©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Shc Isoform-specific Tyrosine Phosphorylation by the Insulin and Epidermal Growth Factor Receptors (*)

(Received for publication, April 11, 1995; and in revised form, June 8, 1995)

Shuichi Okada Keishi Yamauchi (§) Jeffrey E. Pessin (¶)

From the Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IR/ER) resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform with a relatively low extent of 46-kDa Shc tyrosine phosphorylation. In contrast, EGF stimulation resulted in the tyrosine phosphorylation of both the 52- and 46-kDa Shc isoforms. Consistent with these differences, Grb2 predominantly bound to the 52-kDa Shc isoform following insulin stimulation, whereas Grb2 associated with both the 52- and 46-kDa Shc isoforms after EGF stimulation. Further, in vitro kinetic analysis demonstrated that the insulin receptor has a 4-fold greater V(max) with no significant difference in the K for the purified 52-kDa Shc isoform compared with the 46-kDa Shc isoform. However, the EGF receptor displayed the identical V(max) and K for tyrosine phosphorylation of both of these species. In direct contrast to the EGF receptor, we also observed significant differences in binding interactions between the insulin receptor with the 52- and 46-kDa Shc isoforms in vitro. These data demonstrate that the predominant insulin-dependent Shc signaling pathway occurs via the 52-kDa Shc isoform, whereas the EGF receptor can effectively use both the 52- and 46-kDa Shc species.


INTRODUCTION

Autophosphorylation of tyrosine kinase growth factor receptors results in the formation of recognition motifs for various Src homology 2 (SH2) (^1)domain containing effector proteins(1, 2, 3, 4) . The association of tyrosine-phosphorylated receptors with these effector molecules generates distinct receptor-signaling complexes responsible for downstream biological responsiveness. However, unlike most receptor tyrosine kinases, the insulin receptor itself does not persistently associate with these effector molecules but instead tyrosine-phosphorylates intermediate proteins responsible for the formation of multisubunit-signaling complexes(5, 6) . One proximal target of the insulin receptor was originally identified as a series of proteins (66, 52, and 46 kDa), termed Shc for Src homology 2/alpha-collagen-related(7, 8) . The cloning of cDNAs for this family of proteins has demonstrated that the 46- and 52-kDa species arise from the use of alternative translation initiation sites within the same transcript. This results in an amino-terminal 59-amino acid truncation of the 46-kDa isoform compared with the 52-kDa isoform. In contrast, the 66-kDa species most likely arises from an alternatively spliced message since there is only one Shc gene and carboxyl-terminal antibodies cross-react with all three molecular weight species(7, 8, 9, 10, 11) .

The Shc proteins are tyrosine-phosphorylated on a single tyrosine residue (Tyr), which serves as a docking site for Grb2 (8, 12) . Grb2 was originally identified as a 25-kDa growth factor receptor binding protein that contains a single SH2 domain flanked by two Src homology 3 (SH3) domains(13, 14) . The SH3 domains of Grb2 direct the association with several proteins containing proline-rich motifs including the 150-kDa guanylnucleotide exchange factor for Ras, termed SOS for the Drosophila gene Son-of-Sevenless (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . Thus, the tyrosine kinase receptor association of the Shc-Grb2-SOS complex targets guanylnucleotide exchange activity to the plasma membrane location of Ras, thereby providing a molecular switch for Ras activation(26, 27) .

Similar to many SH2 domain-containing proteins, the carboxyl-terminal SH2 domain of Shc was originally assumed to provide the appropriate binding motif for the association of Shc with tyrosine-autophosphorylated receptors(7, 28) . However, recent studies have demonstrated that the amino-terminal 84 amino acids are essential for the association of Shc with the tyrosine-phosphorylated EGF and fibroblast growth factor receptors(29, 30) . Although the tyrosine-phosphorylated insulin receptor does not persistently associate with Shc, the insulin-stimulated tyrosine phosphorylation of Shc appears to function as the major pathway mediating insulin activation of Ras GTP binding and Ras-dependent downstream signaling(31, 32, 33, 34) . In this paper, we demonstrate that the 52-kDa Shc isoform is the predominant substrate for the insulin receptor tyrosine kinase. This is in marked contrast to the EGF receptor, which displays similar substrate specificity for both the 52- and 46-kDa Shc species.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

