The Role of the Shc Phosphotyrosine Interaction/Phosphotyrosine Binding Domain and Tyrosine Phosphorylation Sites in Polyoma Middle T Antigen-mediated Cell Transformation*

The phosphotyrosine interaction (PI)/phosphoty-rosine binding (PTB) domain of Shc binds specific ty- rosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT). Phenylalanine 198 (Phe 198 ) has been identified as a crucial residue involved in the interaction of the Shc PI/PTB with phosphopeptides. In NIH 3T3 cells expressing MT, p52 Shc carrying the F198V mutation is weakly phosphorylated and does not bind MT or Grb2. Overexpression of the PI/PTB domain alone as Shc amino acids 1–238 acted in a dominant interfering fashion blocking MT-induced transforma- tion. However, expression of a slightly longer construct, Shc 1–260, which encompasses Tyr 239 /Tyr 240 , a novel Shc tyrosine phosphorylation site, did not block transforma- tion. This was found to be due to the ability of Shc 1–260 to become tyrosine-phosphorylated and bind Grb2. Fur- thermore, full-length

Shc is a ubiquitously expressed adaptor protein that exists in three different isoforms, p46 Shc, p52 Shc, and p66 Shc, which differ only in the extent of their amino-terminal sequences. The p46 and p52 isoforms arise by alternate translational start sites, whereas the p66 form is generated by alternate splicing (1). Shc becomes tyrosine-phosphorylated after cell stimulation with a wide variety of growth factors and cytokines (1)(2)(3)(4)(5). It is also phosphorylated in cells expressing activated nonreceptor tyrosine kinases (6,7) and other tyrosine-phosphorylated proteins (8,9). p52 Shc contains an amino-terminal PI 1 /PTB do-main (10 -13), a central collagen homology domain (CH1), and a carboxyl-terminal SH2 domain (1). The CH1 domain contains what was previously thought to be the principal Shc tyrosine phosphorylation site, Tyr 317 (1), and the more recently identified tyrosine phosphorylation sites, Tyr 239 /Tyr 240 (14,15). Shc overexpression results in transformation of fibroblasts (1) as well as differentiation of PC12 cells and is implicated in activating Ras via its association with Grb2/Sos (16).
Shc has a unique ability to interact with tyrosine-phosphorylated proteins bearing the sequence ⌿XNPXpY (9,17,18), a sequence that is present in many Shc-associated proteins. This interaction is unusual for SH2 domains that usually select specificity based on amino acids carboxyl-terminal to the phosphotyrosine (19,20). This contradiction was resolved when it was determined that Shc bound to the ⌿XNPXpY motif through the amino-terminal PI/PTB domain rather than through its carboxyl-terminal SH2 domain (21)(22)(23)(24)(25)(26). The PI/ PTB domain represents a novel protein binding domain that has been identified in other proteins (27) suggesting a general role for this domain in protein-protein interactions and signal transduction.
Random mutagenesis of the Shc PI/PTB identified phenylalanine residue 198 of the Shc PI/PTB domain as essential for binding to the ⌿XNPXpY motif. Mutation of this residue to valine abrogated both the ability of the Shc PI/PTB domain to bind to the activated epidermal growth factor receptor (28) and the ability of p52 Shc to undergo phosphorylation by the insulin receptor (29). The importance of this residue in mediating Shc PI/PTB domain binding was confirmed by a recent structural analysis of the Shc PI/PTB domain which revealed the importance of the phenylalanine 198 in directly contacting the ⌿XN-PXpY motif by interacting with the hydrophobic amino acid at position Ϫ5 and the asparagine at Ϫ3 relative to the phosphotyrosine (30). It is interesting to note that phenylalanine 198 is conserved in the majority of other PI/PTB domain-containing proteins identified to date (28). Studies by other groups have indicated that in the case of the Shc PI/PTB domain, arginine 175, which is also evolutionarily conserved, is important for ligand binding and is involved in phosphotyrosine recognition (24,30).
