPI-linked protein

CD4

Alntervening ~ ' ~ olecule

outside inside

p561ck ptyr : S H I . ~ Fig. 4. Interaction of tyrosine kinase oncoproteins with other membrane proteins. Association with membrane-bound proteins can occur via SH2-phosphotyrosine interactions, via cysteine motifs (e.g., p561ck), or through contact with an intervening molecule.

316 cient, as it withstands immunoprecipitation and can reproduced by mixing lysates in vitro. Plasma-membrane association of p56 ~ck does not appear to be an absolute prerequisite for interaction with CD4. Association of p56 lCk and CD4 can occur before either protein is transported to the plasma membrane. Complexes can be detected with newly synthesized protein, and may therefore form at the cytoplasmic face of the endoplasmic reticulum [139]. Moreover, non-myristylated p56 Ick, which is not membrane-bound when expressed by itself, is still found in a complex when co-expressed with CD4. However, non-myristylated lck is co-immunoprecipitated with CD4 with reduced efficiency, compared to wild-type lck [140]. Several lines of evidence support a functional importance for l c k / C D 4 complex formation in normal and oncogenic cell processes. Cross-linking of CD4 leads to stimulation of the tyrosine kinase activity of CD4-bound p56 lck, and tyrosine phosphorylation of the zeta chain of the T-cell receptor [171]. Studies with mutant T-cell lines indicate that association between CD4 and p56 lck is needed for T-cell receptor mediated signal transduction [59,155]. In fact, lck may regulate the amount of CD4 present at the cell surface, as p56 Ick has been shown to inhibit CD4 endocytosis by preventing CD4 entry into coated pits [110]. Overexpression of the activated form of p56 l~k induces thymic tumors [1]. Interestingly, lck 'knockout' mice exhibit defects in thymocyte development, but can still signal through the TCR [100]. Under these circumstances, it is possible that another src family member kinase can substitute for Ick. Taken together, the currently available genetic evidence implicates p56 jck as an important element for regulation of signal transduction in T ceils. V-A.2. Other membrane interactions of lck It is important to consider the evidence that p56 lck can function through other lymphoid molecules, independently of CD4. For example, binding of interleukin2 results in rapid tyrosine phosphorylation of the IL-2/3 receptor as well as cellular proteins. The IL-2/3 receptor is a transmembrane protein with no intrinsic tyrosine protein kinase domain. Association between p56 j~k and the interleukin-2 receptor has been documented in lymphoid cells, by immunoprecipitation and immunoblotting experiments [65,70], and the region of interaction maps between the cytoplasmic region of the IL-2 receptor and the amino-terminal portion of the lck tyrosine kinase domain [160]. Interactions of lck with other membrane proteins have been reported (Table II), suggesting that p56 lck may be involved in multiple signalling pathways. The biological action of p56 ~ck is not restricted to lymphoid cells alone. Overexpression of activated (F505) p56 jck induces transformation of 3T3 cell fibrob-

lasts [4,97], and high levels of lck m R N A and p56 jck protein have been observed in metastatic colon carcinoma cell lines [170]. The downstream targets of p561ck's tyrosine kinase activity in non-lymphoid cells are not yet known. V-B. The T-cell antigen receptor interacts with tyrosine kinases An early biochemical response to activation of the T-cell receptor with antigen is an increase in tyrosine phosphorylation of cellular proteins. The T-cell receptor is composed of seven different proteins, but none of the protein subunits contain an intrinsic tyrosine kinase domain [133,176]. The search for interacting tyrosine kinases was rewarded when a small amount of the src family member p59 fyn was found to co-immunoprecipitate with the TCR [132]. The stoichiometry of the in vitro complexes was, however, very low and was sensitive to the type of detergent employed for immunoprecipitation. More recently, Gassman et al. have exploited immunofluorescent microscopy to demonstrate that antibody-induced capping results in efficient co-localization of p59 fyn with cell-surface labelled TCR [57]. The molecular basis for the interaction between fyn and the TCR has recently been elucidated. The CD3 complex of the TCR (which includes the gamma, delta, epsilon, zeta, and eta chains) contains a sequence motif which consists of two tyrosine residues, spaced approximately 10 amino acids apart, and conserved leucine/ isoleucine residues [124]. Signalling information is contained within this motif [128] which requires the presence and correct spacing of the tyrosine and leucine residues [89]. The recognition domain within p59 fyn has been mapped to the first 10 amino acids [58]. However, the exact contact regions between the TCR and p59 fyn have not yet been identified, and could involve phosphorylated tyrosines in CD3 subunits, a n d / o r myristylation or potential palmitylation of p59 fyn (see Section II-C.2). It is clear, however, that T C R / f y n interaction results in important functional consequences. Stimulation of the TCR by antibody crosslinking leads to activation of p59 fyn tyrosine kinase activity [162]. Overexpression of wild-type fyn in transgenic mice leads to enhanced responsiveness to TCR stimulation, whereas a catalytically-inactive fyn functions as a dominant negative mutant, impairing normal signal transduction [33]. Although T-cell development in fyn knockout mice is apparently normal, the mature thymocytes do not respond properly to TCR stimulation [5,153]. Finally, phosphorylation of the zeta chain of the TCR by p59 fyn creates a binding site for another protein, ZAP-70, an SH2-containing tyrosine kinase which associates only with activated TCR [30]. The net result is the forma-