The Shc cDNA encoding for the 52- and 46-kDa isoforms was kindly provided by Dr. Alan Saltiel (Parke-Davis/Warner-Lambert) and was cloned into the bacterial glutathione S-transferase expression plasmid system (Pharmacia Biotech Inc.). The Shc cDNA was cloned in frame with the first ATG to generate the GST-52Shc isoform. In order to obtain the GST-46Shc isoform fusion protein, the downstream ATG was cloned in frame with the glutathione S-transferase (GST) cDNA following amplification of this portion of the Shc cDNA with specific polymerase chain reaction primers generating a unique 5` EcoRI site (GAGAATTCATATGGGACCCGGGGTTT). All of the constructs used in this study were verified by DNA sequencing.

Cell Culture

CHO cells expressing 3 10^6 human insulin receptors/cell (CHO/IR) were obtained as described previously(35) . These cells were maintained in minimal Eagle's medium containing nucleotides plus 10% fetal bovine serum. NIH 3T3 cells expressing the human EGF receptor (3T3/ER) and the expression plasmid (pcDNA1, Invitrogen) containing the cDNA for the human EGF receptor (pcDNA1-ER) were kindly provided by Dr. John Koland (University of Iowa). CHO cells expressing both the insulin and EGF receptors CHO/IR/ER cells were prepared by co-transfection of CHO/IR cells with the hygromycin-resistant plasmid (pRBK, Invitrogen) and the human EGF expression plasmid (pcDNA1-ER). Twenty-two independent CHO/IR/ER clonal cell lines were isolated expressing various levels of the EGF receptor.

Western Blotting and Immunoprecipitation

Whole cell extracts were prepared by detergent solubilization in a lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 3 mM MgCl(2), 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin) for 1 h at 4 °C. Immunoprecipitations were performed by dilution of the detergent-solubilized cell extracts 10-fold in lysis buffer without Triton X-100 and incubation with 4 µg of a Shc polyclonal antibody (Transduction Laboratories) or Grb2 polyclonal antibody (Santa Cruz Biotechnology) for 2 h at 4 °C. The samples were then incubated with protein A-Sepharose for 1 h at 4 °C. The resulting immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and Western blotted (Enhanced Chemiluminescence detection kit, Amersham Corp.) using either a Shc monoclonal antibody (Transduction Laboratories) or the PY20-HRP phosphotyrosine antibody (Santa Cruz) as indicated in the individual figure legends. Quantification of signal intensity was performed in the linear range of the film using NIH Image 1.5 software.

In Vitro Phosphorylation of GST-Shc Fusion Proteins

The insulin and EGF receptors were partially purified from either CHO/IR or 3T3/ER cell lysate by wheat germ-Sepharose chromatography as described previously(36) . The receptors were incubated in 10 mM Tris-HCl, pH 7.4, 10 mM MnCl(2), 5 mM MgCl(2), 0.1% Triton X-100 containing an equal mixture of GST-52Shc and GST-46Shc fusion proteins (1, 3, 10, and 30 µg) and either 100 nM insulin or 100 ng/ml EGF. Following an additional incubation for 1 h at 22 °C, the kinase reaction was initiated by the addition of [-P] ATP (100 µM, 3 µCi/nmol) for various times. The reaction was terminated by addition of Laemmli sample buffer, and the phosphorylated proteins were resolved by SDS-polyacrylamide gel electrophoresis. Following extensive washing of the gel in 10% trichloroacetic acid, the gels were subjected to autoradiography, and the radiolabeled protein bands were excised and quantified by scintillation counting.

