Insulin-dependent Formation of a Complex Containing an 85-kDa ...

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1985,1987; Izumi et al., 1987; Kadowaki el al., 1987). Tyrosine phosphorylation of pp185 was shown to be catalyzed directly by the insulin receptor kinase and ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, lnc.

Vol. 267, No. 36, Issue of December 25, pp. 25958-25966, 1992 Printed in U.S.A.

Insulin-dependent Formationof a Complex Containing an85-kDa Subunit of Phosphatidylinositol 3-kinase and Tyrosine-phosphorylated Insulin Receptor Substrate 1” (Received for publication, August 3, 1992)

Kazuyoshi Yonezawag, Hiroo UedaS, Kenta Hara$, KazuyukiNishidaS, Akifumi Ando$, Alain ChavanieuQ, Hiroshi Matsuban, Kozui Shiill, Koichi Yokono$, Yasuhisa Fukuill , Bernard CalasQ, Florin GrigorescuQ, Ritu Dhand**, Ivan Gout**, Masayuki Otsu**, Michael D. Waterfield**, and Masato Kasuga$$$ From the $Second Departmentof Internal Medicine, Kobe University Schoolof Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, de la Kobe 650, Japan, the SCRBM of the Centre Nationalde la Recherche Scientifique (UPR8402) and the Znstitut National Sante et dela Recherche Medicale (Unite 249), 1919 Route de Mende, 34033 Montpellier Ceden, France, YHyogo the Institute of Clinical Research, 13-70 Kitaoji-cho, Akashi 673, Japan, the JJLaboratory of Biochemistry, Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo113, Japan, and the **Ludwig Institute for Cancer Research, 91 Riding House Street, London W I P 8 BT, United Kingdom

Monoclonal antibodiesraisedagainst the 85-kDa Phosphatidylinositol (PI)’ 3-kinase phosphorylates, at the subunit (p85) of bovine phosphatidylinositol (PI) 3- D-3 position, theinositol ring of PI, PI4-phosphate (PI-4-P), kinase were found to recognize uncomplexed p85 or and PI 4,5-bisphosphate (PI-4,5-Pz) to produce P I 3-phosp85 in the active PI 3-kinase. Immunoprecipitation phate (PI-3-P), PI 3,4-bisphosphate (PI-3,4-P2), and PI trisstudies of Chinese hamster ovary cells, which overex- phosphate(PIP3, probably PI 3,4,5-trisphosphate), respecpress the human insulin receptor when treated with tively (Whitman et al., 1988; Auger et al., 1989). D-3-phosinsulin, showed increased amounts of p85 and PI 3- phorylated inositides may represent novel second messenger classical PI pathway (Auger et al., kinase activity immunoprecipitable with monoclonal molecules distinct from the anti-p85 antibody andno increase in the tyrosine phos- 1989). Purification of PI 3-kinase has demonstrated that the phorylation of p85. Insulin also induced an association kinase is a heterodimer consisting of 85-kDa (p85) and 110of p85 with the tyrosine-phosphorylated insulin recepkDa (p110) subunits (Carpenter et al., 1990; Morgan et al., tor substrate1 (IRS-1)and other phosphorylated pro- 1990; Shibasaki et al., 1991). Three groups,using different teins ranging in size from 100 to 170 kDa butnot with approaches, have recently cloned bovine, murine, and human the activated insulin receptor. In vitro reconstitution p85 (Otsu et al., 1991; Escobedo et al., 1991b; Skolnik et al., studies wereused to show p85 in the active PI 3-kinase 1991). The amino acidsequence of p85showed that the associated with the tyrosine-phosphorylated IRS1but protein contains two Src homology region 2 (SH-2) domains not with the activated insulin receptor. Competition and one SH-3 domain with no apparent ATP binding site studies using synthetic phosphopeptides corresponding (Otsu et al., 1991; Escobedo et al., 1991b; Skolnik et al., 1991). involved to potential tyrosine phosphorylation sites of IRS-1 Compelling evidence suggeststhat SH-2 domains are revealed that phosphopeptides containing YMXM mo- in interactions with tyrosine-phosphorylated proteins (Koch tifs inhibited this association with different potencies, et al., 1991). When thep85 subunit is expressed alone, it binds whereas nonphosphorylated analogues and a phospho- to and is a substrate for tyrosine-phosphorylated PDGF and peptide containing the EYYE motif had no effect. Src EGF receptor kinases and the polyoma virus middle T antihomology region 2 domains of p85 expressed as gluta- gen-pp60“’” complex, but lacks PI 3-kinase activity (Escothione S-transferase fusion proteinsalso bound to ty- bedo et al., 1991b; Otsu et al., 1991). In addition, SH-2 domains of p85 expressed as fusion proteins in Escherichia coli were rosine-phosphorylated IRS- 1. These results suggest colonythat insulin causes the association of PI 3-kinase with found to bind to receptors for the EGF, PDGF-P, et al., 1992; McGlade et al., IRS-1 via phosphorylatedY l ” motifs of IRS-1 and stimulating factor 1, and Kit (Hu 1992; Klippel et al., 1992; Reedijk et al., 1992). These results Src homology region 2 domains of p85. suggest t h a t p l l 0 represents the catalytic subunit of P I 3kinase, and p85 appears to be the subunit that links PI 3kinase to the ligand-activated receptor. The YMXM motif was proposed to be a consensus sequence for tyrosine phosphorylationsitesthatbindtotheSH-2domains of p85 * This work was supported by a grant-in-aid for cancer research (Cantley et al., 1991). A recent report suggests that two P I 3from the Ministry of Education, Science, and Culture of Japan; a kinase recognition sites exist in the kinase insert region on grant for diabetes research from Ohtsuka Pharmaceutical Co., Ltd.; thePDGF receptorwhich have phosphotyrosine residues a grant from Senri Life Science Foundation (to M. K.), Grant ARC 6747,1987 from The Association pour la Recherche contrele Cancer, and a grant from Genie Biologique et Medical (GBM, 1988) (to F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $$ To whom correspondence should be addressed. Tel.: 078-3417451 (ext. 5520); Fax: 078-382-2080.

The abbreviations used are: PI, phosphatidylinositol; SH-2 and -3, Src homology regions 2and 3, respectively;PDGF, platelet-derived growth factor; EGF, epidermal growth factor; CHO, Chinese hamster ovary; IR, insulin receptor; IRS-1, insulin receptor substrate-1; Sf9 cell, Spodopterafrugiperda cell; bp,base pair(s); Fmoc, N49-fluoreny1)methoxycarbonyl; HPLC, high performance liquid chromatography; mAb, monoclonal antibody.

