THE JOURNAL OF Vol. 268, No. 8, of March pp. The for and ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Vol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 8, Issue of March 15, pp. 574&5753,1993 Printed in U.S.A.

Insulin-induced Phosphorylation of the 46- and 52-kDa Shc Proteins*

(Received for publication, July 13, 1992)

Gijsbertus J. Prank$, Jane McGlade#ll, Guiliana PelicciII , Tony Pawson#**, and Johannes L. Bos$ $$ From the $Laboratory for Physiological Chemistry, University of Utrecht, Vondellaan 24A, 3521 GG Utrecht, The Netherlands, the §Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1x5. Canada. and the lllnstituto de Clinica Medica I, Policlinico Monteluce, University of Perugia, 06100 Perugia, Italy

The products of the she gene appear to be substrates for activated oncogenic tyrosine kinases, such as v-Src and v-Fps and activated tyrosine kinase receptors like the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors. We investigated whether the Shc proteins are targets for the activated insulin receptor tyrosine kinase. Here we show that the 46- and 52-kDa Shc proteins are rapidly phosphorylated upon insulin receptor activation in fibroblasts expressing elevated levels of human insulin receptors. Furthermore, we observed insulin-induced association of a 23-kDa protein with the Shc proteins. These effects on Shc proteins are similar to those observed after EGF and PDGF treatment. In contrast to the observed Shc-EGF receptor association, we did not detect association between the Shc proteins and the insulin receptor. We conclude that the Shc proteins are common elements in a signal transduction pathway that is shared by EGF, PDGF, and insulin.

Src homology 2 (SH2)' domains are elements of about 100 amino acids that appear to be involved in protein-protein interactions through high affinity binding to certain tyrosine- phosphorylated sequences (Anderson et al., 1990; Koch et al., 1991; Matsuda et al., 1990; Mayer and Hanafusa, 1990; McGlade et al., 1992a; Moran et al., 1990; Reedijk et al., 1992; Sadowski et al., 1986). In the case of the p6OC-"" kinase, the SH2 domains may participate in an intramolecular interac- tion, regulating its kinase activity (Matsuda et al., 1990), and can associate in trans with a number of phosphotyrosine- containing proteins, including the activated PDGF receptor (Anderson et al., 1990; Koch et al., 1992; Kypta et al., 1990). In the last years a number of proteins containing SH2 domains were identified. Examples of these proteins are: the p85 subunit of phosphatidylinositol (PI) 3"kinase (Escobedo et al., 1991; Otsu et al., 1991; Skolnik et al., 1991), the p21" GTPase activating protein (p120GAP) (Trahey et al., 1988; Vogel et al., 1988), and phospholipase C r (Stahl et al., 1988; Suh et al., 1988a; Suh et al., 198813). These proteins are

* This work was supported by grants from the Dutch Cancer Society and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adver- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

V A fellow of the National Cancer Institute of Canada (NCIC). ** A Terry Fox Cancer Research Scientist of the NCIC. $$To whom correspondence should be addressed. Tel.: 31-30-

880521; Fax: 31-30-888443. The abbreviations used are: SH2, Src homology 2; PI, phospha-

tidylinositol; PY, phosphotyrosine; IRS-1, insulin receptor substrate 1; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).

involved in intracellular signal transduction and interact with specific phosphorylated tyrosine residues of activated growth factor receptor tyrosine kinases and other phosphoproteins (Brott et al., 1991; Ellis et al., 1991; Ellis et al., 1990; Kaplan et al., 1990; Kazlauskas et al., 1990; McGlade et al., 1992a; Molloy et al., 1989; Pronk et al., 1992). The interactions between SH2 domains and sequences containing phosphotyrosine residues appear to be rather specific. The two SH2 domains of the PI 3"kinase p85 subunit interact with two distinct phosphotyrosine-containing sequences in the activated PDGF receptor (Kashishian et al., 1992), and a single site in the CSF-1 receptor (Reedijk et al., 1992). Also, one of the GAP-SH2 domains displayed high affinity binding to one specific phosphorylated tyrosine residue out of 29 potential PDGF receptor autophosphorylation sites tested (Fantl et al., 1992). Binding of SH2-containing proteins to activated tyrosine kinases often coincides with tyrosine phosphorylation of the respective protein (Cantley et al., 1991; Koch et al., 1991). For instance, coinciding with binding to the respective tyrosine kinase, the p85 subunit of PI 3"kinase and p120GAP are tyrosine-phosphorylated upon PDGF receptor activation and in cells transformed by oncogenic tyrosine kinases such as p60""" (Brott et al., 1991; Ellis et al., 1990; Kaplan et al., 1990; Molloy et al., 1989; Pronk et al., 1992).