In Vitro Receptor Binding to GST-Shc Fusion Proteins

CHO/IR and 3T3/ER cells were incubated in either the absence or presence of 100 nM insulin or 100 ng/ml EGF for 5 min at 37 °C. Total cell detergent lysates were prepared by solubilization in 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 100 mM NaF, 10 mM sodium pyrophosphate, 10 mM ATP, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 µg/ml aprotinin. The cell lysates (2 mg) were then incubated with 10 µg of GST, GST-52Shc, or GST-46Shc bound to glutathione-Sepharose beads in 10 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 5 mM MnCl(2), and 2.5 mM MgCl(2). Following an additional 60-min incubation at 22 °C, the reaction was stopped by the addition of 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, and 10 mM ATP. The samples were microcentrifuged and washed six times with 20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.1% Nonidet P-40, 150 mM NaCl, 2 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride followed by Western blotting using an insulin or EGF receptor antibody (Transduction Laboratories).


RESULTS

Insulin Stimulation Preferentially Results in the Tyrosine Phosphorylation of the 52-kDa Shc Isoform in CHO/IR Cells

During our previous studies examining the relationship between IRS1 and Shc tyrosine phosphorylation(33, 37) , we noticed that the 52-kDa Shc species displayed a greater extent of tyrosine phosphorylation than the 46-kDa species. To further investigate this observation, we initially examined the relative expression levels of the 66-, 52-, and 46-kDa Shc species in CHO/IR cells (Fig. 1A). Shc immunoprecipitation followed by Shc immunoblotting of whole cell detergent extracts from control and insulin-stimulated cells demonstrated that the 52-kDa Shc isoform was approximately 1.5-fold more abundant than the 46-kDa Shc species (Fig. 1A, lanes1 and 2). In these cells, the 66-kDa Shc isoform was also observed to be approximately one-third as abundant as the 46-kDa isoform. Although the relative expression levels of the 66- and 46-kDa species were somewhat lower than that of the 52-kDa isoform, phosphotyrosine immunoblotting following 5 min of insulin stimulation demonstrated a nearly complete absence of 66-kDa Shc tyrosine phosphorylation with a marked reduction in 46-kDa Shc phosphorylation compared with the 52-kDa species (Fig. 1B, lanes1 and 2). Since one established target of tyrosine-phosphorylated Shc is the small adapter protein Grb2, we also determined the insulin-stimulated association of the Shc proteins with Grb2 by co-immunoprecipitation. As expected, in the absence of insulin there were no detectable Shc proteins in the Grb2 immunoprecipitate (Fig. 1A, lane3). However, Grb2 immunoprecipitation of extracts from insulin-stimulated cells demonstrated the presence of the 52-kDa Shc isoform with no detectable 66- or 46-kDa Shc protein isoforms (Fig. 1A, lane4). Similarly, phosphotyrosine immunoblots of the Grb2 immunoprecipitates also demonstrated the predominant co-immunoprecipitation of the tyrosine-phosphorylated 52-kDa Shc isoform with relatively low levels of the 46-kDa Shc species (Fig. 1B, lane4). The band migrating at approximately 180 kDa represents the co-immunoprecipitation of Grb2 with tyrosine-phosphorylated IRS1 (9) . Importantly, the tyrosine-phosphorylated 95-kDa insulin receptor beta subunit was not detected in either the Shc or Grb2 immunoprecipitates (Fig. 1B, lanes2 and 4).


Figure 1: The insulin receptor primarily tyrosine-phosphorylates the 52-kDa Shc isoform in CHO/IR cells. CHO/IR cells were incubated in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100 nM insulin for 5 min at 37 °C. Whole cell detergent lysates were prepared and either immunoprecipitated with a Shc antibody (lanes1 and 2) or a Grb2 antibody (lanes3 and 4) as described under ``Experimental Procedures.'' The immunoprecipitates were then Western blotted with the Shc antibody (A) or the PY20-HRP phosphotyrosine antibody (B). IP, immunoprecipitate; IB, immunoblot.