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located in a 5-amino acid motif with an essential methionine peptide (pep80) corresponding to residues 489-507of the molecule (Sun et al., 1991); and three polyclonal antibodies against p85: (i) at the fourth position C-terminal to the tyrosine (Yu et al., rabbit antiserum against the human p85a produced in E. coli (Ya1991; Kashishian et al., 1992; Kazlauskas et al., 1992; Fantl et manashi et al., 1992), (ii) rabbit antiserum against a synthetic Cal., 1992). Mutation of bothsites was found necessary to terminal peptide of bovine p85a (residues 713-724), and (iii) rabbit eliminate PI 3-kinase association with the receptor and the antiserum against a synthetic C-terminal peptide of bovine p85P (residues 707-724). Rabbit antiserum against the human p85a was PDGF-mediated mitogenic response (Fantl et al., 1992). The insulin receptor (IR) belongs to thefamily of structur- used in the immunoblotting studies, unless otherwise specified. Production and Screening of Monoclonal Antibodiesagainst Bovine ally related transmembrane growth factor receptors with lip85a-Bovine p85a (Otsu etal., 1991) was partially purified from Sf9 gand-activated protein tyrosinekinase activity (Kasuga et al., cells infected with the recombinant virus, using Mono Q anion 1983; Kahn and White, 1988). Several lines of evidence sug- exchange and Superose 12 gel filtration column (Pharmacia, Uppsala, gest thatthis kinase activity is essentialin eliciting the Sweden) chromatography. Female BALB/c mice were immunized by complex cellular response to insulin (Kahn and White, 1988; injection of partially purified p85a mixed with Freund's adjuvant into Becker and Roth,1990;Kasuga et al., 1990).Insulin treatment the hind pad. Animals received booster injections every 3 or 4 days 2 weeks. Four days after final injection, lymphocytes from inguinal of cells has been found to increase PI 3-kinase activity in for lymph nodes were fused with S P 2 / 0 myeloma cells by the polyethimmunoprecipitates made using antibody to phosphotyrosine ylene glycol method (Roth et al., 1982). When the hybrid cells were (Endemann et al., 1990; Ruderman et al., 1990). Unlike the semiconfluent, supernatants were screened by microtiter plate observed responses of other tyrosine kinases, only a small method (Morgan and Roth, 1985) for their ability to precipitate lZ5Ifraction (1-3%) of PI 3-kinase activity which can be precipi- labeled (Bolton-Hunter reagent) partially purified p85a. Positive tated with anti-phosphotyrosine antibody was co-precipitable hybridomas were tested further for their ability to precipitate PI 3activity from cell lysates and p85a or p858 from Sf9 cells with anti-insulinreceptor antibodies (Endemann et al., 1990). kinase infected with the recombinant virus. Positive hybrids were cloned by Thus, the association of PI 3-kinase to the insulin receptor limiting dilution. PI 3-kinase activity was determined as described appears to be too transient or the association is sufficiently previously (Endemann et al., 1990). Generation of Glutathione S-Transferase Fusion Proteins-For p85 weak to be detected by immunoprecipitation (Yonezawa et al., 1991). Treatment of insulin-stimulated intact cells with construct I, the unique NcoI site in the bovine p85p cDNA (1 bp bifunctional cross-linkers has been found to cause a signifi- upstream from the start codon) was filled with Klenow polymerase and modified by the addition of EcoRI linkers. Then a 2.5-kilobase cant increase in PI 3-kinase activity associated with the EcoRI fragment (from the linker site to324 bp downstream from the insulin receptor (Yonezawa et al., 1991). Therefore, an asso- stop codon) including the entirecoding region of p85p wassubcloned ciation of PI 3-kinase with the receptor may be mediated by into the EcoRI site of the pGEX-2T plasmid (Pharmacia). For p85 other tyrosine-phosphorylated proteins. Insulin or insulin- construct 11, the unique StuI site in the bovine p85a cDNA (43 bp like growth factor I treatment of various intact cells causes downstream from the stop codon) was modified by the addition of rapid tyrosine phosphorylation of a high molecular weight EcoRI linkers. A BglII-EcoRI fragment (243 bp downstream from the start codon to thelinker site, 44 bp downstream from the stop codon) = 160,000-185,000) termed pp185 (White et al., was ligated into the BarnHI-EcoRI site of the pGEX-3X plasmid. For protein (M, 1985,1987; Izumi et al., 1987; Kadowaki el al., 1987).Tyrosine p85 construct IV,V, or VII, an EcoRV-EcoRI fragment (1,009 bp phosphorylation of pp185 was shown to be catalyzed directly downstream from the startcodon to thelinker site) of p85a, an AccIby the insulin receptor kinase and was not caused by auto- EcoRI fragment (1,331 bp downstream from the start codon to 324 phosphorylation (Tashiro-Hashimoto et al., 1989). Insulin bp downstream from the stop codon; the AccI site had been filled with Klenow polymerase) of ~ 8 5 0 or , an HincII-EcoRI (1,749bp receptor substrate 1 (IRS-1) was recently purified and its downstream from the start codon to the linker site) of p85a was sequence deduced by cDNA cloning (Rothenberg et ai., 1991; subcloned into theBarnHI site, previously filled with Klenow polymSun et al., 1991). The predicted protein encoded has charac- erase, and theEcoRI site of the pGEX-3X plasmid, respectively. For teristics similar to those of pp185 and contains at least 10 p85 construct VI, a BglII-EcoRI fragment (1,453bp downstream from potential tyrosine phosphorylation sites which have an the start codon to 324 bp downstream from the stop codon) of p85p YMXM motif. In addition, immunoprecipitates made with was subcloned into the BarnHI-EcoRI site of pGEX-3X. For p85 constructs 111 (encompassing nucleotide 988 to the stop codon) and anti-IRS-1 antibodies have been show to contain significant VI11 (encompassing nucleotides 988-1300), oligonucleotides homololevels of PI 3-kinase activity (Sun et al., 1991). gous to the boundaries of the desired sequence within the bovine p85a In the present study, we have examined the insulin-stimu- cDNA were synthesized. These oligonucleotides contained BarnHI latedinteraction of the insulin receptor, PI 3-kinase, and and/or EcoRI sites. The required stretch of DNA was subsequently IRS-1 in cultured cells (in uiuo) and through acell free system amplified by polymerase chain reaction using the bovine p85a cDNA for the reconstitution of enzyme complexes (in uitro). We as a template. The DNA insert was then digested with BarnHI and EcoRI and ligated into the pGEX-3X plasmid. The recombinant found, using a CHO cell line which overexpresses the human plasmids were introduced into E. coli (DH5a)andthe bacterial insulin receptor, that insulin induced the association of p85 transformants analyzed for the presence of inserts. The glutathione in theactive PI 3-kinase (presumably with the p l l 0 catalytic S-transferase-p85 fusion proteins were then expressed by induction Expressed glutasubunit) with tyrosine-phosphorylated IRS-1 butnot with the with 1 mM isopropyl 1-thio-P,D-galactopyranoside. thione S-transferase-p85fusion proteins were isolated from bacterial activated insulin receptor. EXPERIMENTALPROCEDURES

