Interaction between the Phosphotyrosine Binding Domain of Shc and the Insulin Receptor Is Required for Shc Phosphorylation by Insulin in Vivo *

Stimulation of the insulin receptor (IR) results in tyrosine phosphorylation of the intermediate molecules insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which then couple the IR to downstream signaling pathways by serving as binding sites for signaling molecules with SH2 domains. It has been proposed that direct binding of IRS-1, IRS-2, and Shc to an NP X -Tyr(P) motif in the juxtamembrane region of the IR is required for tyrosine phosphorylation of these molecules by the IR. In this regard, Shc and IRS-1 contain domains that are distinct from SH2 domains, referred to as the phosphotyrosine binding (PTB) or phosphotyrosine interaction (PI) domains, which bind phosphotyrosine in the con-text of an NP X -Tyr(P) motif. To further clarify the role of the Shc PTB/PI domain, we identified a mutation in this domain that abrogated binding of Shc to the IR in vitro . Interestingly, this mutation completely abolished Shc phosphorylation by the IR in vivo whereas mutation of the arginine in the FLVRES motif of the Shc SH2 domain did not affect Shc phosphorylation by insulin. In addition, Production of Retroviruses— A chimeric receptor (Trk/IR) was constructed by joining the cDNA encoding the extracellular high affinity NGF binding domain of human TrkA to the cDNA encoding the transmembrane and intracellular domain of the human IR. Briefly, the last amino acid in the extracellular domain of TrkA (glutamic acid 407) was linked in frame with the first hydrophobic amino acid in the membrane-spanning region of the IR (isoleu- cine 918 according to Ullrich et al . (30)) by the polymerase chain reaction using standard techniques. For site-directed mutagenesis, the Trk/IR cDNA was subcloned from pUC118 as an Eco RI- Hin dIII frag-ment into the vector pALTER (Promega), and mutagenesis was per- formed according to the manufacturer’s recommendation. To facilitate screening of mutants, novel restriction sites were incorporated into the oligonucleotides used for mutagenesis, and Trk/IR cDNAs containing the appropriate mutations were identified by restriction digest. To overexpress the Trk/IR receptor in 3T3-L1 cells, the wild type and mutant Trk/IRs were subcloned into the retroviral vector SR (cid:97) using Eco RI- Hin dIII (19). Helper-free infectious retrovirus was produced by transiently transfecting the various constructs into the retroviral pack- aging cell line BOSC 23 using Ca 2 PO 4 precipitation (31). Trk/IR containing retroviruses were used to infect 3T3-L1 fibroblasts, and G418 (1 mg/ml)-resistant pools were selected. Expression of Trk/IR was con-firmed by immunoblotting using an antibody to the IR (cid:98) -chain. To overexpress the IR in NIH 3T3 fibroblasts (NIH/IR), the complete cDNA of the human IR was subcloned into the retroviral expression vector pBABE-puro. Retrovirus was obtained as above and used to infect NIH 3T3 cells, and puromycin (1 (cid:109) g/ml)-resistant

A critical early event necessary for the IR 1 to mediate biological functions is receptor autophosphorylation, which results in increased activity of the receptor's kinase domain. Autophosphorylation also mediates the interaction of the IR, as well as other receptor tyrosine kinases, with several signaling molecules, which must bind autophosphorylated receptors in order to be activated (1). The interaction of signaling molecules with autophosphorylated receptors has now been shown to be mediated by at least two distinct domains on signaling molecules that bind phosphotyrosine. The first domains to be described are the SH2 domains (1). Recently, a second domain that binds phosphotyrosine was identified in the amino terminus of Shc and has been termed the PI or PTB domain (also known as the Shc and IRS-1 NPXY-binding (SAIN) domains or IRS homology 2 (IH2) domains) (2)(3)(4)(5)(6). In contrast to the finding that SH2 domains preferentially recognize amino acids carboxyl-terminal to the phosphotyrosine, the Shc PTB/PI domain recognizes amino acids amino-terminal to the phosphotyrosine that consist of a core NPX-Tyr(P) motif (7)(8)(9)(10)(11)(12).
