Novel functions of phosphatidylinositol 3-kinase in terminally differentiated cells

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Cellular Signalllag Vol. 7, No. 6, pp. 545-557, 1995. Copyright © 1995 Elsevier Science Ltd Pdated in Great Britain. All rights reserved 0898-6568/95 $9.50 + 0.00

1Pergamon

0898-6568(95)00033-X

MINI REVIEW NOVEL FUNCTIONS

OF PHOSPHATIDYLINOSITOL

TERMINALLY

DIFFERENTIATED

3-KINASE IN

CELLS

S. NAKANISHI, H. YANO and Y. MATSUDA* Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. 3-6-6 Asahimachi, Machida-shi, Tokyo 194, Japan (Received 6 March 1995; and accepted 1 April 1995) A b s t r a c t - Importance of phosphatidylinositol 3-kinase (PI 3-kinase) in signalling pathways leading to growth

stimulation has already been reviewed in this journal and others. Evidence has now been accumulating that PI 3-kinase is involved in transmission of activation signals in terminally differentiated cells, especially signals starting from receptors which have no intrinsic tyrosine kinase domain. The pioneer works showed the presence of PI 3-kinase activity and the accumulation of the reaction products of PI 3-kinase correlated with the cell responses. However, these studies were done in only limited cell responses such as respiratory burst in neutrophils and degranulation in platelets. Recent finding of a potent and selective inhibitor of PI 3-kinase, wortmannin, reported from three independent groups including us, gave a new and powerful tool not only to confirm the suggested functions but also to reveal new functions of PI 3-kinase such as histamine release from antigenstimulated mast cells/basophils and glucose uptake in insulin-stimulated adipocytes. Nearly one hundred papers which describe the action of wortmannin on various cells have been reported during one year after the publication of the discovery of wortmannin as PI 3-kinase inhibitor, suggesting possible involvement of the enzyme in the diverse cell responses besides cell proliferation. Key words: Phosphatidylinositol 3-kinase, Wortmannin, LY294002, Inhibitor, Neutrophil, Basophil, Mast

cell, Platelet, Adipocyte.

INTRODUCTION

(PI4P), and PI-4,5-diphosphate (PI4,5P2) to form PI-3-monophosphate (PI3P), PI-3,4-diphosphate (PI3,4P2) and PI-3,4,5-triphosphate (PI3,4,5P3), respectively [I ]. Purification and cloning studies [2-9] have revealed that the enzyme is a heterodimer consisting o f 85,000M, regulatory subunit (p85) and 110,000 Mr catalytic subunit (p110). The regulatory subunit contains two src homology 2 (SH2) domains and a single src homology 3 (SH3) domain. SH2 domain [10] is known to bind to phosphotyrosine residues within a certain consensus sequence in growth factor receptors such as platelet-derived growth factor receptor, oncogene products such as src, and tyrosine kinase substrates such as insulin receptor substrate 1

Phosphatidylinositol 3-kinase (PI 3-kinase) catalyzes the stereoselective transfer of a phosphate group from A T P to Y-position o f inositol in phosphatidylinositol (PI), PI-4-monophosphate

*Authorto whomall correspondenceshouldbeaddressed. Abbreviations: PI 3-kinase--phosphatidylinositol 3-ki-

nase; PI-phosphatidylinositol;PI4P-PI-4-monophosphate; PI4,SP2--PI-4,5-diphosphate; PI3P - PI-3-monophosphate; PI3,4P2--PI-3,4-diphosphate; PI3,4,5P3-- PI-3,4,5-triphosphate; PIP3- PI-triphosphate; SH2--src homology2; SH3src homology3, IRS-1--insulin receptorsubstrate 1; FceRIhigh affinityreceptor for IgE; GLUT4-- glucosetransporter type 4.