Middle tumor antigen (MT), the principal transforming protein of polyoma virus, has no intrinsic enzyme activity and exerts its transforming effect on cells by associating with and modulating the activities of various cellular proteins involved in cell proliferation such as c-Src, c-Fyn and c-Yes (32)(33)(34). Genetic analysis has revealed that the tetrameric sequence NPTY is an essential requirement for MT-mediated transformation (35,36). This NPTY motif is required for the interaction between MT and Shc resulting in Shc tyrosine phosphorylation and association with Grb2/Sos, leading to Ras activation and cellular growth (9). Initially, the SH2 domain of Shc was implicated in this interaction (8). However, more recent data tend to suggest that the Shc PI/PTB domain is involved (22). In this study, our goal was to determine the exact role of the Shc PI/PTB domain in MT-mediated signal transduction.

EXPERIMENTAL PROCEDURES
DNA Constructs and Expression Vector-Using murine p52 Shc cDNA as a template, p52 ShcF198V was created by site-directed mutagenesis using the appropriate oligonucleotides and standard polymerase chain reaction (37). The Shc deletion mutants Shc PI/PTB (Shc 1-238) and Shc PI/PTBϩY239/Y240 (Shc 1-260) were generated by polymerase chain reaction using standard techniques (37). Shc tyrosine mutant constructs p52 ShcY239F/Y240F, p52 ShcY317F, and p52 ShcY239F/Y240F/Y317F were generated using the Transformer Mutagenesis Kit (CLONTECH, Palo Alto, CA). Using the EcoRI site, all constructs were then subcloned into pBabe-Myc vector. pBabe-Myc was generated by cloning a 92-base pair insert encoding the Myc epitope tag amino acid sequence MEQKLISEEDLLEGSPGILD (38, 39) into a retroviral expression vector pBabe, containing a puromycin resistance marker (40). The Myc epitope is preceded by a Kozak sequence and followed by sites allowing cloning of cDNAs. All constructs were sequenced with Sequenase version 2.0 (U. S. Biochemical Corp.).
Generation of Retrovirus-Helper-free infectious retrovirus was produced by transiently transfecting the various wild type and mutant pBabe p52 Shc constructs into the retroviral packaging cell line, Bosc 293, using Ca 2 PO 4 precipitation (40 -43). The resultant virus was used to infect NIH 3T3 cells stably transformed by MT (40,44). Infected cells were then placed under puromycin selection (2 g/ml) for 10 -14 days.
Resistant clones were selected, and expression of the appropriate construct was confirmed by immunoblotting with anti-Myc or anti-Shc antibody. Pooled transformants were used in all experiments. Attempts to obtain stable cells lines expressing the minimum Shc PI/PTB domain as Shc 1-209 were unsuccessful.
Antibodies-Anti-Myc monoclonal antibody 9E10 directed against a peptide with the sequence MEQKLISEEDLN was used for immunoprecipitation and immunoblotting and monoclonal anti-MT (PAb 762) antibody was used for immunoprecipitation. Polyclonal anti-Shc antibody used for immunoprecipitation was a gift from Dr. Ivan Dikic and Dr. J. Schlessinger (NYU Medical Center, New York), and the anti-Shc antibody used for immunoblotting was purchased from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine antibodies were prepared as described previously (45,46). Anti-Grb2 antibody was purchased from Santa Cruz Biotech (Santa Cruz, CA).
Cell Culture-NIH 3T3 cells stably transformed by MT were a gift from Dr. Sarah Courtneidge (Sugen, Redwood City, CA) and have been described elsewhere (47). These cells were grown in Dulbecco's modified Eagle's medium with 10% calf serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 0.5 mg/ml Geneticin.