Cell Cultures and Antibodies-CHO cells and CHO cells overexpressing wild-type human insulin receptor (Yonezawa et al., 1992) were routinely maintained inHam's F-12 medium supplemented with 10% fetal calf serum. Spodoptera frugiperda (Sf9) cells were maintained in IPL-41 medium supplemented with 10% fetal calf serum. The antibodies used were a monoclonal antibody against the human insulin receptor (2F3; Yonezawa et al., 1992); polyclonal antipeptide antibodies specific for amino acids 954-965or1317-1328 at the intracellular domain of the human insulin receptor p subunit (Ullrich et al., 1985); a monoclonal antibody (py20; ICN, Costa Mesa, CA) (Glenney et at., 1988) and a polyclonal antibody (UBI, Lake Placid, NY) against phosphotyrosine residues; a polyclonal antibody against rat IRS-1 that were raised in rabbits immunized with a synthetic

lysates by incubation with glutathione-Sepharose beads. Synthesis and Purification of Phosphorylated and Nonphosphoryluted Peptides-Five synthetic peptides (designated IRP-1, -2, -3, -4, and -5) containing putative tyrosine phosphorylation sites of IRS-1 are: TDDGYMPMSPGV, GNGDYMPMSPKS, DPNGYMMMSPSG, TGDYMNMSPVG, and ARLEYYENEKKW, corresponding to amino acids 604-615,624-635,654-665,724-734, and 42-53 of rat IRS-1, respectively (Sun et al., 1991). Solid phase peptide synthesis was carried out on animatedpolyacrylic resin, PepSyn KA (MilliGen, Bedford, MA) by the Fmoc technique on an automatic 9050 Pepsynthesizer (MilliGen) as described previously in detail (Chavanieu et al., 1991). The Fmoc-Tyr-O-POa-(benzyl)zresidue used for synthesis of phosphorylated peptide was prepared from t-butyloxycarbonylTyr-O-POs-(benzyl)z.After completion of synthesis, the peptide-resin complex wasdried, and then the deprotection of chain blocking groups and peptide cleveage were performed using reagent K (82.5% triflu-

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oroacetic acid, 5% phenol, 5% H20, 5% thioanisole, 2.5% ethanedi- sulintreatmenttoglutathioneS-transferase fusion proteins(p85 thiol) for 2 h a t room temperature (King et al., 1990). Deprotected constructs 11, VII, and VIII), CHO-IR cell lysates with or without peptides were precipitated with cold anhydrous ether, solubilized in insulin treatmentwere incubated for 4 h at 4 "C with fusion proteins water, and then the aqueous phase was extracted five times with ethyl immobilized to glutathione-Sepharose beads. The beads were washed ether. The aqueous phase was rotatory evaporated to remove the and Western blotted with a polyclonal antibody to phosphotyrosine remaining ethyl ether and then freeze dried. The crude peptideswere residues, a polyclonal antibody to IRS-1, and polyclonal antipeptide purified by reverse phase HPLC on a delta-pack C18 column 300A antibodies to thep subunit of the human insulin receptor. (25 X 2.5 cm) in 0.1% trifluoroacetic acid using a linear gradient of To examine binding of baculovirus-expressed bovine p85a to the acetonitrile (0-7096) over 70 min. Following purification,peptide activated insulin receptor, the insulin receptors from CHO-IR cells composition after acid hydrolysis wasconfirmed by amino acid analy- were immunoprecipitated with monoclonal anti-insulin receptor ansisperformed on a 6300 Beckmanapparatus.Thepeptides were tibody bound to protein G-agarose beads and then autophosphorylhydrolyzed in 6 N HCl a t 115 "C for 24 h in evacuated sealed tubes. ated as described previously (Yonezawa et aL, 1992). The activated Gas phase sequencing of phosphorylated peptide showed no phenyl- insulin receptoron the beads was exposed to lysates of Sf9 cells thiohydantoin tyrosine in the appropriate cycles, indicating that all expressing p85a for 4 h a t 4 "C, washed, electrophoresed, and immutyrosine residues in the corresponding positions were phosphorylated noblotted with rabbit antiserum against p85a. in the phosphopeptides. Nonphosphorylated peptides were obtained by dephosphorylation RESULTS with insoluble alkaline phosphatase (Sigma) as described previously Characterization of Monoclonal Antibodies against p85(Zardeneta et al., 1990). The nonphosphorylated peptides were purified further by reverse phase HPLC asdescribed above. The bovine p85aprotein, expressed ininsect cells using Immunoprecipitation, Western Blotting, and Cell Labeling-Conbaculovirus vectors and purified by Mono Q and Superose 12 fluent 100-mm plates of CHO cells overexpressing wild-type human column chromatography, was usedasantigento produce insulin receptor (CHO-IR) were starvedinHam'sF-12 medium monoclonal antibodies (mAbs). Three mAbs (F12, E10, and containing 20 mM Hepes (pH 7.6) for 12 h a t 37 "C. Cells were then incubated withor without insulinat 37 "C, frozen with liquid nitrogen, H1) were found to immunoprecipitate both forms of p85 ( a and stored a t -80 "C until lysis. Cells were then lysed in cold lysis and /3) which were described by Otsu et al. (1991), whereas buffer (137 mM NaCl, 20 mM Tris (pH 7.61, 1 mM MgClz, 1 mM mAb G12 could only immunoprecipitatep85a(datanot CaC12,1 mM dithiothreitol, 10% glycerol, 1mM phenylmethylsulfonyl shown). All mAbs were able to recognize denatured p85 that fluoride, 1% Nonidet P-40, and 1 mM sodium orthovanadate) and was transferredto nitrocellulose paper. To determinethe immunoprecipitated with either control mouse IgG (Cappel, WestChester, PA), monoclonal anti-phosphotyrosine antibody, or mono- regions of p85 recognized by these mAbs, we constructed glutathione S-transferase-bovine p85 fusion proteins as declonal anti-p85a antibody bound to protein G-agarose (15-pl beads; Kurabo, Osaka, Japan) as describedpreviously (Endemann et al., picted in Fig. 1A. An epitope mapping studyshowed that F12 1990). The immunoprecipitates were washed once with phosphateand G12 recognized the bcr homology region (the region buffered saline, 1%Nonidet P-40, once with 0.1 M Tris-HC1 (pH7.4), between SH-3 domain at the N terminus and the first SH-2 0.5 M LiC1, and twice with 50 mM Hepes (pH 7.6)-buffered saline domain). E10 recognized an epitope corresponding to amino containing 0.1% Triton X-100. The samples were electrophoresed on acids 330-337 of bovine p85a (homologous to residues 323SDS-polyacrylamide gels, transferred to Immobilon-Pmembranes (Millipore, Bedford, MA), blocked in 3% gelatin, 10 mM Tris-HC1 330 of bovine p85P). H 1 recognized an epitopecorresponding to amino acids 452-492 of bovine p85a (homologous to resi(pH 7.6), 154 mM NaCl a t 37 "C, andblottedwitheitherrabbit antiserum against p85a, a polyclonal antibody against phosphotyro- dues 445-485of bovine p85p) (Fig. 1A). Furthermore, F12 sine residues, or a polyclonal antibody against IRS-1. Bound antibod- and G12 but not E10 and H1 were also able to immunopreies were detected with horseradishperoxidase-conjugated anti-rabbit cipitate P I 3-kinaseactivity frombovine brain (Fig. l B ) , IgG (Promega, Madison, WI) followed by ECL detection (Amersham, whereas E10 and H1 immunoprecipitated free p85but not the Tokyo, Japan), according tothemanufacturer'sinstructions.For brain(datanotshown), labeling studies, 100-mm plates of CHO-IR cells were washed and P I 3-kinase enzymefrombovine then incubated with phosphate-free RPMI 1640 medium containing indicating that the F12 and G12 recognized P I 3-kinase as a 0.75 mCi of [3ZP]orthophosphatefor 3 h a t 37 "C or incubated with heteromeric form of p85 presumably containing pl10, whereas 0.4 mCi of [35S]methionine and[35S]cysteine(Tran%-label, ICN) in E10 and H1recognized an uncomplexed form of p85. F12 but methionine-free Dulbecco's modified Eagle's medium with 10% dialyzed fetal calf serum for 16 h a t 37 "C. These cells were incubated not G12 was able to immunoprecipitate P I 3-kinase activity shown). from CHOcells (Fig.1B) and hamster brain (data not with or without insulin, lysed, and immunoprecipitated with either E10 but not H1 immunoprecipitated the uncomplexed form control mouse IgG, monoclonal anti-phosphotyrosineantibody,or monoclonal anti-p85aantibodyboundtoprotein G-agarose. The of p85 from hamster brain (data not shown). These results immunoprecipitates were washed three times with a high salt wash showed that F12 and E10 but not G12 or H I were able to buffer (20 mM Na2HP04 (pH 8.6),0.5% Triton X-100, 0.1% SDS, 1 recognize hamster p85. M NaC1,0.1% bovine serum albumin) andtwice with the same wash buffer containingonly 0.15 M NaCl and no albumin. Then the samplesEffect of Insulin on the Association of p85 in the Active PI were electrophoresed on SDS-polyacrylamide gels and analyzed by 3-Kinase with Tyrosine-phosphorylated IRS-1 in Viuo-Deautoradiography. tergent lysates of CHO cells were immunoprecipitated with I n Vitro Binding Assuy-To examine bindingof insulin-stimulated mAb F12 which recognized the active hamster P I 3-kinase. tyrosine-phosphorylated proteins to the p85 protein in the activeP I The immunoprecipitates were then immunoblotted with pol3-kinase, CHO-IRcell lysates withor without insulin treatmentwere yclonal anti-C-terminal peptide serumspecific to either p85a incubated for 4h at 4 "C with the p85 in the active PI 3-kinase immunoprecipitated from CHO cells with monoclonal anti-p85 anti- or p85& showing that in CHO cells the p85a form predomibody (F12) bound to proteinG-agarose. The beads were washed and nates rather than p85p (data not shown).Next, CHO-IR cell immunoblotted with eithera polyclonal antibody to phosphotyrosine (CHO cell overexpressing the human insulin receptor) lysates residues, a polyclonal antibody to IRS-1, or polyclonal antipeptide from cells treated or untreated with insulin were immunopreantibodies to the p subunit of the human insulin receptor. To examine cipitated with F12. The immunoprecipitates were then imthe inhibitory effect of phosphorylated or nonphosphorylated synthetic IRS-1 peptides on the binding of IRS-1 to the p85 protein in munoblotted withpolyclonal antiserum to p85a.F12 was able (Fig. 1B). the active PI 3-kinase, the PI 3-kinaseform of p85 was preincubated to precipitate p85(Fig. 2 . 4 ) and PI 3-kinase activity with synthetic peptides for 2 h at 4 "C and thenexposed to CHO-IR In contrast, mAb E10, which has been shown to interact only cell lysates for 4 h a t 4 "C. The concentrations of synthetic peptides with uncomplexed p85 and not PI 3-kinase, was unable to during preincubation were 5-fold higher than that after the addition immunoprecipitate p85 protein from CHO lysates (data not of CHO-IR cell lysates. After washing, the samples were immunoshown), indicating that in CHO cells p85exists predominantly blotted with a polyclonal antibody to IRS-1. To examine binding of tyrosine-phosphorylated proteins after in- as a heteromeric form presumably containing the p l l 0 cata-