Recently, a new SH2 domain containing protein was identified (Pelicci et al., 1992). The cDNA encoding this protein was cloned on the basis of its homology with SH2 sequences from the human c-fes gene. Besides strong homology to m2 domain sequences the cloned gene had stretches of homology to a glycine/proline-rich region of the a1-collagen gene, and for this reason the gene was called shc. The shc gene codes for three mRNAs of different size that translate into three protein products of about 46, 52, and 66 kDa (p4ek, ~ 5 2 " ~ and ~ 6 6 " ~ ) . ~ 4 6 " ~ and p5Tk, which are ubiquitously ex- pressed, are both encoded by a 3.4-kb mRNA. ~ 6 6 " ~ is ex- pressed from a distinct transcript, and is absent in some hematopoietic cells. In a transfection assay, overexpression of the shc gene causes transformation of NIH3T3 cells, and upon EGF receptor activation the Shc proteins are tyrosine-phosphorylated and associate with the EGF receptor (Pelicci et al., 1992). Furthermore, it was shown that in cells containing temperature-sensitive v-src and v-fps mutants, the Shc proteins are also highly tyrosine-phosphorylated upon activation of the v-Src and v-Fps tyrosine kinases, and, upon induction of Shc tyrosine phosphorylation, a 23-kDa protein coimmu- noprecipitated with the Shc proteins (McGlade et al., 1992b).

Here we report that, upon insulin treatment of fibroblasts expressing elevated levels of the human insulin receptor, ~ 4 6 " ~ and ~ 5 2 " ~ are rapidly phosphorylated on tyrosine and serine residues. In addition, a 23-kDa protein coprecipitates with the Shc proteins after insulin treatment.

5748

Shc Proteins Are Phosphorylated Upon Insulin Receptor Activation 5749

MATERIALS AND METHODS

Cell Culture-A14 is an NIH3T3-derived cell line expressing 7 X lo6 human insulin receptors per cell (Burgering et al., 1991). CH09 is a Chinese hamster ovary cell line expressing 5 X lo3 endogenous insulin receptors, and CHO-IR800 (CH014) is a CHO9-derived cell line expressing 1.6 X 10' human insulin receptors per cell (Osterop et al., 1992). Cells were cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO). For insulin treatment, subconfluent cultures of A14 cells were put on Dulbecco's modified Eagle's medium/0.5% fetal calf serum for 18 h to induce quiescence, after which insulin (5 pg/ml) was added for 5 min. In vivo phosphorylation experiments were performed by incu- bating quiescent A14 cells with 0.5 mCi/ml 32Pi in phosphate-free medium for 4 h, followed by insulin treatment where indicated.

Immunoprecipitations and Immunoblotting-After treatment, cells were washed twice with ice-cold phosphate-buffered saline/l mM sodium orthovanadate, and after lysis in RIPA (30 mM Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Nonidet P-40, 0.5% sodium deox- ycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 0.1 p~ aprotinin, 1 p~ leupeptin, 1 mM sodium orthovanadate) the cell lysates were cleared by centrifugation. Shc immunoprecipitations were performed with a polyclonal rabbit antiserum, raised against a GST-Shc fusion protein as described earlier (McGlade et al., 1992b; Pelicci et al., 1992). Immunoprecipitated proteins were separated by SDS-PAGE, and transferred to nitrocellulose by electroblotting. Phosphotyrosine (PY) immunoblots were performed with a monoclonal antibody against PY (PY20, ICN), and subsequent detection by incubation with goat-anti-mouse IgG linked to horseradish per- oxidase followed by enhanced chemoluminescence (Amersham). Im- munoprecipitations were performed on equal amounts of protein for the phosphotyrosine immunoblots. In in uiuo labeling experiments the incorporation of radioactivity in trichloroacetic acid-precipitable material was determined, and portions of the lysates containing equal amounts of radioactivity were subjected to immunoprecipitation.

