Insulin Stimulates Association of Insulin Receptor Substrate- 1 with the

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 268, No. 15, Issue of May 25, pp. 11167-11171,1993 Printed in U.S.A.

Q 1993 by The American Society for Bioehemistry and Molecular Biology, Inc.

Insulin StimulatesAssociation of Insulin Receptor Substrate-1with the Protein Abundant Src Homology/Growth Factor Receptor-bound Protein 2” (Received for publication, February 1, 1993)

Kazuyuki Tobe$, Koozi Matuokaj, HiroyukiTamernoto$, Kohjiro UekiST, Yasushi Kaburagilll, Shohji Asai$, Tetsuya NoguchillII,Michiyuki Matsuda**, Shinya Tanaka**, Seisuke Hattori$$, Yasuhisa Fukuist, Yasuo Akanuman,Yoshio Yazakil, Tadaomi Takenawaj, and Takashi KadowakiSllll From the $Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, the $Department of Biosignal Research, Tokyo Metropolitan Instituteof Gerontology, Itabashi-ku, Tokyo 173, Japan, the (Institute for Diabetes Care and Research, Asahi Life Foundation, Marunouchi, Chiyoda-ku, Tokyo 100, Japan, the **Division of Molecular Pathology, Department of Pathology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan, the $$Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187, Japan, and the $§Departmentof Biochemistry, Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

Insulinactivatesthe ras proto-oncogeneproduct p21” (Ras) by stimulating conversion of the inactive form. GDP-bound form of Ras to the active GTP-bound The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U.S. A. 89, 90159019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples atyrosine kinase toa Ras protein.We have studiedan interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (aASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosineantibodyand an anti-insulin receptor substrate 1 antibody (aIRS-1) from the insulin-stimulated [32P]orthophosphate-labeledCHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS- 1*ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulared CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated170-kDaproteinthatwas recognized by aIRS-1. We also demonstrated that fusion protein of ASHwas ableto bind thefusion protein of tyrosine-phosphorylated IRS-1fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1*ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phos-

* This work has been supported by Grant 190831from the Juvenile Diabetes FoundationInternational (to T. K.) and by agrant for diabetes research from the Ohtsuka Pharmaceutical Co., Ltd. (to T. K.). 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 this fact. 11 Present address: Second Dept. of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe-shi 650, Japan. (VTo whom all correspondence and reprint requests should be addressed Third Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-3815-2087.

phatidylinositol (PI) 3-kinase activity inaASH immunoprecipitates, suggesting the formation of an ASH*IRS-l*PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin’s signal multidirectionally.

One of the earliest cellular responses to stimulation by insulin is the activation of insulin receptor kinase and tyrosine phosphorylation of insulin receptor p subunit and pp185, a minor cytoplasmic phosphoprotein found in most cells and tissues (Kasuga et al., 1982,1983;White et al., 1985; Kadowaki et al., 1987). Recently, a cDNA for pp185was cloned and referred to as insulin receptor substrate-1 (IRS-1)’ (Sun et al., 1991). IRS-1 contains at least 10 potential tyrosine phosphorylation sites, nine of which are in theTyr-X-X-Met motifs, suggesting that IRS-1 binds signal transducing molecules containing Src homology 2 (SH2) domains. Indeed, tyrosine-phosphorylated IRS-1 binds the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase (p85) both in vitro (Lavan et al., 1992) and in intact cells (Backer et al., 1992; Yonezawa et al., 1992). So far, it seemed that other SH2-containing proteins such as phospholipase C-y, and the Ras GTPaseactivating protein bind poorly to the IRS-1. Considering the multidirectionality of insulin’s action, it has been postulated that several other SH2 proteins may bind to the tyrosinephosphorylated IRS-1 (Backer et al., 1992), thereby linking the insulin receptor kinase and enzymes regulating cellular growth and metabolism via several mechanisms including protein-protein interactions (Pawson et al., 1992; Cantley et al., 1991) and kinase cascades (Krebs et al., 1964; Sturgill et al., 1988 Dent et al., 1990; Ahn et al., 1992). The abbreviations used are: IRS-1, insulin receptor substrate-1; SH2, Src homology 2; PI, phosphatidylinositol; p85, phosphatidylinositol3-kinase 85-kDa subunit; Ras, p21’”; EGF, epidermal growth factor; EGFR, EGF receptor; ASH, abundant Src homology; GRB2, growth factor receptor-bound protein 2; SH3, Src homology 3; PAGE, polyacrylamide gel electrophoresis; aIRS-1, anti-insulinreceptor substrate-1 antibody; aPY, anti-phosphotyrosine antibody; ap85, antip85 antibody; aASH, anti-ASH antibody; GST, glutathione S-transferase; CHO-HIR cell(s), Chinese hamster ovary cell(s) overexpressing human insulin receptor; IRP, insulin receptor @ subunit.