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FIG. 2. Immunoprecipitation of p85 from lysates of CHOIR cells. Cells were treated (lanes b) or not (lanes a ) with 10” M insulin for 1 min a t 37 “C, frozen with liquid nitrogen, and then lysed. The lysates were immunoprecipitated withamonoclonal anti-p85 antibody, F12 (panels A, C, and D )or with an anti-phosphotyrosine antibody (py20) (panel B ) bound to protein G-agarose. The immunoprecipitates were washed, electrophoresed on SDS-polyacrylamide gels, transferred to Immobilon-P membranes, and thenblotted with rabbit antiserum against p850 (panels A and R ) , a polyclonal antiOri Ori phosphotyrosine antibody (panel c),or a polyclonal anti-IRS-1 antibody (panel D ) as described under“Experimental Procedures.” F12 GI2 El0 H I F12 G I 2 E10 H I Bound antibodywas detected with horseradish peroxidase-conjugated FIG. 1. Characterization of monoclonal antLp85ia antibod- anti-rabbit IgG followed by ECL reading. In panels A and R , p85 is ies. Panel A, epitope mapping of glutathione S-transferase-bovine indicated by an arrow. In panel C, the 160-kDa protein (pp185) and p85a or p85@fusion proteins expressed in bacterial systems obtained the position of the insulin receptor @-subunit areindicated by arrows. by anti-p85nmonoclonal antibodies. Each p85 construct was obtained In panel D,IRS-1 isindicated by an arrow. The positions of prestained as shown under “ExperimentalProcedures.” The presumed functional high and/or low molecular mass markers (in kDa) are indicated. domains SH-3 and SH-2 are represented by hatched and black boxes, respectively. The amino acids of each fusion protein are numbered according to theirrelative positions within wild-type bovine p85a or to insulin, immunoprecipitates with anti-p85 F12 antibodies from nontreated and insulin-treated cells were analyzed by 1).Fusion proteinswere precipitated p85@ (first methionine is number on glutathione-Sepharose beads, runonSDS-polyacrylamide gel, Western blotting with a polyclonal anti-phosphotyrosine antransferred to Immobilon-P membrane, and blotted by F12, G12, E10, tibody. As illustrated in Fig. 2C, mAb F12 did not precipitate and H I antibodies. + and - represent fusion proteins recognized or phosphoproteins such as the95-kDa /3 subunit of the insulin not, respectively, by the antibody. Panel B, immunoprecipitation of receptor or p85. In contrast, in insulin-treated cells (lane b ) PI 3-kinase activity with monoclonal anti-p85 antibodies. Detergent lysates of CHO cells or bovine brain were immunoprecipitated with but not in untreated cells (lane a ) , F12 precipitated a 160each monoclonal anti-p85 antibody (F12, G12, E10, or H1) bound to kDa phosphoprotein aswell as other phosphoproteinsof 110, protein G-agarose. The immunoprecipitates were washed, and PI 3- 120, and 140 kDa. T o confirm that the 160-kDa phosphoprokinase activity was determined as described under “Experimental teinco-immunoprecipitated withp85 was IRS-1, we perProcedures.” Ori and PIP markers indicate the positionsof the TLC formed Westernblotting of F12 immunoprecipitates with origin and migration of a PI-4-P standard, respectively. PIP