Phosphoamino Acid Analysis-For phosphoamino acid analysis, labeled phosphoproteins were immunoprecipitated, separated by SDS-PAGE, and electroblotted to Immobilon-P (Millipore) as described. After autoradiography, proteins of interest were excised from the blot and hydrolyzed to single amino acids in 200 pl of 6 N HC1 at 110 "C for 1 h. The amino acid mix was lyophilized, and subsequently phosphoamino acids were resolved by two-dimensional electrophoresis a t pH 1.9 and 3.5, respectively (Boyle et al., 1991; Pronk et al., 1992). The thin-layer cellulose plates were exposed to x-ray film, and the amount of incorporated radioactivity in each phosphoamino acid was determined by liquid scintillation counting of excised spots.

In Vitro Kinase Assay-Immunoprecipitations were performed as described above except for a minor change in the washing procedure. The immunoprecipitates were washed with RIPA (4 X) and kinase buffer (30 mM Tris-HC1, pH 7.5, 5 mM M&l2, 5 mM MnC12, 1 mM phenylmethylsulfonyl fluoride, 0.1 p~ aprotinin, and 1 p~ leupeptin) (2 X). Kinase reactions were performed in 20 pl of kinase buffer containing 10 pCi of [y3'P]ATP (5000 Ci/mmol, Amersham) and incubated for 15 min at 20 "C. Reactions were stopped by the addition of ice-cold RIPA, washing of the beads and boiling in sample buffer (10% glycerol, 60 mM Tris-HC1, pH 6.8, 2% SDS, 0.3 M P-mercapto- ethanol) for 5 min, after which the proteins were separated by SDS- PAGE and electroblotted to nitrocellulose.

Tryptic Phosphopeptide Analysis-Immunoprecipitations and in uitro kinase reactions were performed as described above. Proteins were separated by SDS-PAGE and electroblotted to nitrocellulose. After exposing the blot to x-ray film, bands of interest were excised for tryptic peptide analysis. Tryptic peptide analysis was performed as described earlier (Boyle et al., 1991; Pronk et al., 1992).

RESULTS

To investigate whether the Shc proteins become phosphorylated upon insulin treatment, insulin receptor overexpressing NIH3T3 cells (A14 cells) were treated with insulin as indicated and Shc proteins were immunoprecipitated from the cell lysates with a polyclonal Shc antiserum. The Shc immunoprecipitates were analyzed for the presence of phosphotyrosine by immunoblotting with a monoclonal antibody against phosphotyrosine. Clearly, p52", and to a lesser extent p4ek, are rapidly tyrosine phosphorylated upon insulin treatment (Fig. lA, lanes 4 and 6). The same was observed for a

A Insulin: - 30" 5 ' 5 ' ""

IgG heavy chainl 1 b 5 2 k D a 46kDa

L 1 A14 cells

B

"- - Insulin PDGF

r 1 2 3 4 5 6 I

FIG. 1. Shc proteins are tyrosine phosphorylated upon insulin receptor activation. A, phosphotyrosine immunoblot of Shc immunoprecipitates after insulin treatment of A14 and CH014 cells; A14 and CH014 cells were treated with 5 pg/ml insulin for the times indicated. After treatment, cells were lysed, and Shc proteins were precipitated with a Shc antiserum (lanes 2, 4, 6, and 8). Control immunoprecipitations were performed with a nonimmune rabbit serum (lanes I, 3,5, and 7). Precipitated proteins were separated by SDS-PAGE, and transferred to nitrocellulose. Phosphotyrosine levels were analyzed by immunoblotting with antiphosphotyrosine (PY20). ~ 4 6 ' ~ and ~ 5 2 ' ~ ' are indicated by arrows. Note that the migration of the Shc proteins is somewhat distorted by the IgG heavy chains of the Shc antiserum. B, phosphotyrosine immunoblot of Shc immunoprecipitates from growth factor-stimulated NIH3T3 cells; NIH3T3 cells were stimulated with insulin (5 pg/ml) or PDGF (25 ng/ml), and Shc proteins were immunoprecipitated from the cleared lysates (lanes 2, 4, and 6). Nonimmune rabbit serum was used as a control in parallel precipitations (lanes 1,3, and 5). Precipitated proteins were analyzed for phosphotyrosine levels as described. Shc proteins are indicated by arrows.