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Association of IRS-1 with ASHIGRBB

Insulinactivatesthe ras proto-oncogene product,~21'"" @ASH antibodywas preadsorbed to glutathione S-transferase (GST) and failed to recognize the GST portionof the fusion protein. (Ras), by stimulating the conversion of the inactive GDPbound form of Ras to the active GTP-bound form (Burgering Western Blotting-Chinese hamster ovary cells overexpressinghuman insulin receptors3 (CHO-HIR cells) were usually used. Lysates et al., 1990), and thereby activates the downstream kinases of CHO-HIR cells treated with or without insulin were incubated (de Vries-Smitset al., 1992) such as mitogen-activated protein with aIRS-1, and the immunoprecipitates were subjected to SDSby using kinases and S6 kinase I1 (Sturgill et al., 1988; Tobe et al., PAGE followed by Western blotting with up85 and detection anti-mouse IgG alkaline phosphatase conjugate system (Sigma). 1992) or aninsulin-sensitiveprotein kinase. Thiskinase Immunoprecipitation from ~2P10rthophosphate-labeledCellscascade ispresumably involved inthe insulin's biological Serum-starved CHO-HIR cells were prelabeled with [32P]orthophosactions such as glycogen metabolism (Dent et al., 1990) and phate (1 mCi/ml) on a 60-mm plate in3 ml of phosphate-free RPMI mitogenesis. Thus, the current investigations have been fo- 1640 medium (Flow Laboratories, United Kingdom) (Kadowaki et al., cused on the search of the molecular link that couples the 1987; Tobe et al., 1991). Lysates of the cells incubated without or with lo-' M insulin for 1 min a t 37 "C were subjected to immunopreinsulin receptor kinase to Ras activation. were Recently, the cDNA of a mammalian counterpart of Cae- cipitation with control serum or aASH. The bound proteins eluted with 1%SDS, diluted to bring the SDS concentration to 0.1%, norhabditis elegans sem-5protein, which couples the EGF and immunoprecipitated with nPY. The nPY immunoprecipitates receptor (EGFR)-like let-23 protein to Ras-like let-60 protein were eluted with 20 mM p-nitrophenyl phosphate, and the eluates (Clark et al., 1992), has been cloned independently by two were subjected to SDS-PAGE or to the immunoprecipitation with groups utilizing different methods, and iscalled as ASH (for aIRS-1. Bacterial Fusion Proteins-Glutathione S-transferase fusion proabundant Src homology) (Matuoka et al., 1992) or GRB2 (for tein of ASH fragments were prepared as described (Matuoka et al., growth factor receptor-bound protein 2) (Lowenstein et al., 1992). Briefly, GST-ASH(A+B+C) contains amino acids 15 to -217 1992). The protein ASH/GRB2 is composed of one SH2 and of ASH, a part of N-terminal SH3 region (SH3(N))-SH2 region-Ctwo SH3 domains, and lacks any catalytic domains like the terminal SH3 region (SH3(C)), and GST-ASH(A) contains amino c-Crk and c-Nck. It hasbeen demonstrated that ASH/GRB2 acids 15 to -57 of ASH, a part of N-terminal SH3 region (SH3(N)). is associated with tyrosine-phosphorylated EGFR and plate- We have cloned acDNA of IRS-1 by screeningrat livercDNA libraries (Stratagene), which contains a t least 10 potential tyrosine let-derived growth factor receptors. Importantly, an expres- phosphorylation sites depicted in the present report asfollows: Tyr" sion of an antisense ASH cDNA led to a reduction in cell + Y1; Tyr4M)-.+ Y2; Tyr546+ Y3; Tyr6* + Y4; T y P R+ Y5; T y P R Y6; Tyr7*'+ Y7; Tyrg3' + Y8; Tyrg8' Y9; Tyr''"o +YIO. Regions growth (Matuoka et al., 1992), and comicroinjection of GRB2 proteintogether with H-rasproteinstimulatedthe DNA of IRS-1 cDNA corresponding to amino acids 3-67 (I) and 530-768 synthesis (Lowensteinet al., 1992), suggestingthat the protein (111) of IRS-1, respectively, were prepared as described el~ewhere.~ Tyrosine Phosphorylationof ZRS-1 by the InsulinReceptor KinaseASH/GRBZ/Sem-5 plays a role that couples the membrane- Fusionproteins of IRS-1 (I) and (111) coupledwith glutathionebound growth factor receptor tyrosine kinase toa Ras protein. Sepharose beads were incubated with insulin,Mn", [-y-32P]ATP,and In this study, we have examined the association of ASH/ wheat germ agglutinin fraction from CHO-HIR cells. The reactions GRBZ with insulin-stimulated tyrosine-phosphorylated pro- were stopped by adding buffer B. The fusion protein samples conteins. We have shown that insulin stimulates association of taining comparable amounts of phosphotyrosines were eluted with 100 mM glutathione (pH 7.4) in buffer B and incubated with GSTASH/GRB2 with tyrosine-phosphorylated IRS-1 both in in- ASH(A+B+C). This mixture was subjected to immunoprecipitation that IRS-1 tact cells and in uitro, suggestingapossibility with aASH followed by SDS-PAGE. couples the insulin receptor kinase to ASH/GRB2 through In Vitro Binding of GST-ASH Fusion Proteinsto Phosphotyrosineformation of IRS-1 .ASH/GRB2complex, thereby regulating containing Proteins-GST-ASH fusion proteins on glutathione-agathe Ras signaling pathway. We have also demonstrated that rose beads were incubatedwith cell lysates from CHO-HIR cells (prelabeled or notwith [32P]~rthopho~phate) treated or untreated IRS-1, which bindsASHIGRB2,isassociated with P I 3- with insulin. The fusion proteinsonthe beads were washed and kinaseactivity.This is thefirstreportthatsupportsthe subjectedto: (i) elution with 100 mM glutathione followed by the notion that IRS-1 bindsseveral signal transducing molecules immunoprecipitation with aPY, (ii) SDS-PAGE followed by Western PI 3-kinase activity assay by the method containing SH2 domains, thereby serving as an SH2 docking blotting with nPY, (iii) protein, which mediatesmultidirectionalsignals from the previously described (Fukui etal., 1989). insulin receptor. --$