Insulin and EGF Stimulation of Shc Tyrosine Phosphorylation in CHO/IR/ER Cells

To determine whether the predominant tyrosine phosphorylation of the 52-kDa Shc isoform was specific for the insulin receptor, we isolated CHO cells expressing both the insulin and EGF receptors (CHO/IR/ER) by co-transfection of CHO/IR cells with the human EGF receptor expression plasmid. Following clonal isolation of multiple stable cell lines, we determined the insulin- and EGF-stimulated tyrosine phosphorylation of the Shc isoforms within the identical cell context (Fig. 2). In this clonal isolate, immunoprecipitation of Shc followed by Shc immunoblotting demonstrated the relative proportion of the Shc isoforms in an approximate 1:2:1 ratio for the 66-, 52-, and 46-kDa Shc isoforms, respectively (Fig. 2A, lanes1-3). Insulin treatment of these cells resulted in the predominant tyrosine phosphorylation of the 52-kDa Shc isoform, whereas EGF stimulation displayed a marked increase in the extent of 46-kDa Shc tyrosine phosphorylation (Fig. 2B, lanes1-3). In addition, insulin was also relatively ineffective in the tyrosine phosphorylation of the 66-kDa Shc species compared with EGF.


Figure 2: Differential tyrosine phosphorylation of the 52- and 46-kDa Shc isoforms by insulin and EGF in CHO/IR/ER cells. CHO/IR/ER cells were incubated in the absence (lanes1 and 4) and in the presence of either 100 nM insulin (lanes2 and 5) or 100 ng/ml EGF (lanes3 and 6) for 5 min at 37 °C. Whole cell detergent lysates were prepared and either immunoprecipitated with a Shc antibody (lanes1-3) or a Grb2 antibody (lanes4-6) as described under ``Experimental Procedures.'' The immunoprecipitates were then Western blotted with the Shc antibody (A) or the PY20-HRP phosphotyrosine antibody (B). IP, immunoprecipitate; IB, immunoblot.



Similarly, Shc immunoblotting of the Grb2 immunoprecipitates demonstrated the predominant association of the 52-kDa Shc isoform with Grb2 following insulin stimulation (Fig. 2A, lane5). In contrast, EGF stimulation resulted in a greater extent of 46-kDa Shc association with Grb2 compared with insulin-stimulated CHO/IR/ER cells (Fig. 2A, lane6). In parallel, phosphotyrosine immunoblotting of the Grb2 immunoprecipitates also demonstrated the predominant insulin-stimulated co-immunoprecipitation of the tyrosine-phosphorylated 52-kDa Shc isoform (Fig. 2B, lane5), whereas EGF stimulated the co-immunoprecipitation of the tyrosine-phosphorylated 52- and 46-kDa Shc species (Fig. 2B, lane6). In addition, we also observed a relatively small extent of EGF-stimulated tyrosine phosphorylation and Grb2 co-immunoprecipitation of the 66-kDa Shc isoform (Fig. 2B, lanes3 and 6). Furthermore, both Shc and Grb2 immunoprecipitations resulted in the co-immunoprecipitation of the tyrosine-phosphorylated EGF receptor (Fig. 2B, lanes3 and 6), whereas insulin stimulation only resulted in the Grb2 co-immunoprecipitation of tyrosine-phosphorylated IRS1 (Fig. 2B, lane5). As observed in the CHO/IR cells, we were unable to detect the presence of the tyrosine-phosphorylated insulin receptor in either the Shc or Grb2 immunoprecipitates (Fig. 2B, lanes2 and 5).

Insulin and EGF Receptor Phosphorylation of the Shc Isoforms in Vitro

To further examine the substrate specificity of insulin and EGF receptor-dependent Shc phosphorylation, we prepared GST fusion proteins of the Shc isoforms (Fig. 3). In the absence of insulin, the partially purified insulin receptor was unable to phosphorylate either of the GST-Shc isoforms (data not shown). In the presence of 100 nM insulin, the insulin receptor displayed a greater rate and extent of 52-kDa Shc phosphorylation compared with the 46-kDa Shc isoform (Fig. 3A). This difference in Shc isoform phosphorylation was not due to the phosphorylation of the GST fusion itself, since the GST protein was not a substrate for the insulin receptor (data not shown) and both the GST-52Shc and GST-46Shc fusions contained the identical GST protein. Interestingly, although the GST fusion proteins were precipitated by the glutathione-Sepharose resin and were extensively washed prior to SDS-polyacrylamide gel electrophoresis, the insulin receptor beta subunit was consistently observed to be co-precipitated (see Fig. 5).