545

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0

( (

HaCOH2C

o2

0

H Wortmannin

LY294002

Fig. 1. Chemical structure of wortmannin and LY294002. (IRS-1). The association of PI 3-kinase to tyrosine-phosphorylated proteins via SH2 domains of p85 greatly enhances the catalytic activity of p 110 as well as translocating the enzyme close to the membrane where its substrates are present. It is widely accepted that PI 3-kinase is involved in the growth signals elicited by growth factors and activated oncogene products. Knowledge is also accumulated in the field of studies on cell responses expressed in terminally differentiated cells. Wortmannin (Fig. 1) which has recently been found to be a powerful and selective inhibitor of PI 3-kinase [11-13], boosted the studies of PI 3-kinase in this field (Table 1), especially on cell responses transduced through receptors having no intrinsic tyrosine kinase activity. In this review, we will describe the biological and chemical characteristics of wortmannin, summarize the new functions of PI 3-kinase revealed with wortmannin, and discuss the usefulness and limitation of application of wortmannin. The enzymology, mechanism of regulation, and function in cell growth of PI 3-kinase are summarized in detail in recent reviews [14-17]. PI 3-KINASE INHIBITORS History o f wortmannin studies

In 1957 wortmannin was first reported by Brian et al. as antifungal antibiotic isolated from

a culture of a fungus, Penicillium [18]. Sandoz's group in Switzerland has found an anti-inflammatory agent with a weak antifungal activity from a culture of the same family of fungus and identified it as 11-desacetoxy-wortmannin [19, 20]. They also showed that some of its analogues, including wortmannin itself, had antiinflammatory activities in experimental animal models [20]. Pharmacological effects including toxicity of wortmannin on animals are summarized in Table 2. One of the most exciting findings was reported by Baggiolini et al. in 1987 that wortmannin and some of its analogues blocked the induction of respiratory burst in neutrophils, monocytes and macrophages with tremendously low concentrations (10-9-10 -a M) of the compounds [21]. They suggested that the compounds interfered with the signal transduction pathway starting from the particulate stimulus leading to the respiratory burst oxidase, since 17-hydroxywortmannin, one of the most potent analogues, did not inhibit phagocytosis induced by the same stimulus, had no effect on NADPH oxidase itself, and was not cytotoxic at higher concentrations. However, the direct target site of wortmannin had not been determined at that time in spite of intensive works with neutrophils [22]. We have been searching for inhibitors of myosin light chain kinase, which is known to be a trigger enzyme of smooth muscle contraction,

P I 3 - K i n a s e in t e r m i n a l l y d i f f e r e n t i a t e d cells

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T a b l e 1. C e l l u l a r r e s p o n s e s a f f e c t e d b y w o r t m a n n i n Cell type

Species

Response

Stimulant

fMLP human respiratory burst fMLP human respiratory burst human respiratory burst opsonized zymosan TNF-ct human respiratory burst human respiratory burst C D 1 8 / C D I la-mediated human respiratory burst crystal guinea pig respiratory burst fMLP guinea pig respiratory burst fMLP fMLP human PIP~ production fMLP guinea pig PIP3 production fMLP human phospholipase D activation fMLP human phospholipase D activation human tyr osine-phosphorylation fMLP guinea pig protein kinase activation fMLP guinea pig M A P kinase activation platelet-activating factor guinea pig phagocytosis FetR-mediated platelet human serotonin release thromboxane A2 human serotonin release thromhin human serotonin release collagen human aggregation phorbol ester human aggregation ADP human Ca signalling thrombin human Ca signalling thapsigargin adrenal glomerulosa cell bovine Ca signalling angiotensin It bovine inositol trisphosphate production angiotensin It aorta rat contraction KCI rat myosin light chain phosphorylation KCI rabbit contraction phenylephrine saphenous vein rabbit contraction UK 14304 (a2 agonist) pheochromocytoma PC12 rat neurite outgrowth NFG rat phospholipase D enhancement calbachol adrenal chromaffin cell bovine catecholamine release KCl superior cervical ganglion rat synaptic transmission spontaneous monocyte human respiratory burst zymosan monocyte U937 human phagocytosis Fe'tR-mediated macrophage human respiratory burst zymosan mouse respiratory burst zymosan mouse respiratory burst zymosan mouse antigen presentation M H C class II-restricted natural killer cell human degranulation FcyR-mediated human antibody-dependent cell toxicity FcyR-mediated basophil human histamine release Fc~RI-mediated human histamine release FceRI-mediated basophilic leukemia RBL-2H3 rat histamine release Fc~RI-mediated rat histamine release Fc~RI-mediated rat leukotriene release Fc~RI-mediated rat serotonin release Fc~RI-mediated adipocyte rat 2-deoxyglucose uptake insulin rat antilipolysis insulin rat antilipolysis insulin rat lipogenesis and antilipolysis growth hormone rat phosphodiesterase activation insulin adipocyte 3T3-L 1 rat 2-deoxyglucose uptake insulin rat GLUT1,4 translocation insulin brown adipocyte rat 2-deoxyglucose uptake insulin rat G L U T 4 translocation insulin oocyte Xenopus 2-deoxyglucose uptake IGF-1 C H O / I R / G L U T 4 *l Chinese hamster G L U T 4 translocation insulin C H O / I R .2 Chinese hamster GSK-3 *~ inactivation insulin Chinese hamster GSK-3 .5 inactivation serum skeletal muscle L6 rat GSK-3 .5 inactivation IGF- I gastric mucosal cell rabbit wound repair cell denudation T-lymphoblastoid H 9 human budding of HIV *~ spontaneous T-lymphoblastoid Jurkat human AP-I activation anti CD3 antibody neutrophil