IR autophosphorylation results in tyrosine phosphorylation of several intermediate molecules, including IRS-1, IRS-2, and Shc (6,(13)(14)(15). These molecules, rather than the IR itself, then couple to downstream signaling pathways by serving as binding sites for SH2 domain-containing signaling molecules (16). For example, binding of GRB2-Sos to tyrosine-phosphorylated Shc is thought to couple the IR to Ras activation (14,(17)(18)(19). The recognition that Shc interacts with autophosphorylated receptors through its PTB/PI domain, coupled with the finding that IRS-1 and IRS-2 contain a similar domain that binds phosphotyrosine, have helped clarify how the IR likely activates these signaling molecules. These results have led to the suggestion that IRS-1, IRS-2 and Shc share a common NPX-Tyr(P) binding motif on the autophosphorylated insulin and IGF-1 receptors that functions to juxtapose these molecules adjacent to the IR kinase domain, thereby enabling IRS-1 and Shc to become phosphorylated (5,16,20).
While this has become an attractive model to explain the mechanism whereby the IR phosphorylates IRS-1 and Shc, it is still not clear whether phosphorylation of Shc by the IR requires a functional PTB/PI domain or whether binding of Shc directly to the IR is required for Shc phosphorylation in vivo. For example, epidermal growth factor stimulation of a truncated EGFR that does not bind Shc still results in Shc tyrosine phosphorylation (21). Moreover, although the PTB/PI domain of Shc has been clearly shown by co-immunoprecipitation to associate in vivo with an NPX-Tyr(P) motif on a variety of receptor tyrosine kinases, middle T antigen, and the phosphoprotein p145 (4,8,9,(22)(23)(24), the IR has been shown to bind Shc only in vitro (5,14,15,25). The fact that a mutation in tyrosine 960 in the NPXY motif of the IR impairs tyrosine phosphorylation of Shc by insulin (26) does not prove that Shc is a direct substrate of the IR; this mutation also abrogates tyrosine phosphorylation of IRS-1 by the IR (26,27), thereby raising the possibility that activation of a signaling pathway downstream from IRS-1 or binding of an unidentified signaling molecule to the IR NPX-Tyr(P) motif mediates Shc phosphorylation.
In order to clarify the role of Shc's PTB/PI domain in mediating Shc phosphorylation by insulin, we determined whether a single amino acid substitution in the PTB/PI domain of Shc that impairs binding of Shc to the IR in vitro also impairs the tyrosine phosphorylation of Shc by the IR in vivo. In addition, we determined whether changes in the amino acids surrounding the NPXY motif in the juxtamembrane region of the IR modulate the ability of insulin to stimulate Shc phosphorylation in vivo. The findings reported in this paper support a model in which binding of the PTB/PI domain of Shc directly to the NPX-Tyr(P) motif on the IR is required for Shc phosphorylation by the IR in vivo.

MATERIALS AND METHODS
Cell Lines, Growth Factors, and Cell Stimulation-Murine 3T3-L1 fibroblasts and NIH/IR cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum (Life Technologies, Inc.). For stimulation with growth factors, cells were serum starved overnight in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum and then stimulated with either 100 nM or 1 M insulin or 100 ng/ml nerve growth factor (NGF, Harlan Bioproducts, Madison, WI).
Cell Lysis, Immunoprecipitation, Immunoblot, and Antibodies-Cell lysis, immunoprecipitation, and immunoblotting were performed as described (19). Antibodies to IRS-1, GRB2, Shc, and phosphotyrosine are rabbit polyclonal antibodies and have been described previously (19). Antibody to the IR is a rabbit polyclonal antibody that recognizes the kinase domain of the human and murine IRs (28). Antibody 9E10 was used for immunoprecipitation of the epitope-tagged Myc constructs (29).
Mutagenesis, Expression Vectors, and Production of Retroviruses-A chimeric receptor (Trk/IR) was constructed by joining the cDNA encoding the extracellular high affinity NGF binding domain of human TrkA to the cDNA encoding the transmembrane and intracellular domain of the human IR. Briefly, the last amino acid in the extracellular domain of TrkA (glutamic acid 407) was linked in frame with the first hydrophobic amino acid in the membrane-spanning region of the IR (isoleucine 918 according to Ullrich et al. (30)) by the polymerase chain reaction using standard techniques. For site-directed mutagenesis, the Trk/IR cDNA was subcloned from pUC118 as an EcoRI-HindIII fragment into the vector pALTER (Promega), and mutagenesis was performed according to the manufacturer's recommendation. To facilitate screening of mutants, novel restriction sites were incorporated into the oligonucleotides used for mutagenesis, and Trk/IR cDNAs containing the appropriate mutations were identified by restriction digest.