Cell Lysis, Immunoprecipitation, and Western Blotting-Confluent dishes of cells expressing the various Shc proteins were washed with ice-cold phosphate-buffered saline and lysed in 1% Triton X-100 lysis buffer containing protease and phosphatase inhibitors (45). Lysate protein content was normalized using the Bio-Rad protein assay. Cell lysates were incubated for 2 h at 4°C with the appropriate antibody that had been covalently bound to protein A-Sepharose beads (48). The beads were then washed three times in HNTG (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100), boiled in 1 ϫ sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose. Immunoblotting was performed as described (45,46). Blots were visualized using chemiluminescence (NEN Life Science Products).
Soft Agar Assay-This assay was carried out in standard six-well tissue culture plates. Approximately 3 ϫ 10 4 cells were seeded into an upper layer containing modified Eagle's medium, 0.36% agar (Difco) supplemented with 10% calf serum. The colonies were photographed (magnification ϫ 20) after 14 days.

FIG. 1. Schematic diagram depicting Shc constructs used in these studies.
Phe 198 is an amino acid residue crucial for the binding of the PI/PTB domain to phosphopeptides which, when mutated to valine (F198V), abrogates this binding ability. Tyr 239 /Tyr 240 and Tyr 317 are the major Shc tyrosine phosphorylation sites. These constructs were cloned into pBabe-Myc, a retroviral expression vector that allowed an in-frame amino-terminal Myc tag. Helper-free retrovirus was produced as described under "Experimental Procedures" and used to infect NIH 3T3 cells stably transformed by MT. Clonal cell lines were selected using puromycin (2 g/ml, Sigma) as the selectable marker.

The Shc PI/PTB Domain Is Crucial for Shc Phosphorylation in MT-transformed Cells-
To determine the importance of the Shc PI/PTB domain in MT signal transduction we undertook a comparative analysis of Shc tyrosine phosphorylation and Grb2 binding using wild type p52 Shc and p52 ShcF198V tagged with the Myc epitope ( Fig. 1). High titer p52 Shc and p52 ShcF198V retroviruses were generated and used to infect NIH 3T3 cells stably transformed by MT. Clonal cell lines were developed and maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Cell lines were selected which had equal expression of transfected Shc wild type and mutant proteins as determined by immunoblotting. To determine the tyrosine phosphorylation status of wild type versus mutant Shc, cell lysates expressing either p52 Shc or p52 ShcF198V were immunoprecipitated with the anti-Myc antibody, resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with anti-phosphotyrosine antibody and anti-Grb2 antibody. Wild type p52 Shc became heavily tyrosine-phosphorylated and bound to Grb2, whereas p52 ShcF198V failed to become tyrosine-phosphorylated and did not bind Grb2 (Fig. 2, A and B). In both cases, membranes were stripped and reprobed with the anti-Myc antibody to ensure equal and efficient imunoprecipitation (Fig. 2C). To determine the ability of wild type and mutant Shc to bind MT, cells lysates expressing the respective proteins were lysed and immunoprecipitated with monoclonal anti-MT antibody. Immune complexes were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the anti-Shc antibody. Wild type Shc coimmunoprecipitated with MT, whereas p52 Shc carrying the F198V mutation did not (Fig. 2D).
Shc 1-260 Becomes Tyrosine-phosphorylated and Binds to Grb2-The CH1 domain of Shc apparently provides a scaffold for further protein-protein interactions. Tyr 317 is contained within the pYVNV consensus binding motif for the Grb2 SH2 domain (19,20) and has traditionally been regarded as the major tyrosine phosphorylation and Grb2 binding site of Shc (1). More recent data have identified Tyr 239 /Tyr 240 (which are located in the CH1 domain just downstream of the Shc PI/PTB domain) as two novel and major tyrosine phosphorylation sites of Shc (14,15). It is of interest to note that CH1 residues homologous to ShcY239 and ShcY240 are present in Drosophila Shc, whereas ShcY317 is absent (49). The fact that Tyr 239 / Tyr 240 are highly conserved in evolution suggests that the phosphorylation of these residues is of fundamental importance. In an effort to develop a dominant interfering Shc PI/ PTB domain to study the role of the Shc PI/PTB domain in MT-mediated signal transduction, we overexpressed Shc 1-238 (Shc PI/PTB) and Shc 1-260 (Shc PI/PTBϩY239/Y240) tagged with the Myc epitope. Shc PI/PTB and Shc PI/PTBϩY239/Y240 retroviruses were generated and used to infect NIH 3T3 cells stably transformed by MT which were then placed under puromycin (2 g/ml) selection.