CH014 cHo9 1 2 3 4 5 6

52kDa 46kDa

Vanadate: - - - - + + Insulin: - + - + - +

FIG. 2. ''P in vivo labeling of Shc proteins in CHO cells. CH09 and CH014 cells were labeled in uiuo for 4 h with 32Pi, and treated with 5 pg/ml insulin for 5 min where indicated (lanes 1-4). CH09 cells were also pretreated with sodium orthovanadate (1 mM) before treatment with insulin where indicated (lanes 5 and 6). Im- munoprecipitations were performed with a Shc antiserum as described. Shc proteins are indicated by arrows. Note that the exposure time of the right panel (CHO9) is 10 times longer than that of the left panel (CH014).

Chinese hamster ovary cell line expressing elevated levels of the insulin receptor, CH014 (Fig. lA, lanes 7 and 8). We did not observe an effect of insulin treatment on Shc tyrosine phosphorylation in NIH3T3 cells, but we did observe PDGF- induced tyrosine phosphorylation of p4ek and ~ 5 2 ' ~ in these cells (Fig. 1B). The fact that we do not observe an effect of insulin treatment in NIH3T3 cells can be explained by the relatively low level of insulin receptors in this cell type. Therefore we used CH09 cells, the parental cell line of the insulin receptor overexpressing CH014 cells, that has about 5000 insulin receptors per cell. Both the CH09 and CH014 cells were labeled in vivo with 32Pi, and after a 5-min treatment with insulin Shc proteins were immunopurified. Clearly, the

5750 Shc Proteins Are Phosphorylated Upon Insulin Receptor Activation A " 1

I/ Ins: - 30" 1 ' 2 ' 5' 15'

52kDa

, I I I

n 5 IO 15

time of insulin treatment (min)

B

'-1

2oooo-

Ins: - 30" 1 ' 2 ' 5' 15'

&P 0 5 In 1s

time of insulin treatment (min)

C

- electrophoresis at pH 1.9 + FIG. 3. Time course and phosphoamino acid analysis of in-

sulin-induced Shc phosphorylation. A , time course of insulin- induced Shc phosphorylation; A14 cells were labeled with 32Pi and treated with insulin for various periods of time. Shc proteins were immunoprecipitated and analyzed by SDS-PAGE followed by electroblotting to Immobilon-P and subsequent autoradiography. ~ 5 2 " ~ ' was excised from the blot, and "P incorporation was determined by liquid scintillation counting. Shown are the area of the blot where the Shc proteins are located (insert) and a graphic representation of the amount of incorporated radioactivity. B, time course of insulin- induced receptor autophosphorylation; A14 cells were treated as

Shc proteins are phosphorylated in CH014 cells upon insulin treatment (Fig. 2, lunes 1 and 2, 10-fold increase in incorporated radioactivity). In CH09 cells this effect is less dramatic, but there is a significant increase in p5Yk phosphorylation detectable (Fig. 2, lanes 3 and 4, 1.8-fold increase in incorporated radioactivity, mean of two experiments). Furthermore, when CH09 cells were pretreated with the tyrosine phospha- tase inhibitor sodium orthovanadate, the insulin-induced tyrosine phosphorylation of the Shc proteins was further aug- mented (Fig. 2, lunes 5 and 6 ) . These results indicate that also in cells with low levels of insulin receptors, insulin treatment can induce the phosphorylation of Shc.

To estimate the rate of insulin-induced Shc phosphorylation we performed a time course of insulin treatment after in vivo labeling of A14 cells with 32Pi. As shown in Fig. 3A, after 30 s of insulin treatment there is already a substantial increase in the phosphorylation of ~ 5 2 ' ~ . The rate of phosphorylation seems to slow down after the first 30 s. A similar, rapid phosphorylation was observed for ~46'~', and the insulin receptor P-chain, which was used as a control for insulin receptor activation (Fig. 3B) .