Figure 3: The insulin and EGF receptor display Shc isoform-specific substrate kinase activity in vitro. The insulin and EGF receptors were isolated from CHO/IR and 3T3/ER cells, respectively, by detergent solubilization and wheat germ agglutinin affinity chromatography as described under ``Experimental Procedures.'' The insulin (A) and EGF (B) receptors were incubated with a mixture of 10 µg of the GST-52Shc and 10 µg of the GST-46Shc fusion proteins in the presence of 100 nM insulin or 100 ng/ml EGF, respectively. The reaction was initiated by the addition of [-P]ATP (100 µM, 3 µCi/nmol) for 0 (lane1), 2 (lane2), 5 (lane3), 15 (lane4), 30 (lane5), and 60 (lane6) min. The reaction was terminated by the addition of Laemmli sample buffer, and the samples were then subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.




Figure 5: Differential in vitro binding of the insulin and EGF receptors to the 52- and 46-kDa Shc isoforms. CHO/IR (A) and 3T3/ER (B) cells were incubated with 100 nM insulin or 100 ng/ml EGF for 5 min at 37 °C. The cells were detergent-solubilized, and the lysates (2 mg) were incubated with 10 µg of GST (lanes1 and 2), 10 µg of GST-52Shc (lanes3 and 4), or 10 µg GST-46Shc bound to glutathione-Sepharose beads for 60 min as described under ``Experimental Procedures.'' The samples were then precipitated by microcentrifugation, and the resulting precipitates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was then Western blotted for the presence of the insulin (A) or the EGF (B) receptors.



Consistent with the EGF-dependent Shc phosphorylation in vivo, the EGF receptor demonstrated essentially an identical rate and extent of 52- and 46-kDa Shc phosphorylation in vitro (Fig. 3B). As observed for the insulin receptor, the EGF receptor was unable to phosphorylate the GST fusion protein itself (data not shown). In addition, the autophosphorylated EGF receptor was also found to co-precipitate with the GST-Shc fusion proteins (Fig. 3B) but not with GST alone (data not shown).

To determine if the enhanced insulin receptor phosphorylation of the 52-kDa Shc isoform was due to a differences in V(max) or K, we next determined the substrate concentration dependence of Shc tyrosine phosphorylation (Fig. 4A). In the presence of insulin, the insulin receptor had essentially an identical K (0.45 versus 0.40 µM) for both the 52- and 46-kDa Shc isoforms. In contrast, the insulin receptor had an approximate 4-fold increase in V(max) for the 52-kDa Shc isoform (4.0 pmol/min) compared with the 46-kDa Shc species (1.0 pmol/min). In comparison, we also determined the kinetic properties of the EGF receptor-mediated tyrosine phosphorylation of the Shc proteins (Fig. 4B). As expected, there was no significant difference in either the K (0.70 versus 0.85 µM) or V(max) (4.4 versus 5.0 pmol/min) values between the 52- and 46-kDa Shc isoforms as substrates for the EGF receptor.


Figure 4: Kinetic analysis of insulin and EGF receptor tyrosine phosphorylation of the 52- and 46-kDa Shc isoforms. The initial rates of hormone-stimulated insulin (A) and EGF (B) receptor tyrosine phosphorylation of the GST-52Shc () and GST-46Shc (bullet) fusion proteins were determined as described in the legend to Fig. 3. The initial rate of substrate phosphorylation was determined at various fusion protein concentrations and graphically analyzed in the form of a Lineweaver-Burke plot.