317 tion of a multi-protein complex at the membrane, which allows signal transduction to be propagated from the extracellular milieu to the cell interior.

V-C. Supermolecular complexes form with GPI-linked proteins at the cell surface The discussion so far has been limited to interaction of tyrosine kinases with cytoplasmic domains of transmembrane proteins. However, it is important to consider another class of membrane-bound signalling proteins which are linked to the extracellular surface of the plasma membrane by a glycosylphosphatidyl inositol (GPI) anchor [94]. GPI-linked molecules, such as CD59, CD55 (decay accelerating factor), and CD48, contain neither transmembrane nor intracellular domains, yet are capable of causing leukocyte activation. A key to understanding how signals are transduced by these molecules was provided by the observation that cellular tyrosine kinases are co-immunoprecipitated with these lymphoid GPI-linked proteins [152]. At least two of the kinases have been identified as p56 lck, and p59 fun [45,152]. The association of GPI-linked proteins with intracellular src family kinases poses a biophysical dilemma, as the GPI-linked proteins are located on the outer leaflet of the plasma-membrane bilayer and the src kinases are found on the inner leaflet (Fig. 4). Thus, direct physical contact is not possible without an intervening molecule. It is therefore of interest to note that CD59, CD55, and CD48 are found in large membrane complexes containing glycolipids and tyrosine kinases

[32]. Evidence has been mounting that GPI-linked proteins are preferentially sorted to glycosphingolipidenriched patches in the membrane [18,147]. Cell surface macromolecular subdomains may provide the necessary intervening molecules to form a physical link between the outside and inside of the cell. VI. Signal transduction: from plasma membrane to the nucleus

Activation of tyrosine kinase oncoproteins and growth factor receptors at the plasma membrane is the initial step in signal transmission to the cell interior. Subsequent steps must occur which link the signal(s) generated at the membrane to proteins in the cytoplasm and ultimately to the nucleus. Several different mechanisms that enable membrane-bound proteins to transmit intracellular signals are depicted in Fig. 5. Examples of each type of interaction are now known. The dramatic progress that has been made within the past several years towards elucidating the identities of the linking signalling molecules is summarized below.

VI-A.1. Intermolecular communication at the plasma membrane: link to p21ras In 1986, Smith et al. reported that transformation by v-src could be prevented by microinjection of a monoclonal anti-ras antibody that neutralized c-ras protein [151]. A potential link was therefore established between these two membrane-bound signal transducing molecules. Subsequent genetic and biochemical studies have confirmed that ras functions downstream of

II

Lateral Diffusion in plane of membrane Components remain membrane associated

"X"

III

"X"

IV

"X" Recruitment to the membrane via protein:protein interaction

Shuttle from membrane to cytoplasm

Fig. 5. Mechanisms of signal transduction at the membrane. Signals ( X ) can be generated from membrane-bound complexes in a variety of ways. In Scheme I, membrane-bound components become associated in complexes, and generate a signal. In Scheme II, one of the membrane-bound components undergoes lateral diffusion in the plane of the lipid bilayer, thereby contacting a second molecule, resulting in signalling. Alternatively, recruitment of cytosolic signalling proteins to the membrane can occur via protein-protein interaction (Scheme III). One can also envisage shuttling of components from the membrane to the cytosol, resulting in signal transduction (Scheme IV).