ICs0 (riM) 7 12 13-28 500 *7 500 *7 100*7 50 46 5 50 50 *7 110 50 *7 10-100 200-300 30 1000*7 10-100 ca. 100 ca. 10 6000*7 6000*7 6000*7 3000 2000 300-1000 ca. 300 1000 100 100*7 1500*8 1000*7 1200*7 3 10-I00 10 7 0.8-1.5 10000-1000000 ca. 2 ca. 3 3-30 30 20 2 3 1000*7 30-100 1000*7 100

ca. 20 10-30 6.4 1000*7 100*7 100*7 ca. 20 10-50 100*7 100.7 100*7 1000-10000 300 100*7

Reference 100 13 21 101 101 102 12 103 13 12 104 100 105 103 106 107 29 29 29 29 98 26 118 99 99 23 23 119 119 120 121 24 25 21 107 21 21 21 108 109 109 68 28 28 11 11 110 30 30 86 111 86 82 82 81 81 80 83 112 112 113 122 27 123

(Table continued)

S. N a k a n i s h i et al.

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T a b l e 1. c o n t i n u e d Cell type hepatoblastoma HepG2 epidermoid carcinoma KB

foreskin fibroblast AGI518 foreskin fibroblast AG1523 endothelial cell endothelial cell/kit .3 endothelial ceII/PDGFR .4 megakaryoblast CHRF-288

Species human human human human human human human porcine porcine porcine human

Response $6 kinase activation $6 kinase activation membrane ruffling membrane ruffling PIP3 production membrane ruffling actin rearrangement membrane ruffling proliferation proliferation proliferation

Stimulant PDGF insulin insulin IGF- 1 PDGF PDGF PDGF PDGF SCF PDGF serum

IC~o (nM) ca. 10 ca. 10 ca. 10 ca. 10 100*7 I0 50-100 *7 25 ca. 20 50-100 15-20

Reference 114 114 115 115 116 116 117 116 124 124 125

*tinsulin receptor and GLUT4-expressing; *2insulin receptor-expressing; *3kit-expressing; *4pDGF receptor-expressing; *~glycogen synthase kinase-3; *~h u m a n immunodeficiency virus; ,7 concentration used for the study; *~ECs0 for stimulation.

in order to obtain new leads of pharmaceuticals like vasodilator or bronchodilator. During the course o f this study, we found wortmannin in the culture broth o f a fungus, Talaromyces wortmannii, as new myosin light chain kinase inhibitor in 1992 [23]. Wortmannin, indeed, suppressed phosphorylation of myosin light chain and the contraction o f isolated aortic rings without affecting several other protein kinases in purified enzyme systems. Based on these effective and selective features of wortmannin, Nonomura's group together with us have used this inhibitor to reveal new functions of myosin light chain kinase in non-muscle cells, especially in exocytotic processes such as catecholamine release from adrenal chromaffin cells [24, 25], platelet activation [26], and budding of viral particles from host cells [27]. We noticed that although micromolar concentrations of wortmannin were needed to inhibit such exocytotic processes and myosin light chain kinase, it inhibited antigen-stimulated histamine release from RBL-2H3 cells [28], human basophils [28] and rat peritoneal mast cells [unpublished observation] with nano-molar concentrations as it did respiratory burst o f neutrophils, and that this might suggest another target molecule of wortmannin in basophils, mast cells and neutrophils. Ui's group has also found the effect of wortmannin on platelet activation [29] and metabolic action of insulin, and has been working on identification of its real target in platelets, adipocytes

and neutrophils [11, 30]. Their important discovery was presented at the annual meeting of Japan Biochemistry Society in September 1992; wortmannin at nano-molar concentrations inhibited the PI 3-kinase activity. We have confirmed the inhibition of PI 3-kinase by wortmannin, and found that the kinase is activated in antigen-stimulated RBL-2H3 cells and that wortmannin inhibits PI 3-kinase in the cells [11]. Arcaro and Wymann have been working on the activation mechanism of neutrophils, and reached the same conclusion about the effect of wortmannin [13]. Publications from three independent groups finally appeared within three months [11-13, 30].