In Vitro Association of the 52- and 46-kDa Shc Isoforms with the Insulin and EGF Receptors

The co-precipitation of the insulin receptor beta subunit during in vitro phosphorylation of the 52- and 46-kDa Shc isoforms suggests that the insulin receptors could potentially associate with Shc (Fig. 3). We therefore examined the ability of the insulin receptor to directly bind the 52- and 46-kDa Shc isoforms in vitro (Fig. 5). Detergent-lysed cell extracts were prepared from control and insulin-stimulated CHO/IR cells. These extracts were then incubated with GST, GST-52Shc, and GST-46Shc fusion proteins and precipitated with glutathione-Sepharose beads (Fig. 5A). As expected, the insulin receptor isolated from control or insulin-stimulated cells did not associate with the GST protein itself (Fig. 5A, lanes1 and 2). However, incubation of control cell extracts with the GST-52Shc fusion protein demonstrated barely detectable levels of co-precipitated insulin receptor beta subunit (Fig. 5A, lane3). However, extracts from insulin-stimulated cells demonstrated a significantly increased association of the GST-52Shc species with the insulin receptor (Fig. 5A, lane4). In contrast, extracts from both control or insulin-stimulated cells associated relatively poorly with the GST-46Shc fusion protein (Fig. 5A, lanes5 and 6).

In comparison, the EGF receptor isolated from either control or EGF-stimulated 3T3/ER cells also did not associate with the GST protein alone (Fig. 5B, lanes1 and 2). In addition, control cell extracts did not significantly associate with either the GST-52Shc or GST-46Shc fusion proteins (Fig. 5B, lanes3 and 5). However, extracts from EGF-stimulated cells demonstrated the co-precipitation of the EGF receptor to a similar extent with both the GST-52Shc and GST-46Shc fusion proteins (Fig. 5B, lanes4 and 6). Thus, although tyrosine phosphorylation of both the insulin and EGF receptor was required for in vitro association, the EGF receptor does not strongly discriminate between the 52- and 46-kDa Shc isoforms compared with the insulin receptor.


DISCUSSION

It has been well established that hormone-stimulated tyrosine kinase activity of growth factor receptors is a necessary event in the propagation of growth factor-dependent downstream signaling (for reviews see (38, 39, 40) ). Although several specific combinations of proximal effectors have been identified for numerous growth factor tyrosine kinase receptors, they all appear to have in common the tyrosine phosphorylation and/or association with the Shc proteins. Since tyrosine-phosphorylated Shc directs its association with the Grb2-SOS complex, this provides a pathway directly linking tyrosine kinase receptors to Ras activation(14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) . Consistent with this model, several studies have demonstrated that Shc is the predominant route for the Ras-dependent activation of the mitogen-activated protein kinase pathway(31, 32, 33, 34) .

In the case of EGF signaling, Shc directly associates with the tyrosine-phosphorylated EGF receptor, targeting the Shc-Grb2-SOS ternary complex to the plasma membrane location of Ras(12, 21, 25, 41, 42, 43, 44) . This model is consistent with studies demonstrating that EGF has no effect on SOS or RasGAP activity and that expression of SOS proteins that are directly targeted to the plasma membrane via myristoylation or prenylation constitutively activate Ras GTP loading(26, 27) . Although this is an appealing model, tyrosine kinase receptor docking of Shc cannot account for the ability of insulin to target the Shc-Grb2-SOS complex to the plasma membrane since the insulin receptor does not persistently associate with Shc in vivo(8, 41, 42, 45) . Consistent with this conclusion, we have not been able to observe any significant association of Shc with the insulin receptor under identical conditions that readily detect Shc association with the EGF receptor and Grb2 with IRS1. Furthermore, point mutations and carboxyl-terminal deletions of the EGF receptor that completely abolish Shc binding have no effect on EGF receptor-mediated activation of the Ras-dependent mitogen-activated protein kinase pathway(46, 47, 48, 49) . Thus, it appears that the tyrosine phosphorylation of Shc is the essential feature necessary to mediate growth factor tyrosine kinase receptor-dependent Ras activation.