PIP

polyclonal anti-IRS-1 antibodies. As shown in Fig. 2 0 , F12 lytic subunit. Therewas a significant increase in the amount precipitated IRS-1 from insulin-treated cells (lane b ) but not of p85 (Fig. 2 A ) precipitated withF12 after insulin treatment. from untreated cells (lane a ) . T o detect other proteins associated with p85 and IRS-1 as The PI 3-kinase activity precipitated with F12 also showed a consequence of insulin stimulation, cells were labeled with a n apparent 2-fold increase after insulin treatment (data not 3sS(Fig. 3A) or 32P(Fig. 3B), treatedor untreated with insulin, shown). Treatment of cells with insulin increased the PI 3-kinase lysed, andimmunoprecipitation was performed withthe monoclonal antiphosphotyrosine antibody py20 (lanes a and activity immunoprecipitablefrom cell extractswithantiphosphotyrosine antibodies (Endemann et al., 1990; Ruder- b in Fig. 3, A and B ) or the mAb F12 (lanes c and d in Figs. 3, A and B ) . As shown in Fig. 3A, lane c, the analysis of the man et al., 1990). T o examine whether immunoprecipitates immunoprecipitates from 3sS-labeledcells revealed that, commade usingamonoclonal anti-phosphotyrosineantibody (py20) from lysates of insulin-treated CHO-IR cells contain pared with controls in which mouse IgG was used (data not p85, Western blotting of these immunoprecipitates was per- shown), F12 specifically immunoprecipitated an 85-kDa proformed with polyclonal antiserum against p85a.As shown in tein anda doublet containing 110-kDa and 120-kDa proteins, p85 and p110. In Fig. 2B, the monoclonal anti-phosphotyrosine antibody was which may contain the PI 3-kinase subunits found to precipitate p85 from insulin-treated cells (lane b ) some cases adoublet a t 110 kDahas been observed in purified P I 3-kinase (Carpenteret al., 1990). Insulin treatmentof cells but not from untreated cells (lane a ) . The presence of p85 in anti-phosphotyrosine immunopre- (Fig. 3A, lane d ) caused detectable co-immunoprecipitation cipitates would not seem to be aresult of a physical association with p85 of a 160-kDa protein (identified as IRS-1) and a of 210,135, and 95 kDa (the of PI 3-kinase with the autophosphorylated insulin receptor 170-kDa protein but not proteins because immunoprecipitates with monoclonal antibodies insulin receptor precursor, LY subunit, and /3 subunit of the against the insulin receptor have been shown previously not insulin receptor,respectively), which were immunoprecipit o contain significant amounts of P I 3-kinase activity (En- tated with py20 after insulin treatment (Fig. 3A, lane b). In demann et al., 1990). Therefore, to test whether p85 or other py20 immunoprecipitates the 170-kDa protein was also deassociated proteins are tyrosine-phosphorylated in response tected (Fig. 3A, lane b). From insulin-treated “P-labeled cells

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FIG. 3. Immunoprecipitationof p85 from "S-labeled (panel A ) or "P-labeled CHO-IR cells (panel B ) . CHO-IR cells were labeled with Tran"S"labe1 or [32P]orthophosphateas shown under "Experimental Procedures." Cells without treatment (lanes a and c) or treated with (lanes b and d ) lo-' M insulin for 1 min a t 37 "C were frozen with liquid nitrogen and then lysed. The lysates were immunoprecipitated with an anti-p85 antibody (F12) (lanes c and d ) or a monoclonal anti-phosphotyrosine antibody (py20) (lanes a and b ) bound to protein G-agarose. The immunoprecipitates were washed, electrophoresed on SDS-polyacrylamide gels, and autoradiographed. The 160-kDa protein (pp185), p110, p85, and the insulin receptor precursor (IRpre),(Y subunit (IRa)and p subunit (IRB)of the insulin receptor are indicated by arrows. The positions of prestained high and low molecular mass markers (in kDa) are indicated.

(Fig. 3B), F12 co-immunoprecipitated 170- and 160-kDa phosphoproteins as well as several phosphoproteins of 100, 110, 120, and 140 kDa and a 85-kDa protein (Fig. 3B, lane d ) but not the95-kDa phosphoprotein corresponding to /3 subunit of the insulin receptor identified in py20 immunoprecipitates (Fig. 3B, lane b). Insulin-dependent Association of p85 in the Active PI 3Kinase with IRS-1 in Vitro-As described above, p85 was detected in CHO cells only with antibodies which recognize p85in the active enzyme complex. To define further the interaction of the p85 protein in the active PI 3-kinase with tyrosine-phosphorylated proteins after insulin treatment, the p85 protein inthe active PI 3-kinase was immunoprecipitated from CHO cell lysates with the mAb F12. To avoid immunoprecipitating PI 3-kinase from CHO-IR cell lysates in the following incubation, the mAb-protein G-agarose complex was saturated with PI 3-kinase by incubating with an excess of CHO cell lysates, then washed and incubated with lysates from nontreated and insulin-treated CHO-IR cells. Proteins bound to p85 in the active PI 3-kinase were immunoblotted with either anti-insulinreceptor or anti-IRS-1antibodies. As shown in Fig. 4, A and B, IRS-1 bound to p85 in the active PI 3-kinase in an insulin-dependent fashion; however the insulin receptor did not. A 140-kDa band immunoblotted with anti-IRS-1 antibody was also detected in an insulin-dependent fashion (Fig. 4B). Inhibition of the Association ofp85 in the Active PI 3-Kinase with IRS-1 by Phosphopeptides Corresponding to IRS-1 Putatiue Tyrosine Phosphorylation Sites-IRS-1 has multiple putative tyrosine phosphorylation sites (Sun et al., 1991). To determine which tyrosine phosphorylation sites of IRS-1 are critical for the association with p85, tyrosine-phosphorylated synthetic peptides and nonphosphorylated analogues corresponding to putative tyrosine phosphorylation sites of IRS-1 were prepared as described under "ExperimentalProcedures." The p85 protein in the active PI 3-kinase immobilized on the

IRP-I

s

IRP-5 Insulin Peplide 0

f "

0 US 1.0

ARLE?YENEKKW

+

2.11 5.0 I U 20 30 50 I W 1 0 0

I

I U

Phosphopeplide

NP

FIG.4. In vitro association of the p86 protein in the active PI 3-kinase with IRS-1. Panels A and B, In uitro association of the PI 3-kinase form of p85 with IRS-1 but not with the activated insulin receptor. The PI 3-kinase form of p85 immunoprecipitated from CHO cells with a monoclonal anti-p85 antibody (F12) bound to protein G-agarose (anti-p85) was incubated with CHO-IR cell lysates treated with (Is(+)) or without (Insf-)) M insulin for 1 min at 37 "C. The beads were washed and immunoblotted with polyclonal anti-insulin receptor (panel A ) or anti-IRS-1 (panel B ) antibody. In panel A , as control (Anti-P-Tyr),the insulin receptor was immunoprecipitated with a monoclonal anti-phosphotyrosine antibody, py20, from insulin-stimulated CHO cell lysates. The IRS-1 and @ subunit of the insulin receptor are indicated by arrows. The positions of prestained high molecular mass markers (in kDa) are indicated. Panel C, the inhibitory effect of phosphorylated or nonphosphorylated peptides (IRP-1, -2, -3, -4,and -5) corresponding to putative tyrosine phosphorylation sites of IRS-1 on in uitro association of the PI 3kinase form of p85 with IRS-1. The PI 3-kinase form of p85 from CHO cell lysates, immobilized on the protein G-agarose-boundmonoclonal anti-p85 antibody (F12) was preincubated for 2 h at 4 "C with various concentrations of either phosphopeptides or nonphosphorylated peptide ( N P )and thenexposed for another 4 h a t 4 "C to CHOM IR cell lysates without treatment (-) or treated with (+) insulin for 1 min a t 37 "C in the absence or presence of the phosphopeptides (final concentrations, 0.5-100 PM) or nonphosphorylated peptides (final concentration, 100 PM). The beads were washed and then immunoblotted with anti-IRS-1 antibody.