To determine the phosphoamino acid composition of the Shc proteins, the ~ 4 6 " ~ ' and ~ 5 2 " ~ ' phosphoproteins were excised from the Immobilon-P blot (see Fig. 3A, insert), and hydrolyzed to single amino acids as described. Phosphoamino acids were separated by two-dimensional electrophoresis and "P incorporation was determined. Upon insulin treatment ~ 5 2 " ~ ' is clearly phosphorylated on tyrosine as well as serine residues (Fig. 3C). The phosphorylation on tyrosine residues is initially very rapid (10-fold increase in the first minute), and slows down during longer exposures to insulin (2-fold increase at 15 min as compared to the level a t 1 min, mean values of three experiments). The increase in serine phosphorylation on the contrary seems to be more linear in time. A similar pattern of phosphorylation was observed for ~ 4 6 " ~ ' (not shown).

It has been shown that upon EGF treatment the Shc proteins associate with the EGF receptor (Pelicci et ul., 1992). This led us to investigate whether the Shc proteins could be found in a complex with the activated insulin receptor. A14 cells were stimulated with insulin, and insulin receptors were immunoprecipitated with a monoclonal antibody recognizing the insulin receptor a-chain (Amersham). Precipitated proteins were analyzed for the presence of Shc by immunoblotting. By this method we were not able to detect insulin- induced association between the insulin receptor and the Shc proteins (data not shown). Therefore, Shc immunoprecipitates were analyzed for the presence of the insulin receptor &chain. For this purpose, Shc proteins were immunoprecipitated, and subsequently incubated with [Y-~~PIATP for 15 min a t 20 "C. By this procedure the insulin receptor /3-chain, when present, would be detected by autophosphorylation. Clearly, there is an insulin-induced kinase activity coprecipitating with the Shc proteins which phosphorylates both the

described above, and autophosphorylated insulin receptors were precipitated with antiphosphotyrosine. The labeled proteins were separated by SDS-PAGE, and the insulin receptor P-chain was analyzed as described for ~52"~ ' . Shown are the area of the blot where the insulin receptor &chain is located (insert) and a graphic representation of 92P incorporation. C, phosphoamino acid analysis of ~52"~'; in uioo labeled ~ 5 2 " ~ ' (see A ) was acid-hydrolyzed to single amino acids, and the phosphoamino acid composition was determined as described. Shown are the results obtained for the time points 0, 1, and 15 min, and a schematic representation of the positions of the different phosphoamino acid standards (upper left panel). The identity of the fourth spot, indicated in black, is unknown. The results are repre- sentative for three separate experiments.


Shc proteins and a 100-kDa protein (Fig. 4A). Phosphoamino acid analysis of both the Shc, and the 100-kDa phosphoproteins revealed that this in vitro phosphorylation occurs pre- dominantly on tyrosine residues (not shown). The 100-kDa phosphoprotein has an electrophoretic mobility similar to that of the insulin receptor @-chain. Therefore, we compared the two-dimensional tryptic phosphopeptide pattern of this 100- kDa phosphoprotein with that of the autophosphorylated insulin receptor @-chain from an insulin receptor immunoprecipitate. The tryptic phosphopeptide pattern obtained with the 100-kDa phosphoprotein is remarkably different from the one obtained with the insulin receptor @-chain (Fig. 4B). None of the major phosphopeptides had an identical mobility, as was verified by mixing of the two samples (not shown). We therefore conclude that the 100-kDa phosphoprotein is most likely not the insulin receptor @-chain. In conclusion, our results do not reveal an association between the Shc proteins and the insulin receptor.

Earlier studies showed that a 23-kDa protein copurifies with the Shc proteins after EGF receptor activation and upon induction of v-Src and v-Fps activity in cell lines containing ts mutants of these kinases (McGlade et al., 1992b). This 23- kDa protein appeared not to be phosphorylated in these cases, and thus could only be detected after [35S]methionine labeling.