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RESULTS

EXPERIMENTALPROCEDURES

Materials-PI(bovineliver) was purchasedfromAvantiPolar Lipids, Inc. Protein A-Sepharose, pGEX-PT, and glutathione-Sepharose beads were from Pharmacia LKB Biotechnology Inc. [-y-"PI ATP (6,000 Ci/mmol) was from Du Pont-New EnglandNuclear, lZ51protein A (30 mCi/mg) was fromAmersham Corp. (Buckingham, United Kingdom), and [32P]orthophosphate (285 Ci/mg)was obtained fromICN Biomedicals (Irvine, CA). Prestained molecular weight markers for sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) was from Bio-Rad. Porcine insulin was a gift from Eli Lilly Co. (Indianapolis, IN). Buffers-Buffer A consisted of 25 mM Tris-HC1 (pH 7.41, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, and 1 mM EDTA. Buffer B consisted of buffer A + 1% Nonidet P-40. Antibodies-An antiserum directed to IRS-1 peptide YIPGATMGTSPALTGDE (B-56; correspondingto residues 489-505; Sun et al. (1991)) was preparedand was used asananti-insulin receptor substrate-1 antibody (aIRS-1).An anti-phosphotyrosine antibody (aPY) was prepared as described (Tobe et al., 1990). Another aPY, PY20,from ICN Biomedicals was alsoused in some experiments. Monoclonal antibodiesagainsthuman ~8501,mainly AB6, were as described (Tanaka et al., 1993). A rabbit polyclonal antibody against ASH (aASH) was raised by a conventional method.' This K. Matuoka andT. Takenawa, submitted for publication.