318 growth factor receptor and src family tyrosine protein kinases [23,83]. The next connection came with the identification of conserved SH2 and SH3 domains, first noted in src and fps [131], which have now been found in several dozen normal and oncogenic proteins [81]. The function of the SH2 domain is to mediate protein-protein interactions by binding to phosphotyrosine residues contained within a defined sequence context [81]. SH3 domains apparently recognize specific proline-rich sequences in target proteins [119]. Many proteins contain both SH2 and SH3 domains, and several have been found associated with specific phosphotyrosine containing regions of activated growth factor receptors. Using the tyrosine phosphorylated tail of the E G F receptor as a probe, Schlessinger and colleagues identified one such protein, GRB2 [95], which was proposed to serve as a bridge or 'adaptor' between tyrosine kinases and intracellular signal transducers. A recent flux of papers [19,48,98,146] has identified the missing link in the pathway between E G F receptor, GRB2 and ras as sosl ('son of sevenless'), a guanine nucleotide releasing protein first identified in Drosophila. The SH3 domain of GRB2 recognizes and binds to a proline-rich region in sosl. Sosl then enhances nucleotide exchange on p21 ras, converting ras from the inactive GDP-bound form to the activated, GTP-bound form. Since ras itself is membrane bound, a macromolecular signal transduction complex is established at the inner surface of the plasma membrane (Fig. 6).

ligand

A similar cascade is utilized in v-src-transformed cells, where the adaptor protein SHC becomes phosphorylated on tyrosine and then binds to GRB2 [48]. The G R B 2 / s o s l / r a s pathway has been remarkably conserved in evolution, with analogous genes functioning in signal transduction in Drosophila melanogaster and Caenorhabditis elegans. The net effect of complex formation is to increase the effective local concentration of sosl at the membrane interface, thereby increasing accessibility to its membrane-bound substrate, p21 ras. The lipid bilayer thereby functions as a 'scaffold' or organizing center, providing a concentrating effect for efficient protein-protein interactions.

VI-A.2. Other signal transducers associate with the PDGF receptor The signals generated by activation of tyrosine kinase oncoproteins and growth factor receptors are pleiotropic, and in some cases can be ras-independent. Study of the P D G F receptor has revealed that multiple tyrosine residues in the cytoplasmic domain are phosphorylated following ligand binding [26,168]. These residues function as magnets for a variety of SH2 containing proteins which link the P D G F receptor to intracellular signalling pathways. At least two enzymes involved in phosphoinositide metabolism are associated with activated P D G F receptor. Phospholipase Cgamma is an S H 2 / S H 3 containing protein which binds to tyrosine 1021 in the C-terminal tail of the P D G F receptor [126,167]. As a result of cleavage of the PLC

i

extracellular intracellular raf

I

'Y-

Receptor

sos

p21 ras

GRB2

MEK/MAPKK

~

MAPK

Gene activation

Fig. 6. Signal transduction from membrane to nucleus. A schematic representation of the protein-protein contacts that are formed during receptor-mediated signal transduction (see text). Activation of a membrane-bound receptor with ligand stimulates tyrosine phosphorylationof the receptor, which recruits a complexof GRB2-sos from the cytosolto the membrane. The increase in local concentration of sos in the proximity of membrane-bound p21ras allows activationof ras, and interaction with raf. Signals are then transduced into the cytoplasm,via MEK and MAP kinase, and ultimatelyinto the nucleus.