Clones expressing the desired protein were selected for further analysis. The Shc PI/PTB domain alone did not become tyrosine-phosphorylated (Fig. 3A) and did not bind to Grb2 (Fig. 3B). In contrast, the Shc PI/PTBϩY239/Y240 became heavily tyrosine-phosphorylated (Fig. 3A) and bound to Grb2 (Fig. 3B). In both cases, immunoblots were stripped and reprobed with the anti-Myc antibody to ensure equal and efficient immunoprecipitation (Fig. 3C). These findings suggest that Tyr 239 /Tyr 240 is an important Shc tyrosine phosphorylation site and a Grb2 binding site in MT-transformed fibroblasts.
Overexpression of Either the Shc PI/PTB or Shc PI/ PTBϩY239/Y240 Inhibits the Binding of Endogenous Shc to MT and Grb2-To analyze further the role of the Shc PI/PTB domain in MT-mediated signal transduction, we investigated the ability of the Shc PI/PTB domain alone and the Shc PI/ PTBϩY239/Y240 to interfere with endogenous p52 Shc signaling in MT-transformed NIH 3T3. Lysates from NIH 3T3 cells transformed by MT expressing either the Shc PI/PTB alone or Shc PI/PTBϩY239/Y240 were immunoprecipitated with anti-MT antibody (Fig. 4, A and B) or anti-Shc antibody (Fig. 4C). Immune complexes were separated by SDS-PAGE and immunoblotted with anti-Shc antibody (Fig. 4A), anti-phosphotyrosine antibody (Fig. 4B), and anti-Grb2 antibody (Fig. 4C). The anti-Shc antibody is directed against the Shc SH2 domain and does not immunoprecipitate the Shc PI/PTB proteins. Overexpression of the Shc PI/PTB domain alone or Shc PI/ PTBϩY239/Y240 inhibited the binding of endogenous p52 Shc to MT (Fig. 4, A and B) and Grb2 (Fig. 4C). These data suggest that the Shc PI/PTB domain is directly involved in interaction with MT. When overexpressed in cells transformed by MT, it displaces endogenous Shc from MT and reduces the binding of endogenous Shc to Grb2.

Differential Effects of the Shc PI/PTB versus Shc PI/ PTBϩY239/Y240 on Growth of MT-transformed Cells in Soft
Agar-In an attempt to investigate the dominant interfering effect of the Shc PI/PTB domain on MT-induced transformation, we investigated the effect of overexpression of either the Shc PI/PTB or Shc PI/PTBϩY239/Y240 on the behavior in soft agar of NIH 3T3 cells stably transformed by MT. Cells were seeded at a density of 3 ϫ 10 4 /well in a six-well dish and supplemented every 5 days with 10% calf serum. Colonies were photographed after 14 days. Overexpression of the Shc PI/PTB domain acted in a dominant interfering fashion to inhibit MT-induced transformation (Fig. 5B). Overexpression of the Shc PI/PTBϩY239/Y240 did not inhibit transformation (Fig. 5C), presumably because of the ability of the Shc PI/PTBϩY239/ Y240 domain to become tyrosine-phosphorylated and bind Grb2 (Fig. 3). Shc PI/PTBϩY239/Y240 overexpression resulted in colonies similar in size to those seen in uninfected cells (Fig.  5A). The inhibitory effect of the Shc PI/PTB domain on colony formation in soft agar was abrogated by the introduction of the F198V mutation (Fig. 5D, Table I  and the recently identified Tyr 239 /Tyr 240 , in MT signal transduction, the appropriate tyrosine residues were mutated to phenylalanine and incorporated into full-length p52 Shc. The resultant Myc epitope-tagged Shc constructs, p52 