It hasbeen reported that insulin stimulates a stable complex formation of IRS-1 with p85 in intact cells (Backer et al., 1992; Yonezawa et al., 1992). Westudiedthe kinetics of insulin-stimulated association of IRS-1 with p85. CHO-HIR M insulinfor 10 s, 30 s, 1 min, 5 min, cells treatedwith and 10 min were lysed and immunoprecipitatedwith aIRS-1, and the immunoprecipitateswere subjected to Western blotting with ap85. IRS-1 interacted with p85 as early as 10 s after insulin stimulation (Fig. 1A). The amount of p85 in the GIRS-1 immunoprecipitates, the tyrosine phosphorylation of IRS-1, and thePI 3-kinase activityin aIRS-1 immunoprecipitates were alldetectedwithin 10 s, andthese reacheda maximal level within 1 min after exposure of cells to insulin and remained at an elevated level for at least 30 min (Fig.

1B). Next, we examined the interaction of another SH2-containing protein, ASH/GRB2, with insulin-stimulated tyrosinephosphorylated proteins. [32P]Orthophosphate-labeledCHOHIR cells were immunoprecipitated with control serum (Fig. 2 A , lanes a and b ) or aASH (Fig. 2 4 , lanes c-f). This antibody The sequence of the human insulin receptorusedhere sponds to thatof Ebina etal. (1985). K. Tobe and T. Kadowaki, manuscript in preparation.