Inhibitory mechanism o f wortmannin and other PI 3-kinase inhibitors Wortmannin at low nano-molar concentrations inhibits the activity of PI 3-kinase purified from bovine thymus, guinea pig and human neutrophils, rat adipocytes, and various cell lines (Table 3). The inhibition is irreversible and, therefore, is time- and temperature-dependent [11, 23]. The furane-ring of wortmannin is chemically active and can be attacked nucleophilically by amino and thiol groups of proteins to form a covalent bond (Fig. 2). Tight binding of wortmannin with the p110 subunit o f PI 3-kinase was demonstrated by immunological and radiometric detection of wortmannin at 110,000 Mr

PI 3-Kinase in terminally differentiated cells

549

Table 2. Pharmacological effects of wortmannin on animals Activity

Animal

acute toxicity hemorrhage of myocardium and gastrointestinal tract anti-inflammation immunosuppression

Effective dose

rat

3-10 mg/kg

rat rat rat, mouse

4 mg/kg 0.1 mg/kg 3.1-4.2 ppm in diet

Reference 20 126 20 127

Table 3. Effects of wortmannin on PI 3-kinase from various sources Source (Partially) Purified calf thymus guinea pig neutrophil human neutrophil* 1 rat adipocyte rat basophilic leukemia RBL-2H3 cell *n rat adipocyte 3T3-LI cell.1 human epidermoid carcinoma KB cell *n human fihrohlast AG1523 cell .1 human myeloid derived U937 cell.2 human platelet .2 bovine adrenal reticulosa cell .3 plant (Daucus carota) cell Cloned and Expressed yeast Vps34p bovine p110a

ICso (riM)

Reference

3 5 1 ca. 10 3 2.6 ca. 10 1 43 2 100 >200

11 12 13 30 11 82 115 117 33 34 35 38

>400 6.2

32 32

.limmunoprecipitated with anti-p85 antibody; *2activated by [}y-subunit of G protein; *3PI-specific enzyme.

protein band in Western blots [11, 31]. The irreversible inhibition of the enzyme activity is due to covalent binding of wortmannin to the catalytic subunit of PI 3-kinase, since derivatives of

H3COH2 C

o--

wortmannin cleaved at the furane ring are 3000 times less potent than wortmannin [11]. Although two isosubunits (a and [}) of both p85 and p110 have been cloned [5-9], the effect

P

!

2_

R-NIt E

O

--o

O

H+ H

P

NH

\

R Fig. 2. C h e m i c a l reaction o f w o r t m a n n i n and proteins. R-NH2 : protein, H + : acid.

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s. Nakanishiet al.

of wortmannin has been reported only on p 110a [32]. However, it seems unlikely that the wortmarmin sensitivity of p 11013 or any combination of p110a, 13, and p85a, 13is very different, since the activity immunoprecipitated from several cell sources with anti p85 antibody which immunoprecipitates both p85et and p851~ is inhibited by wortmannin in a similar concentration range (Table 3). Recent reports [33-38] have demonstrated the presence of new types of PI 3-kinase activity distinguished from those of p85/p110 complex type. Such activity shows obvious difference in wortmannin-sensitivity(Table 3). The yeast PI 3-kinase, Vps34p [36], and the PI 3-kinase activity [38] detected in plant cells are insensitive to wortmannin as high as 100 nM. The PI-specific activity detected in bovine adrenal reticulosa cells which does not utilize PI4P or PI4,5P2 as substrate and is not immunoprecipitated with anti p85 antibody is less sensitive to wortmannin (IC~0value : 100nM) [35]. The activity detected in human myeloid-derived U937 cells which is not immunoprecipitated with anti p85 antibody and is activated by 13~/subunitsof G-proteins is inhibited by wortmannin with an ICso value of 43 nM, whereas the activity (p85/p110 type) stimulated with phosphotyrosine-containing peptide is with an IC5o value of 17 nM [33]. However, the fly-stimulated activity in human platelets is inhibited with an ICso value of 3 nM [34]. It is necessary for precise comparison of wortmannin sensitivity to measure ICso values under the same conditions, since the effect of wortmannin is considerably affected by concentrations of ATP and proteins, incubation time, sequence of addition, pH and temperature [11, 23]. Judging from the experiments with purified enzymes, mammalian PI 3-kinase is the species which is most sensitive to wortmannin amongst several protein kinases and lipid kinases examined [11]. However, myosin light chain kinase in smooth muscle [23] and a soluble form of PI 4-kinase in bovine adrenal cortex [39] and rat adipocytes [30] are inhibited with ICso values as high a s 1 0 - 7 - 1 0 -6 M wortmannin. It is possible that wortmannin binds to amino groups of these protein kinases and lipid kinases, the activities