ShcY239F/ Y240F, p52 ShcY317F, and p52 ShcY239F/Y240F/Y317F, were used to generate high titer retrovirus that was then used to infect NIH 3T3 cells stably transformed by MT. Cells were placed under puromycin selection (2 g/ml) and screened for protein expression. Confluent dishes of cells were lysed and lysates subjected to immunoprecipitation with anti-Myc antibody. Immune complexes were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibody (Fig. 6A), anti-Grb2 antibody (Fig. 6B), and anti-Myc antibody (Fig. 6C) to ensure equal and efficient immunoprecipitation. p52 ShcY317F behaved in a fashion similar to that of wild type p52 Shc, becoming heavily tyrosine-phosphorylated (Fig. 6A) and binding to Grb2 (Fig. 6B). In contrast, p52 ShcY239F/Y240F failed to become tyrosine-phosphorylated (Fig. 6A) and did not bind to Grb2 (Fig. 6B). p52 ShcY239F/ Y240F/Y317F did not become tyrosine-phosphorylated (Fig.  6A) and did not bind to Grb2 (Fig. 6B). These data suggest that Y239F/Y240F is an important tyrosine phosphorylation site of p52 Shc and is essential for Grb2 binding and downstream signaling in the MT-mediated signal transduction.

Differential Effects of p52 ShcY239F/Y240F versus p52 ShcY317F on Growth of MT-transformed Cells in Soft
Agar-To determine the effect of the Shc tyrosine mutations on cellular growth, we investigated the growth in soft agar of NIH 3T3 cells stably transformed by MT and expressing wild type p52 Shc, p52 ShcY239F/Y240F, p52 ShcY317F, or p52 Shc Y239F/Y240F/Y317F. Cells were seeded in soft agar and supplemented with 10% calf serum. Growth was assessed every 2 days and colonies photographed after 14 days. Cells overex-pressing p52 Shc formed colonies in soft agar similar in size to those seen in cells infected with vector alone (Fig. 7, A and B). Cells overexpressing p52 ShcY317F also formed colonies similar in size to those seen with wild type p52 Shc (Fig. 7D). However, cells expressing p52 ShcY239F/Y240F either formed very small colonies or failed to form colonies at all (Fig. 7C). Cells expressing p52 Shc carrying all three tyrosine mutations also failed to form colonies in soft agar (Fig. 7E, Table I). These data suggest that the Tyr 239 /Tyr 240 tyrosine phosphorylation site of p52 Shc is crucial for signaling in the MT-transformed cells, and mutation of these residues to phenylalanine results in growth inhibition. DISCUSSION It is now well established that the Shc PI/PTB domain is a protein module that can mediate the formation of protein complexes via its recognition of specific phosphotyrosine-containing motifs (10,21,22,27,50). Amino acid residue Phe 198 of the Shc PI/PTB domain has been identified as a crucial residue for binding of the Shc PI/PTB domain, and its mutation to valine abrogated the ability of p52 Shc to bind activated epidermal growth factor receptor (28) and inhibited Shc phosphorylation by the insulin receptor (29). The importance of this residue has been confirmed by structural analysis of the Shc PI/PTB domain complexed with the TrkA phosphopeptide, which revealed that Phe 198 is important for establishing contact with the hydrophobic residue at the Ϫ5 position and the asparagine at the Ϫ3 position relative to the phosphotyrosine (30).