A shc n i Ins: - + + "

1 OOkDa

52kDa 46kDa

Insulin receptor p-chain

FIG. 4. Insulin-induced association of a tyrosine kinase activity with Shc proteins. A, in vitro kinase reactions of Shc immunoprecipitates; A14 cells were treated with insulin for 5 min where indicated, and Shc proteins were immunoprecipitated. Immu- noprecipitates were incubated with [-p3*P]ATP, and phosphorylated proteins were analyzed by SDS-PAGE followed by electroblotting and autoradiography. As a control an immunoprecipitation with nonimmune rabbit serum was also performed (ni) . ~ 4 6 " ~ ' and ~ 5 2 " ~ ' are indicated by arrows. The phosphoprotein of about 100 kDa is indicated as well. B, tryptic phosphopeptide maps of the insulin receptor 8-chain and the 100-kDa Shc-"associated" phosphoprotein; the in vitro phosphorylated 100-kDa phosphoprotein from Fig. 4A and the in vitro phosphorylated insulin receptor @-chain from an insulin receptor immunoprecipitate (not shown) were digested with trypsin as described. Phosphopeptides were separated by two-dimensional thin-layer electrophoresis/liquid chromatography. Shown are the autoradiographs of the thin-layer cellulose plates.

To investigate whether this association also occurs after insulin receptor activation, A14 cells were labeled with [35S] methionine and treated with insulin for different periods. Clearly a 23-kDa protein coprecipitates with the Shc proteins upon insulin treatment (Fig. 5, lanes 2, 4, 6, and 8). The 23- kDa protein was not observed in parallel immunoprecipitations with nonimmune rabbit serum (lanes 1, 3, 5, and 7). This experiment also reveals another protein that is only observed in Shc immunoprecipitates. This protein has an electrophoretic mobility of about 33 kDa and is present in all Shc immunoprecipitates, independent of insulin treatment. The insulin-induced association between the Shc proteins and the 23-kDa protein is very rapid, after 1 min of insulin treatment the 23-kDa protein can already be detected in Shc immunoprecipitates. In 32Pi in vivo labeling experiments we detected no phosphorylation of the 23-kDa protein upon insulin treatment (not shown).

DISCUSSION

Recently considerable progress has been made toward un- derstanding some of the early events that occur after insulin receptor activation. For instance, it was shown that insulin receptor stimulation leads to activation of p21" (Burgering et dl., 1991; Osterop et al., 1992), and this subsequently leads to activation of the extracellular regulated kinases (de Vries- Smits et al., 1992). Furthermore, the insulin receptor substrate 1 (IRS-1) has been cloned, and it appeared that IRS-1 binds PI 3"kinase upon insulin treatment, leading to the observed insulin-induced activation of PI 3"kinase (Sun et al., 1991; Backer et al., 1992). Therefore it seems that insulin receptor signaling in some aspects resembles signaling from other tyrosine kinase receptors like, e.g. signaling from the EGF and PDGF receptors, which also involves activation of p21" (Gibbs et al., 1990; Satoh et al., 1990), and PI 3"k' mase (Cantley et al., 1991). In this report, we show another simi- larity between insulin, EGF, and PDGF receptor signaling. Upon insulin treatment of fibroblasts expressing elevated levels of the insulin receptor, ~ 4 6 " ~ and p52""' become rapidly phosphorylated. Also in cells expressing lower levels of insulin receptors, like the CH09 cells, insulin treatment can induce phosphorylation of the Shc proteins, indicating that this effect is not restricted to cells overexpressing the insulin receptor.

The insulin-induced Shc phosphorylation is similar to what was reported for EGF and PDGF signaling (Pelicci et al.,

Ins: - 1 ' 5' 15 '

FIG. 5. Insulin-induced association between Shc and a 23- kDa protein. A14 cells were labeled with [%]methionine and treated with insulin as indicated. Cells were lysed, and immunoprecipitations with the Shc antiserum (lanes 2, 4, 6 and 8) and nonimmune rabbit serum (lanes 1,3,5 and 7) were performed. Precipitated proteins were separated by SDS-PAGE and visualized by autoradiography. Indicated are ~ 4 6 ' ~ ' and p5ThC, a coprecipitating protein of about 33 kDa (open arrow), and the Shc-associated protein of about 23 kDa.