of which are not affected by wortmannin. Of the kinases whose activities are unaffected by wortmannin, it is possible that the rate of binding could be negligiblyslow or, in those instances where that rate is fast, such binding o f wortmannin is unable to affect the enzyme activities. Very recently, another selective inhibitor of PI 3-kinase, LY294002 (Fig. 1) has been reported [40]. LY294002 is a derivative of quercetin and is improved in potency (ICso : 1.4 ~tM)and enzyme selectivity. The inhibitory mechanism of LY294002 is of competitive mode with respect to ATP, and therefore, the inhibition is reversible. There are some other reports which show that a cyclic peptide [41] and an ether lipid [42] inhibit PI 3-kinase with ICso values of 1.0 ~tM and 35 lxM, respectively, and that lovastatin, a cholesterollowering agent, suppresses the PI 3-kinase activity in cells but not of the isolated enzyme [43]. NEW FUNCTIONS OF PI 3-KINASE

The respiratory burst o f neutrophils induced by chemotactic agents The essential function of neutrophils is the defence of the organism against invading microbes. One of the major responses to kill invaders is the respiratory burst, a sudden increase in oxygen consumption to produce microbicidal oxidants. The products of the burst are also a cause of tissue damage and inflammation. The activation of the respiratory burst has been studied most intensively in neutrophils stimulated with chemotactic agents, such as N-formylated peptides (e.g., fMLP), a complement fragment C5a, platelet-activating factor and leukotriene B4 [for detailed review, see ref. 44]. The receptors for such chemotactic agents are members of G-proteincoupled types [45-49]. Extensive data indicate that two well-characterized second messengers, inositol-l,4,5-trisphosphate and diacylglycerol are involved in signalling starting from chemotactic agonist-stimulation [44]. These messenger molecules increase cytoplasmic Ca 2÷ concentration and activate protein kinase C. It has been suggested that there a r e C a 2+dependent and -independent processes in signal

PI 3-Kinasein terminallydifferentiatedcells transduction sequences activated by receptor agonists [50, 51]. Receptor agonists cannot induce the respiratory burst in Ca2+-depleted cells, but the preceding activation of protein kinase C by a phorbol ester permits the receptor-mediated oxidant production in Ca~+-depleted cells [51]. Excellent work done by Dewald et al. [22] has clearly distinguished the two processes in terms of sensitivity to 17-hydroxy-wortmannin; the Ca2+-dependent process is insensitive to the inhibitor, whereas the Ca2+-independent process is blocked by the inhibitor. Possible involvement of PI 3-kinase in the respiratory burst has been suggested by TraynorKaplan et al. [52, 53]. They showed that PI3,4P2 and phosphatidylinositol triphosphate (PIP3) were produced in human neutrophils and increased upon stimulation. Good correlation is observed in the time course and intensity of the lipid generation and oxidant production when cells are stimulated with different agents and varied concentrations of a chemotactic peptide. They also showed that the lipid generation was stimulated even in Ca2+-depleted cells, suggesting that this process is Ca2+-independent. The reports connecting these findings have been published by two groups [12, 13]. Wortmannin inhibits the activity of PI 3-kinase purified from guinea pig [12] and human [ 13] neutrophils, and suppresses the production of PIP3 induced with a chemotactic peptide. The concentrations of wortmannin needed to inhibit both the PI 3-kinase activity and the respiratory burst are in the same range. These data strongly suggest that PI 3-kinase is involved in the Ca2+-inde pendent and wortmannin-sensitive process. However, it is still unclear how PI 3-kinase is activated in agonist-stimulated neutrophils and which molecules receive and transduce the signal of activated PI 3-kinase. There are several reports indicating that a chemotactic peptide induces tyrosine phosphorylation within 1 min, and that tyrosine kinase inhibitors blocked the phosphorylation and the respiratory burst [5458]. It is easily speculated that PI 3-kinase is activated by the unknown tyrosine-kinases via direct phosphorylation of p85 or association with