protein G-agarose-bound mAbs waspreincubated with various concentrations of these peptides, exposed to lysates from insulin-treated CHO-IR cells in thepresence of the peptides, and thenimmunoblotted with anti-IRS-1 antibody. As shown in Fig. 4C, immunoblotting withanti-IRS-1 antibody revealed an upper band corresponding to IRS-1 and also another band of 140 kDa bound in aninsulin-dependent fashion. These two bands were also detected with anti-phosphotyrosine antibodies (data not shown). The phosphorylated peptides, except IRP-5, blocked the association of p85 in active PI 3-kinase with IRS-1 and the140-kDa band in a dose-dependent manner. The relative inhibitory potencies of these peptides were

Insulin-dependent Association

of p85 and IRS-1

25963

IRP-1 > IRP-4 > IRP-3 > IRP-2. In contrast, IRP-5 aswell form of p85a bound to the insulin receptor only when the as nonphosphorylated peptidesfailed to block the association receptor had been incubated with insulin and ATP. of p85 in theactive P I 3-kinase with both IRS-1 and the 140DISCUSSION kDa band. Involvement of SH-2 Domains in the Associationof p85 with In this study, we prepared four mAbs (F12, G12, E10, and IRS-1-To determine whether SH-2 domains of p85 are in- H1) against recombinant bovine p85a. F12 and G12 recogvolved intheinteraction of PI3-kinasewith IRS-1, we nized active P I 3-kinase presumably in itsheterodimeric form constructed three glutathione S-transferase fusion proteins composed of p85 and pllO subunits as reported previously expressing individual SH-2 domains (85N and85C: VI11 and (Carpenter et al., 1990; Shibasaki et al., 1991; Otsu et al., VI1 in Fig. lA, respectively) or both SH-2 domains together 1991). E10 and H1 recognized the uncomplexed form of p85. (85N+C: I1 in Fig. lA). Binding of these immobilized fusion The simple explanation is that epitopes recognized by E10 proteins to native IRS-1was investigated by incubation with and H1 are buried when p l l 0 associates with p85. The epitope CHO-IR cell lysates from untreatedcells or cells treated with mapping studies revealed that in bovine p85a E10 and H1 insulin. As shown in Fig. 5A, all three fusion proteins were recognized epitopes corresponding to amino acids 330-337 found tobind tyrosine-phosphorylated proteinsfrom insulin- and 452-492, respectively. Therefore, these regions may be stimulated cell lysates, including IRS-1 and the insulin recep- involved in the interaction between p85 and pllO subunits. tor p subunit. The identity of these proteins was confirmed Alternatively, when p85 and pllO associate, conformational by Western blotting with either anti-IRS-1 antibodies or changes anti- occur in the epitopes recognized by E10 and H1. insulin receptor (data not shown). Binding was insulin-de- Although free p85 was detected inbovine and hamster brains, pendent, and glutathione S-transferase alone bound no tyro-the immunoprecipitation studies with mAbs indicated that in sine-phosphorylated proteins. CHO cells p85 was present only in the complex as the active Association of the Uncomplexed Form of p85 with the Acti- PI 3-kinase. Further analysis with E10 or H1could reveal the vated Insulin Receptor in Vitro"SH2 fragments of p85 bound distribution andsignificance of the free form of p85 in differto the activated insulin receptor but the p85 protein in the ent tissues and cell lines. active P I 3-kinase did not.T o determine whether the uncom- mAb F12 immunoprecipitated only the p85 protein as part in vitro, of the active PI 3-kinase in CHO cells. Immunoprecipitation plexedform of p85 binds to the insulin receptor insulin receptors immunoprecipitated with monoclonal anti- studies with this antibody using CHO-IR cells revealed three insulin receptor antibodies from unstimulated CHO-IR cells main conclusions. (i) Insulinincreased the amountof p85 and were incubated in the presence or absence of 1O"j M insulin P I 3-kinaseactivity immunoprecipitable with monoclonal and 1mM ATP, washed, and exposed to baculovirus-expressed anti-p85 antibody. (ii) Insulin did not induce tyrosine phosp85a a t 4 "C for 4 h. As shown in Fig. 5B, the uncomplexed phorylation of p85. (iii) Insulin induced an association of p85 as partof the PI 3-kinasecomplex with tyrosine-phosphorylA B ated IRS-1 aswell as with other phosphorylated proteins but not with the activated insulin receptor. Previous studieshave demonstratedthatinsulintreatment of cells can causea dramatic increase in PI 3-kinase activity detectable in antiphosphotyrosine antibody immunoprecipitatesof cell lysates (Endemann et al., 1990). The data presentedhere suggestthat this is not because of an association of PI 3-kinase with the activated insulinreceptor or increased phosphorylation of p85 ontyrosine.Insteaditis likely to bearesult of a tight association of PI 3-kinase with tyrosine phosphoproteins including IRS-1. 1"S"li" - + - + - +- + IRS-1 is oneof the major tyrosine-phosphorylated proteins u u u u a b co-immunoprecipitated with p85. We could reconstitute the 85N+C 85N R5C GST physical association of tyrosine-phosphorylated IRS-1 with FIG. 5. In vitro association of free form of p85 or its truncated fragments with IRS-1or the activatedinsulin receptor. the F12-immunoprecipitated p85 in the active PI 3-kinase Panel A, in vitro association of bacterially expressed truncated frag- complex in vitro assay system. This implies thatnot all ments of p85 with IRS-1 and theactivated insulin receptor. CHO-IR tyrosine-phosphorylated IRS-1 isoccupied by p85 in insulincell lysates with or without insulin stimulation (lo" M insulin for 1 treated CHO-IRcell lysates. IRS-1 containsa t least 10 potenmin at 37 "C) were incubated for 4 h a t 4 "C with glutathione S- tial tyrosine phosphorylation sites; six contain the YMXM transferase fusion proteins immobilized to glutathione-Sepharose motif, three others have the YXXM motif, and one site has beads. The association of tyrosine-phosphorylated proteins with bacterial fusion proteins was followed by Western blotting with poly- the EYYEsequence (Sun et al., 1991). The YMXM motif has clonal anti-phosphotyrosine antibodies. From left to right, CHO-IR been proposed as a consensus sequence for tyrosine phosof p85 (Cancell lysate mixed with p85a SH-2-N+C (construct II), SH-2-N (con- phorylation sites that bind to the SH-2 domain struct VIII), SH-2-C (construct VII) or glutathioneS-transferase tley et al., 1991). Tyrosine phosphopeptides IRP-1,-2, -3, and alone. The IRS-1 and 0 subunit of the insulin receptor are indicated -4 containing themotif YMXM all blocked the association of by arrows. Panel B, i n vitro association of free p85a from Sf9 cells tyrosine phosphorylated IRS-1 with the p85 protein in the with the activated insulin receptor. The insulin receptor from CHOIR cells was immunoprecipitated with a monoclonal anti-insulin active P I 3-kinase. The simplest explanation is that these receptor antibody bound to protein G-agarose and then incubated peptides are able to mimic IRS-1 sequences and thus act as M insulin and 1 mM ATP in with (lane b ) or without ( l a n e a ) competitive inhibitors. Sincenonphosphorylated peptides the presence of 10 mM MgCI2 and 3 mM MnCI2. The treated.insulin were unable to inhibit the IRS-l-p85 association, phosphorylreceptor on the beads was exposed to lysates of Sf9 cells expressing ated tyrosine appears be to an essential structural recognition p85a for 4 h a t 4 "C, washed, electrophoresed, and immunoblotted mowith rabbit antiserum to p85a. The p85a is indicated by an arrow. element. However, the phosphopeptide IRP-5 containing The positions of prestained low molecular mass markers (in kDa) are tif EYYE did not block this association, suggesting that p85 indicated. molecules recognize phosphotyrosine ina quite specific struc-