Several studies have observed that activation of various growth factor receptor tyrosine kinases results in the tyrosine phosphorylation of both the 52- and 46-kDa Shc isoforms and, in some studies, the 66-kDa isoform(8, 11, 43, 50) . In these studies, we have not detected significant insulin or EGF-stimulated tyrosine phosphorylation of the 66-kDa Shc isoform by phosphotyrosine immunoblotting. However, there was a marked decrease in electrophoretic mobility of the 66-kDa Shc protein, most likely reflecting serine/threonine phosphorylation. In any case, we have observed that insulin stimulation predominantly resulted in the tyrosine phosphorylation of the 52-kDa Shc isoform compared with the 46-kDa Shc species. This difference in substrate specificity was also recapitulated in vitro, demonstrating that the 52-kDa Shc isoform was a significantly better substrate for the insulin receptor compared with the 46-kDa Shc isoform. Although we have been unable to detect the co-immunoprecipitation of Shc with the insulin receptor, in vitro binding demonstrated that the 52-kDa Shc isoform was more efficiently associated with the insulin receptor than the 46-kDa Shc isoform. Similarly, binding interactions between the insulin receptor beta subunit and Shc have been observed by high level expression in the yeast two-hybrid system(51) . We therefore hypothesize that the observed in vitro binding between the insulin receptor and Shc was indicative of association at the level of substrate-enzyme interactions but does not reflect a stable interaction in vivo.

In contrast, the tyrosine-phosphorylated EGF receptor was stably associated with Shc, which was directly detected by co-immunoprecipitation. Since Shc contains a carboxyl-terminal SH2 domain, it was previously assumed that this domain was responsible for the docking of Shc to the EGF receptor. However, recent studies have demonstrated that the amino-terminal 84 amino acids of Shc provide this function independent of the carboxyl-terminal SH2 domain(29, 30) . Consistent with these findings, we also have not observed any significant difference in the binding of the tyrosine-phosphorylated EGF receptor to the 52- and 46-kDa Shc isoforms, the latter differing by the deletion of 59 amino-terminal residues. Together, these data suggest that amino acid residues 59-84 define the Shc protein tyrosine binding domain for the EGF receptor. This is in contrast to the insulin receptor, which preferentially utilized the 52-kDa Shc isoform as a tyrosine kinase substrate both in vivo and in vitro. Thus, although the amino-terminal 84 residues of Shc were necessary for receptor association and tyrosine phosphorylation, specific determinants within this domain are apparently necessary to impart substrate specificity between the EGF and insulin receptors.

To date, there does not appear to be any specific difference in the signaling role played by the 52- and 46-kDa Shc isoforms. Both species, when tyrosine-phosphorylated, associate with Grb2 and thereby presumably link SOS to the activation of Ras. However, since all cells express both the 52- and 46-kDa Shc isoforms, albeit at different levels, the contribution of these complexes to downstream signaling events has not been quantitatively determined. Based upon the apparent specificity of the insulin receptor, we speculate that the 52- and 46-kDa Shc isoforms have defined signaling and/or regulatory roles.

In summary, we have observed that the EGF receptor associates with and tyrosine phosphorylates both the 52- and 46-kDa Shc isoforms with equal efficacy both in vivo and in vitro. In contrast, the insulin receptor preferentially tyrosine phosphorylates the 52-kDa Shc isoform, resulting in the predominant targeting of Grb2 to this species. Whether or not the tyrosine phosphorylation and/or receptor association of the 52- and 46-kDa Shc isoforms underlies physiological specificity in the signaling pathways between the EGF and insulin receptors remains to be determined.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK33823 and DK25295. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Endocrinology and Geriatrics, Shinshu University, School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: SH2 and SH3 domains, Src homology 2 domain and Src homology 3 domain, respectively; EGF, epidermal growth factor; IRS1, insulin receptor substrate-1; CHO, Chinese hamster ovary; IR, insulin receptor; ER, epidermal growth factor receptor; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We thank Dr. Alan Saltiel for providing the Shc cDNA and Dr. John Koland for the human EGF receptor cDNA and the NIH 3T3/ER cell line.


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