To overexpress the Trk/IR receptor in 3T3-L1 cells, the wild type and mutant Trk/IRs were subcloned into the retroviral vector SR␣ using EcoRI-HindIII (19). Helper-free infectious retrovirus was produced by transiently transfecting the various constructs into the retroviral packaging cell line BOSC 23 using Ca 2 PO 4 precipitation (31). Trk/IR containing retroviruses were used to infect 3T3-L1 fibroblasts, and G418 (1 mg/ml)-resistant pools were selected. Expression of Trk/IR was confirmed by immunoblotting using an antibody to the IR ␤-chain. To overexpress the IR in NIH 3T3 fibroblasts (NIH/IR), the complete cDNA of the human IR was subcloned into the retroviral expression vector pBABE-puro. Retrovirus was obtained as above and used to infect NIH 3T3 cells, and puromycin (1 g/ml)-resistant pools were selected. Wild type (wt) and mutant murine p52 Shc proteins containing a point mutation in either the PTB/PI domain (replacement of phenylalanine at position 198 by valine, F198V) or the SH2 domain (replacement of arginine at position 397 for lysine in the highly conserved FLVRES motif, R397K) were tagged with the myc epitope and subcloned into the retroviral expression vector pLEN (32). Helper-free infectious retroviruses were obtained and used to infect NIH/IR cells.
Yeast Two-hybrid Analysis-The yeast two-hybrid system was used to determine whether the F198V Shc bound the IR in vitro (33). The cytoplasmic domain (amino acids 941-1343) of the IR was expressed as a fusion with the LexA DNA binding domain using the vector pBTM116. Wild type and F198V Shc were joined to the nuclear localized VP16acidic activation domain using the vector pVP16. All constructs were generated by polymerase chain reaction using custom primers containing convenient restriction sites to facilitate cloning. All experiments were done in the yeast strain L40 (his3 leu2-3 trp1). Routine growth, maintenance, and transformation of yeast was as described previously (33,34). Interaction was determined by selecting for growth on medium lacking histidine in the presence of 5 mM 3-aminotriazole (Sigma).

RESULTS AND DISCUSSION
Shc requires an intact PTB/PI domain to be tyrosine-phosphorylated in insulin-stimulated cells. To identify residues in the PTB/PI domain of Shc that are important in binding phosphorylated peptides, random mutagenesis of Shc's PTB/PI domain was performed, and the ability of these mutants to bind the tyrosine-phosphorylated EGFR was determined (35). One of the PTB/PI mutants identified that decreased binding to the autophosphorylated EGFR contained valine in place of phenylalanine at amino acid position 198 (F198V) of p52 Shc. This residue, which is highly conserved in most PTB/PI domains described so far, has recently been shown by NMR to interact with the NPXY asparagine (36). We found that F198V Shc also impairs the binding of Shc to the IR using the yeast two-hybrid system (Fig. 1). In agreement with previous studies (7), wt Shc bound the IR as demonstrated by the selection for growth of yeast on medium lacking histidine in the presence of 5 mM 3-aminotriazole. However, while the efficiency of yeast transformation of the F198V Shc is similar to that of wt Shc, F198V Shc did not support growth on medium lacking histidine. These results indicate that the F198V mutation impairs the binding of Shc to the IR in vitro.
After determining that F198V Shc does not interact with the IR in the yeast two-hybrid system, we determined whether this mutation affected tyrosine phosphorylation of Shc by the IR in vivo. We reasoned that if binding of Shc to the IR is required for Shc phosphorylation in cells, then F198V Shc would not undergo tyrosine phosphorylation in response to insulin stimulation. NIH/IR cells were infected with retroviruses containing either Myc epitope-tagged wt or F198V Shc, and G418-resistant pools of cells expressing the various Shc proteins were isolated. Cells were then stimulated with insulin, and Myc epitope-tagged Shc was immunoprecipitated using the anti-Myc antibody 9E10. Insulin stimulated tyrosine phosphorylation of wt p52 Shc (Fig. 2, lanes c and d) but did not stimulate tyrosine phosphorylation of F198V Shc (Fig. 2, lanes e and f ) although a similar amount of F198V Shc as compared with wt Shc was immunoprecipitated. We also determined whether mutation of the arginine in the highly conserved FLVRES motif of the Shc SH2 domain (R397K) impaired tyrosine phosphorylation of Shc. This mutation has previously been shown not to inhibit the binding of Shc to the IR in vitro (7). In contrast to the results obtained with F198V Shc, insulin stimulated increased tyrosine phosphorylation of R397K Shc (Fig. 2, lanes g  and h). GRB2 also co-immunoprecipitated with tyrosine-phosphorylated Shc (Fig. 2, lanes d and h). These findings demonstrate that Shc requires an intact PTB/PI domain to be tyrosine-phosphorylated in insulin-stimulated cells.