5752 Shc Proteins Are Phosphorylated Upon Insulin Receptor Activation

1992). In the case of EGF treatment it was reported that the Shc proteins associate with the activated EGF receptor. How- ever, we could not detect association between the Shc proteins and the activated insulin receptor. Surprisingly, we observe insulin-induced tyrosine kinase activity in Shc immunoprecipitates that phosphorylated both the Shc proteins and a protein of about 100 kDa. This 100-kDa phosphoprotein has an electrophoretic mobility similar to that of the insulin receptor @-chain. However, tryptic phosphopeptide analysis showed that this protein is most likely not the insulin receptor p-chain, because none of the phosphopeptides of the insulin receptor &chain were identical to those obtained with the 100-kDa phosphoprotein, although we cannot formally ex- clude that the differences in phosphorylation patterns are due to Shc-insulin receptor association.

The fact that the Shc proteins do not seem to associate directly with the insulin receptor could point to a mechanism similar to insulin-induced PI 3"kinase activation. Upon EGF and PDGF receptor activation, PI 3'-kinase associates with the EGF and PDGF receptor, respectively (Cantley et al., 1991), whereas upon insulin receptor activation, PI 3"kinase binds to IRS-1, which functions as a docking protein for proteins involved in insulin receptor signaling (Sun et al., 1991). Alternatively, Shc proteins may bind only transiently to the insulin receptor, or Shc proteins may not be direct substrates for the activated insulin receptor. This latter pos- sibility is supported by the observed insulin-induced tyrosine kinase activity present in Shc immunoprecipitates. The 100- kDa phosphoprotein is a good candidate for this kinase. As already discussed, this protein is not the insulin receptor p- chain. Also it is unlikely that it is the p-chain of the insulin- like growth factor 1 receptor since we do not observe insulin- induced Shc phosphorylation in NIH3T3 cells which have an equal number of insulin-like growth factor 1 receptors as the A14 cells. Further study is necessary to investigate the in- volvement of IRS-1, and to determine the identity of the 100- kDa Shc-associated protein and the Shc-associated tyrosine kinase.

The Shc proteins associate with a protein of about 23 kDa upon EGF treatment of EGF receptor overexpressing Rat-1 cells (McGlade et al., 1992b). Here we show that this protein also coimmunoprecipitates with the Shc proteins upon insulin treatment. As was also shown in the case of EGF stimulation, the 23-kDa protein is not phosphorylated upon insulin treatment. Similarly as observed for insulin-induced Shc phosphorylation the insulin-induced association between Shc and the 23-kDa protein is a rapid effect. After 1 min of insulin treatment the 23-kDa protein can already be detected in Shc immunoprecipitates. This 23-kDa protein is not detected when immunoprecipitations are performed with a nonimmune rabbit serum, nor is it recognized by the Shc antiserum (McGlade et al., 199213). This indicates that the 23-kDa protein is associated with the Shc proteins. The 23-kDa protein was recently identified as the mammalian sen-5 homologue, grb-2, that was shown to form a complex with the EGF- receptor upon EGF treatment (Lowenstein et al., 1992; Ro- zakis-Adcock et al., 1993). In Caenorhabditis elegans the sem- 5 gene product appears to be involved in signaling from the activated let-23 receptor to the let-60ras gene product (Clark et al., 1992). Due to homology between sem-5 andgrb-2 it was speculated that the Grb-2 protein might fulfill a similar function in growth factor-stimulated signal transduction pathways in fibroblasts. Indeed it was shown that, upon coinjection of p21" and Grb-2, DNA synthesis is stimulated in fibroblasts (Lowenstein et al., 1992). The observed association between Grb-2 and Shc suggests that the Shc proteins might also

function in signaling from activated growth factor receptors to p21". This is supported by the fact that shc overexpression leads to transformation of NIH3T3 cells (Pelicci et al., 1992). Furthermore, it was shown that overexpression of shc induces p21"dependent neurite outgrowth in PC12 cells (Rozakis- Adcock et al., 1993). It is therefore well possible that in insulin signaling, Shc mediates signaling from the activated receptor to p21". Recently we have shown that insulin-induced activation of p21" occurs through activation of a guanine nucleo- tide exchange factor (Medema et al., 1993). We speculate that Shc and Grb-2 are involved in the insulin-induced activation of this factor.

Acknowledgments-We thank J. A. Maassen and A. Osterop for the insulin receptor expressing cell lines, R. H. Medema, J.-P. Med- ema, and B. M. T. Burgering for critical reading of the manuscript, and M. E. Pronk-May for editorial assistance.

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