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the tyrosine-phosphorylatedproteins. Otherwise, a novel PI 3-kinase [33, 34] may be activated by ~y subunits released from the trimeric G proteins upon agonist-stimulation. FceRI-mediated activation o f basophils and mast cells

Mast cells and basophils express the high affinity Fc receptor (Fc~RI) for IgE on their surface membrane. Cross-linking of FceRI with a combination of IgE and the specific antigen activates the cells to induce the release of allergic mediators, such as histamine, leukotrienes, cytokines and proteases. FceRI is a tetrameric complex composed of an IgE-binding a-chain, a [I-chain, and two y-chains. Since 13 and ~ have extensive cytoplasmic domains without any homologous sequences suggesting intrinsic enzyme activities, it has been assumed that some functional proteins associate with 13and/or "/to transduce the signal inside the cells [for recent reviews, see refs. 59-61]. Many investigators have pointed out the functional coupling of various cellular activities with FceRI-induced degranulation or cytokine production; these include Ca 2÷ mobilization, the activation of protein kinases, adenylyl cyclase, and phospholipases A2, C, and D, PI turnover, membrane depolarization and demethylation of phospholipids [for recent review, see ref. 62]. Physical association of functional proteins with FccRI was first reported by Eiseman and Bolen [63] who showed that src-related tyrosine kinases, p56 tynand pp60~srcwere activated in RBL2H3 cells immediately after Fc~RI crosslinking, and that p56 tynwas co-immunoprecipitated with FceRI. They also showed that in a mouse mast cell line PT-18, another src-related tyrosine kinase p62 c-y~was activated by FcsRI engagement and co-immunoprecipitated with FcsRI. Hutchcroft et al. [64] have confirmed the association of p56 ~y~with Fc~RI in RBL-2H3 cells and found another associated tyrosine kinase, PTK72, which appears to be identical or closely-relatedto p72 syk that is involved in signalling of B lymphocytesand platelets [65]. Benhamou et al. [66] have shown

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that tyrosine phosphorylation of 72,000 Mr proteins, including a p72 syk, is rapid after stimulation, and correlates with the time course and antigen dose for histamine release, suggesting that the phosphorylation is an early signal following the receptor engagement. Several observations have indicated that the phosphorylation of 72,000 Mr proteins is a distinct signal independent of Ca 2÷ responses and of protein kinase C or phospholipase C activation [66, 67]. Early studies with wortmannin have described its profound inhibitory effect on FceRI-mediated histamine release in human basophils [68], RBL-2H3 cells [28] and rat peritoneal mast cells (unpublished data). A recent study [11] has shown that PI 3-kinase activity co-immunoprecipitated with anti-phosphotyrosine antibody or anti-p56 zyn antibody increases in RBL-2H3 cells rapidly after antigen stimulation. Wortmannin inhibits the activity of PI 3-kinase both in RBL-2H3 cells in vivo and in cell-free systems with the enzyme purified from RBL-2H3 cells and calf thymus, but does not suppress the tyrosine phosphorylation of p56tynin vivo. The concentrations of wortmannin needed to inhibit PI 3-kinase activity are similar to those required for inhibition of histamine release and leukotriene production. Studies with some wortmannin derivatives indicate the correlation of their inhibitory potencies towards PI 3-kinase activity and histamine release from RBL-2H3 cells. These data strongly suggest that a part, if not all, of PI 3-kinase is associated with a complex of p56 tynand Fc~RI, is activated upon antigen stimulation, and transduces the signal to the final cellular events [11]. However, it remains unclear how activated PI 3-kinase transduces the signals to its downstream events. Ca2+-independent isozymes (/5, e, rl or ~) of protein kinase C could be the candidates that receive the signals from PI 3-kinase, since PI3,4,5P3 stimulates the activity of ~ isozyme purified from bovine kidney [69], and since PI3,4P2 and PI3,4,5P3 stimulate the activity of /5, e, and ~ isozymes expressed in and purified from insect cells [70]. It is noteworthy that RBL2H3 cells contain/5, e, and ~ isozymes of protein kinase C, and that the addition of/5 isozyme