We have analyzed the role of the Shc PI/PTB domain in MT-mediated signal transduction. By using the retroviral expression system, we have generated high titer retrovirus that was then used to infect NIH 3T3 cells stably transformed by MT. We have demonstrated that p52 Shc carrying the F198V mutation was poorly phosphorylated and failed to bind to Grb2 or MT, whereas wild type p52 Shc became heavily tyrosinephosphorylated and coimmunoprecipitated with both Grb2 and MT. This was presumably the result of the nullifying effect of the F198V mutation on the binding ability of the Shc PI/PTB domain rendering p52 ShcF198V unable to bind to MT and therefore inhibiting its tyrosine phosphorylation and association with Grb2. When we overexpressed the Shc PI/PTB domain alone in MT-transformed, cells we found that it did not become tyrosine-phosphorylated, nor was it able to bind to Grb2. It did, however, inhibit the phosphorylation and binding of endogenous Shc to MT and Grb2. When we overexpressed the Shc PI/PTBϩY239/Y240, a construct encompassing the adjacent, recently identified Shc tyrosine phosphorylation site Tyr 239 /Tyr 240 (14,15), we found that this protein became heavily tyrosine-phosphorylated and formed a complex with Grb2 in MT-transformed cells. To determine the effect on transformation in vivo, we used MT-transformed cells expressing either Shc PI/PTB or ShcPI/PTBϩY239/Y240 in a soft agar assay. Interestingly, we found that overexpression of the Shc PI/PTB alone acted in a dominant interfering fashion, inhibiting MTinduced transformation. However, the ShcPI/PTBϩY239/Y240 did not inhibit MT-mediated transformation. This was because of the ability of Shc PI/PTBϩY239/Y240 to become tyrosinephosphorylated and bind Grb2. In additional studies using p52 Shc in which Tyr 239 /Tyr 240 and Tyr 317 were mutated to phenylalanine either separately or together, we found that p52 ShcY239F/Y240F failed to become tyrosine-phosphorylated, did not bind to Grb2, and inhibited MT-mediated transformation on soft agar. In contrast, p52 ShcY317F behaved in a fashion similar to that of wild type p52 Shc, becoming heavily tyrosine-phosphorylated, binding to Grb2, and forming colonies in soft agar similar to those seen in uninfected cells. Taken together, these data confirm the importance of the Shc PI/PTB domain in MT-mediated signal transduction and, in addition, suggest an important role for Tyr 239 /Tyr 240 in Shc tyrosine phosphorylation and Grb2 binding.
Shc is an important adaptor protein responsible for linking many activated proteins to the Ras pathway via Grb2/Sos. The presence in Shc of both a PI/PTB domain and an SH2 domain presumably gives Shc the ability to interact with a large number of tyrosine-phosphorylated proteins in many different systems. Although we detect a very important role for the Shc PI/PTB domain in MT-induced transformation, we could detect little role for the SH2 domain in this system. A Shc construct containing residues 1-260 (Shc PI/PTBϩY239/Y240) transformed cells nearly as well as wild type p52 Shc, even though it lacked the SH2 domain. The presence of two Grb2 binding sites in mammalian Shc proteins adds to the complexity of this signaling molecule. It is possible that in some systems, such as the MT system, Tyr 239 /Tyr 240 may have the role as the major tyrosine phosphorylation and Grb2 binding site, whereas in other systems, Tyr 317 is the main player (51,52). It is also possible that in some systems, both may play a role. It is of great interest that Tyr 239 /Tyr 240 are conserved among the Shc family members (11,31), suggesting a conservation of function. In contrast to Tyr 317 , which is absent in Drosophila Shc, Tyr 239 / Tyr 240 is conserved in Drosophila Shc (49), suggesting that the function of Tyr 239 /Tyr 240 was established early in evolution and may be conserved between Drosophila and man, whereas the function of Tyr 317 may have evolved more recently.
In conclusion, our data demonstrate that the Shc PI/PTB domain interacts with tyrosine-phosphorylated MT. This interaction is essential for the tyrosine phosphorylation of Shc which occurs predominantly at Tyr 239 /Tyr 240 . This phosphorylation is crucial for Shc binding to Grb2 and MT-induced transformation.