25964

Insulin-dependent Association of p85 and IRS-1

tural context. The relative potency of inhibition was I R P - l > IRP-4 > IRP-3 > IRP-2. The IRP-1 peptide was the most efficient inhibitor and has been shown previously as the best exogenous substrate of the insulin receptor kinase (Shoelson et al,. 1992). We specifically analyzed the YMPM motif since this sequence was also present in the major tyrosine phosphorylation site of murine and hamster polyoma virus middle T antigen induced by pp60""" and was involved in pp6OC-""dependent binding of PI 3-kinase (Auger et al., 1992). Our results cannot entirely explain why peptide IRP-2 containing the same motif was relatively less efficient than IRP-4 and -3 with a slightly different YMNM motif. However, sequences surrounding this motif might possibly affect binding to p85. Nevertheless, these data suggest that tyrosine 608 in IRP-1 can be involved in the IRS-l-p85association although further in vivo studies would be necessary to determine the relative contribution of IRS-1 phosphorylation sites in mediating the p85 association. It is possible that one IRS-1 molecule can associate with several molecules of p85. This could explain the increase in the amount of p85 and PI 3-kinase activity immunoprecipitated with monoclonal antibodies in CHO-IR cells after insulin stimulation. In vitro assays were used to demonstrate the physical association of tyrosine-phosphorylated IRS-1 with SH-2 domains of p85 (85N, 85C, and 85N+C)-glutathione S-transferase fusion proteins expressed in E. coli. The present results indicate that SH-2domains of p85 associate with IRS-1 when it is tyrosine-phosphorylated. Similar datawere obtained with receptors for EGF, PDGF-P, colony-stimulating factor 1, and Kit (Hu et al., 1992; McGladeet al., 1992; Klippel et al., 1992; Reedijk et al., 1992). Our data also showed an association of the activated insulin receptor with both SH-2domains of p85glutathione S-transferasefusion proteins. Thesedata contrast with the present in vivo and in vitro findings showing that the p85 protein in the active PI 3-kinase associated with tyrosine-phosphorylated IRS-1butnot with the activated insulin receptor. The three-dimensional structure of SH-2 fragments expressed as glutathione S-transferasefusion proteins may have differed from SH-2 domains of a free form of p85. However, this is probably not the case, since recombinant p85a expressed alone in insect cells bound to the activated receptor in vitro. These results suggest that in vitro studies on the association of tyrosine-phosphorylated proteins with SH-2 fragments may not reflect the in vivo situation, and these results should be interpreted with caution. There is a difference in specificity of binding between free p85 and PI 3-kinase form of p85 in the interaction of SH-2 domainswith specific sequences containing phosphotyrosine. This is consistent with the previous observation showing that PI3-kinase from bovine brain cannot interact with activated EGF receptors, butfree p85can (Otsu et al., 1991). Thus, theassociation of p85with pllO might cause a conformational change of SH2 domain(s) which would affect this interaction. Several phosphoproteins, including tyrosine-phosphorylated IRS-1, were immunoprecipitated from CHO-IR cells by the monoclonal antibody to p85 after insulin treatment. A 170-kDa phosphoprotein was immunoprecipitated with antip85 antibody. This protein was poorly immunoblotted with polyclonal anti-phosphotyrosine antibody, suggesting that it mainly contained phosphoserine and phosphothreonine. However, it was immunoprecipitated with anti-phosphotyrosine antibody after insulin treatment. In addition, the 170kDa protein was not immunoblotted with anti-IRS-1 antibody. These data suggest that p170 is not IRS-1-related and binds to tyrosine-phosphorylated proteins associating with the p85 protein in the active PI 3-kinase. Two other phospho-

proteins of 120 and 110 kDa appeared to be tyrosine-phosphorylated in response to insulin. Proteins of the same molecular weights were also immunoprecipitated with the monoclonal anti-p85 antibody from 35S-labeled CHO-IR cells without insulin treatment. It has been reported previously that p110, one subunit of PI 3-kinase, was associated with a doublet of 110- and 120-kDa proteins as purified from rat liver (Carpenter et al., 1990). Studies with specific antibodies against pllO willbe necessary to determine how tyrosinephosphorylated 120- and 110-kDa proteins are related to the pllO subunit of PI 3-kinase. 140- and 100-kDa phosphoproteins were also co-immunoprecipitated together with the p85 in the active PI 3-kinase after insulin treatment. The former was tyrosine-phosphorylated, but the 100-kDa protein was not. A protein of a size similar to the 140 kDa was immunoblotted with anti-IRS-1 antibody in an in vitro assay for the association of IRS-1 (from insulin-stimulated CHO-IR cells) with the p85 in the active PI 3-kinase immobilized on beads. This could be an IRS-1-related protein or a proteolytic fragment of IRS-1. However, the 140-kDa protein was only poorly detected in the immunoblots of p85 immunoprecipitates with anti-IRS-1 antibody from insulin-stimulated CHO-IR cells because longer exposure of the immunoblots caused high background around the 140-kDa protein. In conclusion, we report that insulin can induce an association of the p85 in PI 3-kinase with the tyrosine-phosphorylated IRS-1, not with the activated insulin receptor, throughout the tyrosine-phosphorylated YMXM sequence of IRS-1 and the SH-2 domains of p85. Although p85 in PI 3-kinase form is not tyrosine-phosphorylated in response to insulin, the PI 3-kinase form of p85 displayed an insulin-dependent association with other phosphoproteins ranging from 100 to 170 kDa, resulting in the formation of a molecular complex. The signal transduction pathway of the insulin receptor tyrosine kinase differs from other receptor tyrosine kinases, such as those for PDGF and colony-stimulating factor 1, as well as Kit (Kazlauskas and Cooper, 1990; Escobedo et al., 1991a; Kashishian et al., 1992; Fantl et al., 1992; Reedijk et al., 1992; Levet al., 1992), where p85 directly associates with receptor tyrosine kinases. The obvious questions to be answered are why signal transduction via the insulin receptor has thisunique system and what is the role of IRS-1 ininsulin signal transduction. Another question to be addressed is the mechanism of the increase of PI 3-phosphoinositide formation in insulin-treated cells (Ruderman et al., 1990). The cDNA cloning of pllO and the expression of the active protein would help to answer this question. Acknowledgments-We thank Drs. K. Kaibuchi, A. Kikuchi, and Y. Takai (the Department of Biochemistry, Kobe University School of Medicine) for support and useful discussions and Dr.T. Kadowaki (the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo) for critical readingof this manuscript and useful discussions. REFERENCES Au er, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) jell 67,167-175 Au er, K. R., Carpenter, C. L., Shoelson, S. E., Piwnica-Worms, H., and Zantley, L. C. (1992) J. Biol. Chem. 267,5408-5415 Becker, A. B., and Roth, R. A. (1990) Annu. Reu. Med. 41,99-115 Cantley, L. C., Au er, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and $oltoff, S. (1991) Cell 6 4 , 281-302 Carpenter, C. L., Duckworth, B. C., Au er, K. R.,.Cohen, B., Schaffiausen,B. S., and Cantley, L. C. (1990) J. Bwl. &ern. 266,19704-19711 Chavanieu, A., Naharisoa, H., Heitz, F., Calas, B., and Grigorescu, F. (1991) Bworg. Med. Chem. Lett. 1,299-302 Endemann, G.,Yonezawa, K., and Roth, R. A. (1990) J. Biol. Chem. 266,396-