Expression of Trk/IR Chimeras in 3T3-L1 Fibroblasts-Insulin-stimulated tyrosine phosphorylation of both Shc and FIG. 1. Wild type Shc but not F198V Shc interacts specifically with IR in the yeast two-hybrid system. Saccharomyces cerevisiae strain L40 containing plasmid pBTM116 that expresses a fusion protein containing the LexA DNA-binding domain and cytoplasmic domain of the IR was transfected with either the plasmid pVP16 alone or pVP16 containing wt p52 Shc or F198V Shc as fusion proteins with the VP16 activation domain. The efficiency of transformation was determined by selecting yeast transformants on medium lacking tryptophan, leucine, and uracil. The interaction between the IR and Shc was determined by plating yeast on similar plates lacking histidine and containing 5 mM 3-aminotriazole.

Phosphorylation of Shc by Insulin Receptor 3960
IRS-1 in vivo requires phosphorylation of tyrosine 960 in the juxtamembrane NPXY motif of the IR (26,27). To determine whether the amino acids surrounding the NPXY motif impart specificity for tyrosine phosphorylation of either Shc or IRS-1 in cells, we generated a panel of mutant Trk/IRs containing point mutations in the juxtamembrane region surrounding the NPXY motif (Fig. 3A). The amino acids selected for mutagenesis were derived from the alignment of a variety of NPXY motifs on different receptors that were shown to activate either Shc or IRS-1 (Fig. 3B). We reasoned that if direct binding of the PTB/PI domain of Shc to the IR is required for Shc tyrosine phosphorylation, there should be a direct correlation between in vivo tyrosine phosphorylation of Shc by the chimeric receptors and in vitro binding of Shc's PTB/PI domain to NPX-Tyr(P)-containing sequences. Stable pools of 3T3-L1 fibroblasts expressing roughly equal amounts of wild type and mutant Trk/IR receptors were obtained and were autophosphorylated in response to NGF (Fig. 3C).
Amino Acids Surrounding the NPX-Tyr(P) Motif on the IR That Are Critical for Tyrosine Phosphorylation of Shc by the IR-In response to stimulation with NGF, the chimeric Trk/IR behaved similarly to the wt IR. Stimulation of the wt Trk/IR with NGF led to tyrosine phosphorylation of p52 Shc, p66 Shc, and IRS-1, whereas tyrosine phosphorylation of these proteins was markedly reduced by mutating tyrosine 960 to phenylalanine (Y960F) and was completely abrogated by a mutation that inhibited the kinase activity (K1018A) (Fig. 4, lanes a-i). However, we identified three point mutations in the juxtamembrane region of Trk/IR that either inhibited or augmented Shc phosphorylation in response to NGF without affecting the ability of these receptors to phosphorylate IRS-1 (Fig. 4, lanes p-x). Substitution of glycine for serine at position 955 (S955G) and arginine for leucine at position 961 (L961R) inhibited tyrosine phosphorylation of Shc by NGF whereas substitution of leucine for serine at 955 (S955L) augmented Shc phosphorylation. Tyrosine phosphorylation of Shc by the different Trk/IRs also correlated with their ability to stimulate the formation of a complex between Shc and GRB2 (data not shown). Gross changes in structure or failure to autophosphorylate a critical tyrosine on the IR are unlikely to account for the failure of S955G or L961R Trk/IR to phosphorylate Shc since IRS-1 is phosphorylated normally by these receptors (Fig. 4).