back to the permeabilized and washed RBL-2H3 cells restores the lost secretory response to the level at which intact cells can perform [71]. The fact that PI 3-kinase has serine protein kinase activity [72, 73] may indicate the presence of another signalling pathway distinct from one transduced by PI3,4P2 and/or PI3,4,5P3. Metabolic action o f insulin on adipocytes

Insulin induces a variety of responses, such as proliferation, protein synthesis, glycolysis, and lipogenesis via its receptor possessing intrinsic tyrosine kinase activity. The kinase activity is essential for expression of insulin actions [for review, see ref. 74]. A large number of studies have been performed indicating that proteins containing SH2 domains become associated with and activated by the autophosphorylated receptor and/or the receptor substrates, such as IRS-1, upon occupation of the receptor with insulin [for review, see ref. 75]. Early studies with CHO cells overexpressing human insulin receptor have shown that PI 3-kinase is co-immunoprecipitated with anti-phosphotyrosine antibody, and that the activity in the immunoprecipitates increases upon insulin stimulation [76, 77]. Similar results have been obtained with rat adipocytes which are physiological target cells for insulin and in which insulin exerts metabolic action, such as glucose transport and utilization [78, 79]. Application of wortmannin on adipocytes and intensive analysis of its actions have been reported by several groups. Okada et al. [30] showed that wortmannin at concentrations lower than 1 IxM inhibited the activity of PI 3-kinase purified from rat adipocytes, insulin-induced membraneassociation of PI 3-kinase in the cells, insulinstimulated cellular uptake of 2-deoxyglucose, and antagonistic action of insulin on isoproterenolinduced lipolysis. Blockade of glucose uptake by wortmannin is described also in insulin-like growth factor-l-stimulated Xenopus oocytes [80] and insulin-stimulated brown adipocytes [81]. Insulin-stimulated translocation of glucose transporters from intracellular specific vesicles to the plasma membrane, which is a major cause of

PI 3-Kinasein terminallydifferentiatedcells stimulated glucose uptake, is blocked by wortmannin in 3T3-L 1 adipocytes [82] and in CHO cells expressing both the human insulin receptor and a tagged glucose transporter type 4 (GLUT4) [83]. Other ways to inhibit the activity of PI 3-kinase is (i) to overexpress a mutant p85 which lacks a binding site for p 110 and, thus, is unable to activate p110 [84]; and (ii) to microinject a synthetic peptide which is designed to bind to SH2 domains of p85 and to inhibit the interaction of p85 and p 110 [81]. Both ways successfully blocked the insulin-stimulated glucose uptake and translocation of glucose transporters [80, 84]. These results may provide a hint for a mutual function of mammalian PI 3-kinase and yeast one (Vps34p) which is required for protein sorting to the vacuole in yeast [36]. It is thought that a signal for antilipolyticaction of insulin is transduced in the following sequence; activation of unidentified insulin-dependent protein serine/threonine kinase, phosphorylation and activation of cGMP-inhibited cAMP phosphodiesteraseby the kinase, decrease of intracellular cAMP level, inactivation of cAMP-dependent protein kinase, and inactivation of hormonesensitive lipase [85]. Wortmannin with 1C5o values of 10-30 nM blocked the phosphorylation and the activation of cGMP-inhibited cAMP phosphodiesterase, as it does antilipolytic action of insulin [86], suggesting that the activation of PI 3-kinase is an earlier event than such sequential events. Activation o f platelets

Platelets are activated by various stimuli, such as thrombin, ADP, platelet-activating factor, collagen, to result in aggregation and degranulation. It is widely accepted that the activation of phospholipase C-mediated pathway is important for such responses ofplatelets [87, 88]. Possible involvement of PI 3-kinase in human platelets stimulated with the agonists for receptors of the G-protein-coupled types is suggested from the following results. (i) Tyrosine phosphorylation is induced rapidly upon stimulation with thrombin, collagen, or a combination of Ca 2÷