corre-

Association of IRS-1 with ASHIGRBB

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eluates from the aASH aPY immunoprecipitates by p nitrophenyl phosphate were subjected to immunoprecipitation with control serum or aIRS-1 (Fig. 2 A , lanes e and f ) . The 170-kDa phosphoprotein was immunoprecipitated with 106+ aIRS-1 and thereforewas identified as IRS-1. p85 T o study the association of ASH with insulin-stimulated 80 + tyrosine-phosphorylated proteins, lysates of [:'ZP]orthophosphate-labeled CHO-HIR cells treated or untreated with in_.. -.:.:a. sulin were incubated withASHfragments. Theproteins bound tofusion proteins were eluted with 100mM glutathione 49.5+ . .. and were immunoprecipitated with aPY, aIRS-1, or control Insulin (-) 10 30 1 5 10 serum. We observed no significant phosphoproteins associsec sec min min min ated with the GST-ASH(A)in aPY immunoprecipitates (Fig. 2B, lanes a and b). Upon insulin stimulation of the cells, we observed that a 170-kDa phosphoprotein was associated with the GST-ASH(A+B+C)in aPY immunoprecipitates (Fig. 2B, lanes c and d ) . We did not detect a 95-kDa phosphoprotein, suggesting that IRP was not associated with GSTASH(A+B+C). We also observed that GST-ASH(A+B+C) was associatedwith an aIRS-1-precipitable 170-kDaphosP L - 0 phoprotein from the insulin-stimulatedcells (Fig. 2C, lanes a and b). These data suggest that GST-ASH(A+B+C) is able L o to bind tyrosine-phosphorylated IRS-1. For further confirmation,lysates of CHO-HIR cells treated without or with insulin were incubated with GST-ASH(A) (Fig. 2 0 , lanes a and b ) or GST-ASH(A+B+C) (Fig. 2 0 , lunes c and d ) , and the bound proteins were subjected to SDSPAGE followed by immunoblotting with aPY. We observed a 170-kDa band associated with GST-ASH(A+B+C) in insulin-stimulated cells (Fig. 2 0 , lane d ) . We did not detect a 95-kDa tyrosine-phosphorylated protein. Re-immunoblotting of themembrane with aIRS-1 showed thatthe 170-kDa J I I ? protein reacted with aIRS-1 and was thus identified as IRS1 5 10 1 (Fig. 2 0 , lunes e a n d f ) . A fusion protein GST-IRS-1 (I) spans amino acids 3-67 of El IRS-1 containing EY46Y47E sequence. Another fusion protein GST-IRS-l(II1) spans amino acids 530 to 768 containing p 0 0 Tyr54ti, TyrlXN, TYrR'2H, TyrIi.78, and Tyr;"' whose tyrosines were -0 all in the YXXM or YMXM motif. We studied whether GSTASH was able to bind tyrosine-phosphorylated GST-IRS-1 fusion proteins in uitro. We observed that a phosphorylated fusion protein GST-IRS-1 (111) was associated with GSTASH(A+B+C) (Fig. 2E, lane d ) , suggesting thatGSTFIG. 1. Insulin stimulatesassociation of IRS-1 with p85 as ASH(A+B+C) has the capability of direct binding of tyrosineearly as 10 seconds. A, CHO-HIR cells treated with insulin for the phosphorylated IRS-1. indicated times were immunoprecipitated with aIRS-I, and the imIt has been shown that tyrosine-phosphorylated IRS-1 is munoprecipitates were subjected to Western blotting with ap85. B, PI 3-kinaseactivity(Backer et al., 1992; time course of the amount of p85 associated with IRS-1 (panel I ), associatedwith tyrosine phosphorylation of IRS-1 (panel Z), and the PI 3-kinase Yonezawa et al., 1992). We also observed an insulin-stimuactivity in aIRS-1 immunoprecipitates (panel 3 ) after insulin stim- lated increase in PI 3-kinase activity in d R S - 1 immunopreulation. Activitieswere measured by densitometric scanning. cipitates (Fig. 3A, lanes c and d ) . To examine whether ASHbound IRS-1 isassociated with PI 3-kinase activity in intact (aASH) detecteda 28-kDa protein in the aASH immunopre- cells, the aASH immunoprecipitates were subjected to PI 3cipitates by immunoblotting (data not shown). The proteins kinase assay. We observed an increase in PI 3-kinase activity in the immunoprecipitates were eluted and immunoprecipi- in aASH immunoprecipitates after insulin stimulation (Fig. tated with aPY followed by SDS-PAGE (Fig. 2 A , lanes a-d). 3A, lanes aand b ) ,suggesting the presence of IRS-1 molecules We observed no clear bands either in control serum -+ aPY that associate with both ASH and PI 3-kinase in intact cells. immunoprecipitates (Fig. 2 A , lunes a and b ) or in aASH + The increase in PI 3-kinase activity in aASH immunoprecipitates was detected as early as 10 s after insulin stimulation aPY immunoprecipitates from the insulin-untreated CHOHIR cells (Fig. 2 A , lane c ) . We observed a 170-kDa phospho- in intact cells(data not shown). We were not able to detect protein in aASH aPY immunoprecipitates from the insu- the insulin-stimulated PI 3-kinase activity in aASH immulin-stimulated ['"P]orthophosphate-labeled CHO-HIR cells noprecipitates when 12.5 PM phosphopeptide D'"PNGYMM(Fig. 2 4 , lane d ) . We did not observe a 95-kDa phosphopro- MSPSG (containing thephosphotyrosine in theYMXM motein, suggesting that p subunit of the insulin receptor (IRP) tif (Yonezawa et al., 1992)) were included in the lysates for was not in the immunoprecipitates.We didnot detect tyrosine immunoprecipitation, suggesting that IRS-1. ASHbinding phosphorylation of ASH or tyrosine phosphorylation of 46-, may be mediated via phosphotyrosine(s) in the YMXM motif 52-, and66-kDaproteins, suggesting thattyrosine-phoson the IRS-1molecules. However, the affinity of IRS-1 .ASH phorylated shc gene product (Shc) may not be in the aASH binding appears to be lower than that of IRS-laPI 3-kinase immunoprecipitates (Rozakis-Adcock et al., 1992). The binding, since we did not observe detectable decrease in PI 3205 +