In agreement with our results, several studies have shown that, in addition to NPX-Tyr(P), a leucine or hydrophobic amino acid at a position Ϫ5 from the NPXY phosphotyrosine (Tyr(P)Ϫ5) (equivalent to Ser-955 of the IR) is critical for binding the PTB/PI domain of Shc. Deletion of this leucine or substitution of alanine or glycine markedly reduced binding of the PTB/PI domain of Shc to NPXY containing phosphopeptides (7,11,12,22). In addition, Kavanaugh et al. (9) found that a hydrophobic or aromatic residue at the (Tyr(P)ϩ1) position (equivalent to Leu-961 of the IR) was important for the binding of the PTB/PI domain of Shc to c-ErbB-2 (9). The three-dimensional structure of Shc complexed to a phosphopeptide as recently determined by nuclear magnetic resonance (NMR) may help explain the role of the Tyr(P)Ϫ5 and Tyr(P)ϩ1 positions in binding of Shc's PTB/PI domain to NPXY phosphopeptides (36). A hydrophobic amino acid at the Tyr(P)Ϫ5 position contacts a hydrophobic pocket formed by the PTB/PI domain of Shc, and the amino acid at the Tyr(P)ϩ1 position forms a hydrophobic interaction with the aliphatic side chains of arginine 67 of Shc. These structural findings, together with the finding that phosphorylation of Shc by the mutant Trk/IRs correlates with binding of Shc's PTB/PI domain to other NPX-Tyr(P)-containing phosphopeptides, support a model in which binding of Shc via its PTB/PI domain directly to the IR is critical for insulin-

Phosphorylation of Shc by Insulin Receptor 3961
stimulated Shc phosphorylation in cells. In addition, the inability of L961R Trk/IR to tyrosine phosphorylate Shc may explain why the IL-4R binds and mediates tyrosine phosphorylation of IRS-1 but not Shc. The IL-4R ␣ subunit, which phosphorylates IRS-1 but not Shc (19,37), is similar to L961R Trk/IR in that it contains an arginine at the Tyr(P)ϩ1 position rather than the hydrophobic amino acid, which is present in receptor tyrosine kinases that bind the PTB/PI domain of Shc (Fig. 3B). While this paper was in preparation, He et al. (38) reported an analysis of the interaction of Shc and IRS-1 with IRs containing a series of amino acid substitutions surrounding the NPXY motif using the yeast two-hybrid system. We have been able to extend some of their observations to Shc phosphorylation in vivo. However, in contrast to the findings reported here, He et al. (38) did not find that serine at position 955 was important for binding the PTB/PI domain of Shc. Interestingly, He et al. (38) also demonstrated that substitution of leucine at position 952 and tyrosine at 953 with alanine led to reduced IRS-1 but not Shc binding. These findings led the authors to conclude that hydrophobic residues at these two positions are critical for binding and tyrosine phosphorylation of IRS-1 by the insulin and IL-4Rs. Our demonstration that substitution of tyrosine 953 with phenylalanine (Y953F) does not impair tyrosine phosphorylation of IRS-1 (Fig. 4, lanes m-o) is consistent with their conclusion that a bulky hydrophobic residue at this position is critical for in vivo binding and phosphorylation of IRS-1. However, in contrast to their results, substitution of alanine for leucine in Trk/IR (L952A) did not reduce IRS-1 phosphorylation in vivo by NGF (data not shown).
Shc and IRS-1 appear to bind the IR with a relatively low affinity compared with the binding of SH2 domains to their target proteins or the binding of the PTB/PI domain of Shc to other targets such as the EGFR or middle T antigen. For example, as stated above we and others have been unable to co-immunoprecipitate Shc with the IR from insulin-stimulated cell lysates (17,18,25). In addition, we have been unable to demonstrate binding of a GST-Shc fusion protein containing amino acids 1-209 of Shc to either the activated wt IR or the chimeric Trk/IR by far Western analysis, although binding to the activated EGFR could be easily detected (data not shown). The presence of a serine rather than a hydrophobic amino acid at the Tyr(P)Ϫ5 position in the IR may account for the low affinity binding of Shc to the IR. It is possible that this low affinity binding facilitates signaling. The interaction of Shc, IRS-1, and IRS-2 with the IR, while being of a sufficient affinity to localize these molecules adjacent to the IR kinase to promote phosphorylation, may be of a low enough affinity to allow these molecules to rapidly dissociate from the activated receptor following phosphorylation. The binding affinity of Shc and IRS-1 to the IR may be reduced as a result of a rapid dissociation rate from the activated IR. Such rapid dissociation of Shc, IRS-1, and IRS-2 from the IR may ensure that all these molecules that compete for the same binding site on the IR have a chance to bind and become phosphorylated. In addition, the rapid rate of dissociation from the receptor may enable IRS-1 to target active associated signaling molecules such as phosphatidylinositol 3-kinase to specific locations in the cell.
In summary, the findings reported here support the idea that Shc must bind directly to the activated IR via its PTB/PI domain to become tyrosine-phosphorylated in vivo. Analysis of the mutants reported in this study together with those reported by other investigators (38) support a model in which both Shc and IRS-1 recognize a core NPX-Tyr(P) motif on the IR, with amino acids surrounding this motif being critical for binding either IRS-1 or Shc in cells. The mutant IRs described in this report should provide a powerful tool to uncover biological functions that are predominantly mediated by Shc or IRS-1 in insulin-stimulated cells.