553

ionophore and phorbol ester [89-91]; (ii) PI3,4P2 and PI3,4,5P3 are accumulated in platelets stimulated with receptor agonists, phorbol ester, or GTP~/S [92-95]; (iii) PI 3-kinase activity is associated with p60 c-~'~and p59ry~and the associated activity increases quickly upon thrombin-stimulation [96]; (iv) Stimulation of platelet adhesion receptor glycoprotein Ib/IX with von Willebrand factor induces the tyrosine phosphorylation of a set of proteins and the cytoskeletal association of PI 3-kinase and pp60 cs'~ [97]. However, experiments with wortmannin do not clearly indicate the involvement of PI 3-kinase in platelet activation. Yatomi et al. [29] have shown that wortmannin at 1 ~tM does inhibit serotonin release from human platelets stimulated with a stable thromboxane A2, phorbol ester or low concentrations of either thrombin (0.02 unit/ml) or collagen (1 and 2 lxg/mL), but does not the release from human platelets stimulated with higher concentrations of thrombin or collagen. They also described that aggregation induced with phorbol ester was inhibited completely by 100 nM wortmannin, but Hashimoto et al. [98] used 6 ~tM wortmannin to inhibit ADP-induced aggregation completely. The effects of wortmannin observed at higher concentrations of the compound could be a consequence of myosin light chain kinase inhibition as suggested by Hashimoto et ai. [98] or of PI 4-kinase inhibition and resulting blockade of PI turnover [39, 99]. It is possible to speculate that PI 3-kinase is a major determinant of aggregation in weakly stimulated platelets, and that other signalling pathways become dominant in platelets activated with higher concentrations of strong agonists. However, it remains to be determined whether wortmannin suppresses the association of PI 3-kinase activity with the src-related tyrosine kinases and the accumulation of PI 3-kinase products in platelets activated with various stimuli. CONCLUSION Wortmannin is a powerful tool to dissect the physiological functions of PI 3-kinase in cell

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systems. However, caution must be paid in interpreting the cellular effects of this c o m p o u n d in two particular respects. First, the effective concentrations are varied depending on experimental conditions, such as time, temperature, p H , and concentrations of proteins and, thus, cell densities in the medium. This is due to irreversible binding o f wortmannin with PI 3-kinase and surrounding proteins. Serum, for example, in the medium is a causative factor which inactivates wortmannin and raises its effective concentrations. Therefore, performing experiments under the same conditions is essential in order to compare the effective concentrations for PI 3-kinase inhibition in the cells and relate this to inhibition o f cellular responses. The correlation between these data are crucial in analysing the consequent actions caused by wortmannin. In addition, it is recommended that stock solution o f wortmannin in dimethylsulfoxide should be diluted with medium or buffer just before use to minimize the degradation of the compound. Second, high concentrations of wortmannin inhibit myosin light chain kinase and a PI 4-kinase. Myosin light chain kinase is an enzyme regulating some of the cellular kinetic processes elicited by actin-myosin interaction. Since cellular kinetic processes, such as protein sorting, translocation of glucose transporter-containing vesicles and m e m b r a n e ruffling, are some of the suggested functions of P I 3-kinase and are possibly regulated by myosin light chain kinase, close examination o f wortmannin effects on such processes should be recommended to distinguish the contribution o f the two enzymes. On the other hand, irreversible action of wortmannin is beneficial in that the inhibition of PI 3-kinase in cells can be determined, since wortmannin remains bound with the kinase after disruption o f cells, immunoprecipitation, and intensive wash of the immunoprecipitates. In some cell types, such as RBL-2H3 cells, it is difficult to detect PI3,4P2 a n d / o r PI3,4,SP3 in the radiolabelled cells because of poor labelling or small amounts of the polyphosphorylated PI. H o w ever, acid-precipitation of wortmannin-bound proteins should be avoided, since wortmannin

will release f r o m the proteins under acidic conditions (Fig. 2). The combinational use of wortmannin, its derivatives with different selectivity and potency, and a structurally-different inhibitor, LY294002, have and will continue to provide more insight into the action of PI 3-kinase. Acknowledgements-- We thank Drs. YoshiakiNonomura and Yasuhisa Fukui for helpful discussion. We also thank all researchers concerned in our original work in Tokyo Research Laboratories and Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo.

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