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FIG.2. Insulin stimulates association of IRS-1 w i t h ASHIGRBP. A, [:'YP]orthophosphate-labeled CHO-HIR cells treated without (lanes a and c ) or with (lanes b, d, e, and f ) insulin for 1 min were lysed and immunoprecipitated with control serum (lanes aand b ) or aASH (lanes c-f). The eluted proteins in the nASH immunoprecipitates were eluted with 1% SDS. After the dilution of SDS to 0.196, the eluates were subjected to the immunoprecipitationwith tvPY followed by the elution by 20 mM p-nitrophenyl phosphate. The eluates were subjected either to SDS-PAGE(lanes a-d) or to the immunoprecipitationwith control serum (lane e)or trIRS-1 (lane f). B and C, [:"P]orthophosphatelabeled CHO-HIR cells treated without ( B , lanes a and c; C, lane a ) or with ( R , lanes b and d; C, lanes b and c) insulin were lysed, and incubated with GST-ASH(A) ( B , lanes a and b ) or GST-ASH(A+B+C) ( B , lanes c and d; C, lanes a-c) on glutathione-agarose. The bound proteins were eluted with 100 mM glutathione, and the eluates were subjected to the immunoprecipitation with nPY ( R , lanes a-d), nIRS-1 (C, lanes a and b ) , and control serum (C, lane c ) . The immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. D, CHO-HIR cells treated without (lanes a and c) or with (lanes b and d ) insulin were lysed and incubated with GST-ASH(A) (lanes a and b ) or GST-ASH(A+B+C) (lanes c and d ) on glutathione-agarose. The bound proteins were subjected to SDS-PAGE followed by Western blotting with nPY. The bound antibody was detected with '"I-protein A followed by autoradiography. After washing the nitrocellulose sheet, with the solution containing 0.1 M glycine-HCI (pH 2.5), 4 M NaCl for 5 min twice, the sheet was subjected to Western blotting with alRS1. The bound antibody was detected with anti-rabbit IgG alkaline phosphatase conjugate system (lanes eand f). We observed a faint band at 55 kDa in SDS-PAGE, which probably represents non-specifically recognized band by nPY, since it reacted with aASH and was identified as GST-ASH(A+B+C). E, GST-IRS-1 (I) (lanes a and b ) and GST-IRS-1 (111) (lanes c and d ) on glutathione-agarose beads were incubated without (lanes a and c ) or with (lanes b and d ) insulin receptor kinase in the presence of insulin, Mn", and [y:"P]ATP. After equalizing the phosphorylation, phosphorylated fusion proteins were eluted with 100 m M glutathione. The eluates were incubated with GST-ASH(A+B+C), and the mixturewas subjected to the immunoprecipitationwith nASH. In this case, nASH was preincubated with GST on agarose and this aASH was not able to recognize the GST portionof each fusion protein, kinase activity in cuIRS-1 immunoprecipitates under the same kinases andS6 kinase 11, which appear toregulate mitogenesis conditions (data not shown). To further confirm the ASH. and glycogen metabolism. The molecular link that couples IRS-1. PI 3-kinase complex formation, lysates of CHO-HIR the insulin receptorkinase to Ras activation has been incells withoutor with insulin were incubated with GST- tensely investigated, but little has been revealed. IRS-1 has ASH(A), or GST-ASH(A+B+C) on glutathione-agarose been thought to play a crucial role in many, if not all, of beads, and the bound proteins were subjected to PI 3-kinase insulin's intracellular events initiatedby the insulin receptor activity. We observed a marked increase in PI 3-kinase activ- kinase. ity associatedwith the GST-ASH(A+B+C) that had been ASH/GRB2 is amammalian counterpart of s e m d gene incubated with the lysates from the insulin-stimulated cells product in C. elegans that couples EGFR-like let-23 tyrosine (Fig. 3B, lanes c and d ) . kinase to Ras-like let-60protein in the pathway that regulates DISCUSSION vulval development (Clark et al., 1992). Recently,ASHIGRBB has been demonstrated tolink the activatedreceptor tyrosine Insulinactivates a Rasprotein,therebyactivatingthe downstream kinases such as mitogen-activated protein kinase (EGFR, platelet-derived growth factor receptor) (Low-

Association of IRS-1 withASHIGRB:!