Insulin-dependent Association Fantl, W. J., Escobedo, J. A., Martin, G.A., Turck, C.W., del Rosario, M., McCormic, F., and Williams, L. T. (1992) Cell 69,413-423 Glennev. R.. Jr.. Zokas., L.., and KamDs. _ M. . P. (1988) . . J. Imrnunol. Methods 109,"2771285'

Hu, P., Margolis, B., Skolnik, E. Y., Lammers, R., Ullrich, A,, and Schlessinger, J. (1992) Mol. Cell. Biol. 12, 981-990 Izumi, T., White, M. F., Kadowaki, T., Takaku, F., Akanuma, Y., and Kasuga, M. (1987) J. Biol. Chem. 262,1282-1287 Kadowaki, T., Koyasu, S., Nishida, E., Tobe, K., Izumi, T., Takaku, F., Sakai, H., Yahara, I., and Kasuga, M. (1987) J. Biol. Chern. 262,7324-7350 Kahn, C. R., and White, M. F. (1988) J. Clin. Inuest. 82, 1151-1156 Kashishian, A., Kazlauskas, A., and Cooper, J. (1992) EMBO J. 11,1373-1382 Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D., and Kahn, C. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,2137-2141 Kasuga, M., Izumi, T., Tobe,K., Shiba ,T., Momomura, K., Tashiro-Hashimoto, Y., and Kadowaki, T. (1990) Diabetes Care 13,317-326 Kazlauskas, A., and Cooper, J. A. (1990) EMBO J. 9,3279-3286 Kazlauskas, A., Kashishian, A., Cooper, J. A., and Valius, M.(1992) Mol. Cell. Bid. 12,2534-2544 King, D. S., Fields, C. G., and Fields, G. B. (1990) Int. J. Peptide Protein Res. 36,255-265

KliDDel. A,. Escobedo. J. A.. Fantl. W. J.. and Williams. L. T. (1992) . . Mol. Cell. Biol. 12,'1451-1459 ' Koch, C.A,, Anderson, D., Moran, M. F., Ellis, C., and Pawson, T. (1991) Science 262,668-674 Lev, S., Givol, D., and Yarden, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,

_"

6784372

McGlade, C. J., Ellis, C., Reedijk. M., Anderson, D., Mbamalu, G., Reith, A. D., Panayotou, G., End, P., Bernstein, A,, Kazlauskas, A,, Waterfield, M. D., and Pawson, T. (1992) Mol. Cell. Biol. 12,991-997 Morgan, D. O., and Roth, R. A. (1985) Endocrinology 116,1224-1226 Morgan, S. J., Smith, A. D., and Parker, P. J. (1990) Eur. J. Blochem. 191, 761-767

Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A,, Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 66, 91104

Reedijk, M., Liu, X., van der Geer, P., Letwin, K., Waterfield, M. D., Hunter, T., and Pawson, T. (1992) EMBO J. 11, 1365-1372

and of p85

IRS-1

25965

Roth, R. A., Cassell, D. J., Wong, K. Y., Maddux, B. A,, and Goldfine, I. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7312-7316 Rothenberg, P. L., Lane, W. S., Karasik, A., Backer, J., White, M., and Kahn, C. R. (1991) J. Biol. Chern. 266,8302-8311 Ruderman,,N., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sa. U. S. A. 87,1411-1415 Shibasaki, F., Homma, Y., and Takenawa, T.(1991) J. Biol. Chern. 266,81088114

Shoelson, S. E., Chatterjee, S., Chaudhuri, M., and White, M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,2027-2031 Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A,, and Schlessinger, J. (1991) Cell 66,83-90 Sun, X. J., Rothenberg, P.,, Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A,, Cahill, D. A,, Goldstem, B. J., and White, M. F. (1991) Nature 352, 7377 I ,

Tashiro-Hashimoto, Y., Tobe, K., Koshio, O., Izumi, T., Takaku, F., Akanuma, Y., and Kasuga, M. (1989) J. Biol. Chern. 264,6879-6885 Ullrich, A,, Bell, J. B., Chen, E. Y., Herrera, R., Petruzzelli, L. L. M., Dull, T. J. Gra , A,, Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A,, Seeburg, P.'H., Zrunfeld, C., Rosen, 0. M., and Ramachandran,J. (1985) Nature 313, 756-761

White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186 White M. F., Stegmann E. W., Dull, Y. J., Ullrich, A., and Kahn, C. R. (1987) J. B'iol. Chern. 262,9%39-9777 Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988) Nature 332,644-646 Yamanashi, Y., Fukui, Y., Won sasant, B., Kinoshita, Y., Ichimori, Y., Toyoshima, K., and Yamamoto, T. f1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11181122

Yonezawa, K., Pierce, S., Stover, C., Aggerbeck, M., Rutter, W. J., and Roth, R. A. (1991) in Molecular Biology and Physiology of Insulin and Insulin-like Growth Factors (Raizada, M., and LeRoith, D., eds) pp. 227-238, Plenum Press, New York Yonezawa, K., Yokono, K., Shii, K., Ogawa, W., Ando, A,, Hara, K., Baba, S., Kaburagi, Y., Yamamoto-Honda, R., Momomura, K., Kadowaki, T.,and Kasuga, M. (1992) J. Bid. Chem. 267,440-446 Yu, J.-C., Heidaran,M. A,, Pierce, J. H., Gutkind, J. S., Lombardi, D., Ruggiero, M., and Aaronson, S. A. (1991) Mol. Cell. Biol. 11, 3780-3785 Zardeneta, G., Chen, D. L., Weintraub, S. T., and Klebe, R. J. (1990) Anal. Biochern. 190,340-347