319 substrate, PI(4,5)-bisphosphate, two second messengers are formed: diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which promotes the release of intracellular Ca z+ stores. Phosphotyrosines in the kinase insert domain of PDGF receptor promote binding of another PI metabolizing enzyme, PI3 kinase, whose substrates are phosphorylated at the 3 position of the inositol ring [26]. Although the exact function of the products of the PI3 kinase reaction is not known, recent evidence has linked PI3 kinase to regulation of intracellular protein trafficking in yeast [135]. The binding sites for at least three additional types of SH2 containing proteins have been mapped within the C-terminal region of the PDGF receptor [51]. The ras-specific GTPase activating protein, rasGAP binds to phosphotyrosine 771 on the receptor. It is tempting to speculate that this association may serve to sequester rasGAP away from p21ras, thereby preventing ras deactivation. On the other hand, rasGAP is itself phosphorylated in response to PDGF as well as transformation by tyrosine kinase oncoproteins, whereupon two additional proteins, p190 and p62 become tightly bound [50]. p190 encodes GAP activity specific for the rho family of small G proteins [138], which have been shown to regulate cytoskeletal structure [127]. Tyrosine 1009 serves as a binding site for a 64K protein termed syp or SH-PTP2, a tyrosine protein phosphatase [167]. Finally, three members of the src family of tyrosine kinases, src, fyn and yes, have been shown to bind to receptor tyrosine 857 [35,84] via their SH2 domains [165]. The end result is the formation of macromolecular complexes at the intracellular membrane surface.

VI-B. Signalling from membrane to nucleus Many of the signal transducers mentioned above are cytosolic proteins which are recruited to the membrane surface by interaction with activated tyrosine kinase receptors or oncoproteins. In addition, the responses to cellular transformation a n d / o r receptor activation involve increases not only in tyrosine, but also in serine and threonine phosphorylation. The next series of steps in the cascade must therefore involve propagation of the signals to serine/threonine kinases in the cytoplasm and ultimately into the nucleus. A number of potential candidates to perform this function have now been identified. The most proximal protein to p21ras in the signalling cascade seems to be pp74 craf~, a cytosolic serine/threonine protein kinase. Activation of membrane-bound tyrosine kinases results in increased phosphorylation and stimulation of raf-1 kinase activity [102]. Raf-1 activation is prevented by dominant nega-

tive mutants of ras [182], and conversely, dominant negative raf-1 mutants inhibit v-ras transformation [82]. In addition, a physical complex between raf-1 and GTP-ras can be isolated [101]. The kinase cascade is propagated when Raf-1 activates MEK ( M A P / E R K kinase, or MAP kinase kinase), an upstream activator of mitogen-activated protein (MAP) kinase [38,43]. One of MAP kinases substrates is another kinase, pp90rsk, first identified by its ability to phosphorylate ribosomal protein $6. Both MAP kinase and pp90rsk are translocated to the nucleus [31], where their targets include the transcription factors encoded by the myc, fos and jun proto-oncogenes [3,31]. Thus, at least one complete signalling pathway has now been defined (Fig. 6): EGF-R

~ G R B 2 ~ S O S 1 ~ p 2 1 r a s ~ raf-1 ~

MEK

MAP-K'~ pp90rsk~ ~ Nucleus

VI-C. Unanswered questions A number of questions still remain to be addressed. For example, how many signalling molecules are bound to an activated receptor at a given time? Even though SH2 containing proteins bind to distinct subsets of phosphotyrosine sites, does occupancy of the GRB2 site on a receptor preclude binding of GAP, by steric hinderance? Is complex formation timed or sequential, thereby allowing signalling waves to be propagated through the cell interior? Are all of the intervening signalling components identified? Are some of the complexes preformed, eg GRB2-sosl? Many of the described interactions involve two tyrosine kinases. Why would a receptor tyrosine kinase (e.g., PDGF-R) need to use another tyrosine kinase (e.g., src) to propagate a signal? One must presume that the substrate specificities of the two kinases are significantly different. Alternatively, src kinases have the ability to interact with several different membrane proteins, thereby propagating signals through lateral diffusion in the membrane (Fig. 5). Finally, how is the signal transduction cascade turned off? In the case of growth factor receptors, the 'off' switch can be provided by either ligand dissociation, dissociation of receptor dimers, or desensitization. In addition, physical removal from the membrane can occur through receptor down-regulation, i.e. via endocytosis and degradation. Other kinases may be deactivated by re-phosphorylation at the regulatory tyrosine site, e.g., by CSK. It is clear that in the absence of appropriate regulation, constitutive expression of tyrosine kinases leads to malignant transformation.

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