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support thenotion that IRS-1 binds several SH2 proteins and forms a signaling complex, thereby transducing the insulin's signal multidirectionally. It seems that insulin's action depends on the amount and the nature of the IRS-l-bound SH2 proteins that regulate the downstream targets through other domains such as catalytic domain(s) or SH3 domain(s). It is noteworthy that in v-src or v-fps-transformed cells or EGF-stimulated cells, Shc, an SH2 protein and ofone tyrosine kinase substrates, seemed to couple the tyrosine kinase to ASH/GRB2 through theformation of Shc.ASH/GRB2 complex (Rozakis-Adcock et al., 1992). However, when CHO-HIR cells were stimulated with insulin, we did not detecttyrosinephosphorylated 46-, 52-, and 66-kDa proteins in aASH immunoprecipitates. Furthermore, tyrosine-phosphorylated GST-IRS-1 fusion protein can bind GST-ASH(A+B+C) in vitro directly. These data suggest that IRS-1 directly associates with ASH/GRB2, probably not via tyrosine-phosphorylated Shc. Acknowledgments-We thank Dr. Masato Kasuga for helpful discussions and critical reading of this manuscript. We also thankProf. Yamamoto of Institute for Medical Science Attached to TokyoUniversity andpersonnel of the radioisotope center for support.We thank Dr. Kinori Kosaka, Dr. Ryoko Hagura, Director Hajime Kawashima, and Dr. Osamu Koshio of the Institute for Diabetes Care and Research, Asahi Life Foundation. We also thank Dr. Ritsuko Yamamoto-Honda and Dr. MakotoYachi for support.

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FIG. 3. Insulin stimulates PI 3-kinase activity in aASH immunoprecipitates. A, CHO-HIR cells treated without (lanes a and c) or with (lanes b and d ) insulin were subjected to immunoprecipitation with aASH (lanes a and b ) o r d R S - 1 (lanes c and d ) . The immunoprecipitates were washed and subjected to PI3-kinase assay. B, CHO-HIR cells treated without (lanes a and c) or with (lanes b and d ) insulin were lysed and incubated with GST-ASH(A) (lanes a and b ) or GST-ASH(A+B+C) (lanes c and d ) on glutathione-agarose beads or aPY (lanes e and f ) followed by the addition of protein ASepharose. The beads were washed and subjected toPI3-kinase activity.

Sun, X. ;i.,.kothenherg, P., Kahn, C. R., Backer, d . M., Araki, E., Wilden, P. A;, Cahill, D. A,, Goldstein, H.-1.. and White, M.F. (1991) Nature 352, 73, I

Tanaka. S..Matsuda, M., Nagata. S., Shizawa, Y., and Fukui, Y. (1993) Jap. J. Cancer Res., in press Tohe, K.. Koshio, 0.. Tashiro-Hashimoto, Y., Takaku, F.. Akanuma, Y., and Kasuga. M. (1990) Diabetes 39,528-533 enstein et al., 1992; Matuoka et al., 1992) or their substrates Tohe, K., Kadowaki, T., Tamemoto, H., Ueki, K., Hara, K., Koshio, 0.. (Shc) (Rozakis-Adcock et al., 1992) totheRas signaling Momomura, K., Gotoh, Y., Nishida, E., Akanurna, Y., Yazaki, Y., and Kasuga M. (1991) J. Hiol. Chem. 266,24793-24803 process in mammalian cells. In the present study, we have Tohe, K.. Kadowaki, T., Hara, K., Gotoh, Y., Kosako, H., Matsuda, S., Tarnemoto, H., Ueki, K., Akanuma. Y., Nishida, E., and Yazaki, Y. (1992) J . Riol. observed that insulin rapidly stimulates association of ASH/ ('hem. 267,21089-21097 GRB2 with IRS-1 but not with IRP, suggesting that ASH/ White, M. F., Maron, K.,and Kahn, C. R. (1985) Nature 318, 183-186 GRB2. IRS-1complex may be the molecular link that couples Wood, K. W . , Sarnecki, C., Roberts, T. M.. and Hlenis,J. (1992) Cell 68,10411050 the insulin receptor kinase to Ras activation. In this report, Yonezawa, K. Ueda, H., Hara, K., Nishida, K., Ando, A,, Chavanieu, A., Matsuha, H., Shii, K., Yokono, K., Fukui, Y.. Calas, B., Grigorescu, F., we have also shownthat insulin stimulates ASHIGRB2. IRS- Dhand, R., Gout, I., Otsu, M., Waterfield, M. D., and Kasuga. M. (1992) J . Hiol. C'hem. 2 6 7 , 25958-25966 1.PI 3-kinasecomplex formation in intact cells. The findings