(Received for publication, March 21, 1997, and in revised form, May 15, 1997)
From the Department of Internal Medicine and
Biological Chemistry and the
Howard Hughes
Medical Institute, University of Michigan Medical School, Ann Arbor,
Michigan 48109-0650, the § Department of Pharmacology,
New York University Medical Center, New York, New York 10016, the
** Department of Metabolic Medicine, Royal Postgraduate Medical School,
London W12 ONN, United Kingdom, and the ¶ Laboratoire
d'Oncologie Moleculaire, U1119 INSERM, 13009 Marseille, France
The phosphotyrosine interaction (PI)/phosphotyrosine binding (PTB) domain of Shc binds specific tyrosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT). Phenylalanine 198 (Phe198) 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 transformation. However, expression of a slightly longer construct, Shc 1-260, which encompasses Tyr239/Tyr240, a novel Shc tyrosine phosphorylation site, did not block transformation. This was found to be due to the ability of Shc 1-260 to become tyrosine-phosphorylated and bind Grb2. Furthermore, full-length Shc in which Tyr239/Tyr240 had been mutated to phenylalanine did not become tyrosine-phosphorylated or bind Grb2 but did inhibit colony formation in soft agar. Conversely, p52 Shc carrying a mutation in the other tyrosine phosphorylation site, Tyr317, became heavily tyrosine-phosphorylated, bound Grb2, and gave rise to colonies in soft agar.
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 (1-5). 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 PI1/PTB domain (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, Tyr317 (1), and the more recently identified tyrosine phosphorylation sites, Tyr239/Tyr240 (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 (21-26). 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
XNPXpY motif by interacting
with the hydrophobic amino acid at position
5 and the asparagine at
3 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 (32-34). 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.
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/PTB+Y239/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 RetrovirusHelper-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 Ca2PO4 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 µg/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.
AntibodiesAnti-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 CultureNIH 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 µg/ml streptomycin, and 0.5 mg/ml Geneticin.
Cell Lysis, Immunoprecipitation, and Western BlottingConfluent 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 × 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 AssayThis assay was carried out in standard six-well tissue culture plates. Approximately 3 × 104 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 × 20) after 14 days.
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. Tyr317 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 Tyr239/Tyr240 (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 Tyr239/Tyr240 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/PTB+Y239/Y240) tagged with the Myc epitope. Shc PI/PTB and Shc PI/PTB+Y239/Y240 retroviruses were generated and used to infect NIH 3T3 cells stably transformed by MT which were then placed under puromycin (2 µg/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/PTB+Y239/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
Tyr239/Tyr240 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/PTB+Y239/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/PTB+Y239/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/PTB+Y239/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/PTB+Y239/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/PTB+Y239/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/PTB+Y239/Y240 on the behavior in
soft agar of NIH 3T3 cells stably transformed by MT. Cells were seeded
at a density of 3 × 104/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/PTB+Y239/Y240 did not inhibit
transformation (Fig. 5C), presumably because of the ability
of the Shc PI/PTB+Y239/Y240 domain to become tyrosine-phosphorylated
and bind Grb2 (Fig. 3). Shc PI/PTB+Y239/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).
|
In an effort to determine the
relative significance of the two Shc tyrosine phosphorylation sites,
Tyr317 and the recently identified
Tyr239/Tyr240, 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 µg/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 overexpressing 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
Tyr239/Tyr240 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.
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 Phe198 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
Phe198 is important for establishing contact with the
hydrophobic residue at the 5 position and the asparagine at the
3
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 tyrosine-phosphorylated 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/PTB+Y239/Y240, a construct encompassing the adjacent, recently identified Shc tyrosine phosphorylation site Tyr239/Tyr240 (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/PTB+Y239/Y240 in a soft agar assay. Interestingly, we found that overexpression of the Shc PI/PTB alone acted in a dominant interfering fashion, inhibiting MT-induced transformation. However, the ShcPI/PTB+Y239/Y240 did not inhibit MT-mediated transformation. This was because of the ability of Shc PI/PTB+Y239/Y240 to become tyrosine-phosphorylated and bind Grb2. In additional studies using p52 Shc in which Tyr239/Tyr240 and Tyr317 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 Tyr239/Tyr240 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/PTB+Y239/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, Tyr239/Tyr240 may have the role as the major tyrosine phosphorylation and Grb2 binding site, whereas in other systems, Tyr317 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 Tyr239/Tyr240 are conserved among the Shc family members (11, 31), suggesting a conservation of function. In contrast to Tyr317, which is absent in Drosophila Shc, Tyr239/Tyr240 is conserved in Drosophila Shc (49), suggesting that the function of Tyr239/Tyr240 was established early in evolution and may be conserved between Drosophila and man, whereas the function of Tyr317 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 Tyr239/Tyr240. This phosphorylation is crucial for Shc binding to Grb2 and MT-induced transformation.
We thank Dr. Sarah Courtneidge for the generous gift of NIH 3T3 cells stably transformed by MT, and Dr. Ivan Dikic and Dr. Yossi Schlessinger for the polyclonal anti-Shc antibody.