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• W2000343242 abstract "The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the “Grb2-adapter” model of Shp2 action. The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the “Grb2-adapter” model of Shp2 action. Many signaling pathways regulating cell growth, proliferation, differentiation, and migration involve protein-tyrosyl phosphorylation which, in turn, is controlled by protein-tyrosine kinases (PTKs) 1The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; RPTPα, receptor PTP α; RTK, receptor tyrosine kinase; SH2, Src homology; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; IGF-1, insulin-like growth factor 1; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; FGF, fibroblast growth factor; Csw, corkscrew; WT, wild type; SFK, Src family kinase; SYF, cells deficient in Src, Fyn, and Yes.1The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; RPTPα, receptor PTP α; RTK, receptor tyrosine kinase; SH2, Src homology; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; IGF-1, insulin-like growth factor 1; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; FGF, fibroblast growth factor; Csw, corkscrew; WT, wild type; SFK, Src family kinase; SYF, cells deficient in Src, Fyn, and Yes. and protein-tyrosine phosphatases (PTPs). Most peptide growth factor receptors are transmembrane PTKs, termed receptor tyrosine kinases (RTKs). Ligand binding activates the intrinsic PTK activity of RTKs, leading to the phosphorylation of several substrates, most of which have Src homology-2 (SH2) or phosphotyrosine-binding domains. Tyrosyl phosphorylation of such signal relay molecules can alter their structure and/or function, thereby regulating enzyme activity, protein localization, and/or the assembly of multimeric signaling complexes (1Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2231) Google Scholar). These events, in turn, control downstream signaling pathways, such as the Ras/Raf/MEK/ERK cascade (2Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2266) Google Scholar, 3Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1123) Google Scholar). Shp2 is a ubiquitously expressed non-transmembrane PTP that plays an essential role in most, if not all, RTK signaling pathways. In all of these pathways, Shp2 function is required for normal activation of the ERK MAPK and its downstream transcriptional targets (4Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar, 5Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar, 6Van Vactor D. O'Reilly A.M. Neel B.G. Curr. Opin. Genet. Dev. 1998; 8: 112-126Crossref PubMed Scopus (131) Google Scholar, 7Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar). Experiments using dominant negative (catalytically inactive) Shp2 as well as studies of primary fibroblasts and cell lines from Shp2 mutant mice generated by homologous recombination (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar) have shown that Shp2 acts upstream of Ras in insulin/insulin-like growth factor 1 (IGF-1) (9Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar) and epidermal growth factor (EGF) signaling (10Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (184) Google Scholar). Although likely, it has not been shown that Shp2 acts upstream of Ras in other RTK signaling pathways. Moreover, studies of mammalian cell systems as well as genetic analyses of the Drosophila Shp2 ortholog Corkscrew (Csw) suggest that Shp2 also acts downstream of Ras and/or in a parallel signaling pathway (5Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar, 6Van Vactor D. O'Reilly A.M. Neel B.G. Curr. Opin. Genet. Dev. 1998; 8: 112-126Crossref PubMed Scopus (131) Google Scholar, 7Feng G.S. Exp. Cell Res. 1999; 253: 47-54Crossref PubMed Scopus (252) Google Scholar). Like its close relative Shp1, Shp2 contains two N-terminal SH2 domains, a tyrosine phosphatase catalytic (PTP) domain, a C terminus with two tyrosyl phosphorylation sites, Tyr-542 and Tyr-580, and an interposed proline-rich domain (Fig. 1A). Multiple studies have established that PTP activity is required for all actions of Shp2, including its role in the Ras/ERK pathway (5Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar, 6Van Vactor D. O'Reilly A.M. Neel B.G. Curr. Opin. Genet. Dev. 1998; 8: 112-126Crossref PubMed Scopus (131) Google Scholar). In contrast, the molecular details and functional consequences of tyrosyl phosphorylation of Shp2 have remained controversial. Shp2 undergoes phosphorylation at both Tyr-542 and Tyr-580 in response to many (but not all) stimuli, including many (but not all) peptide growth factors (11Feng G.S. Hui C.C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (582) Google Scholar, 12Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (488) Google Scholar, 13Lechleider R.J. Freeman Jr., R.M. Neel B.G. J. Biol. Chem. 1993; 268: 13434-13438Abstract Full Text PDF PubMed Google Scholar). Both of these sites conform to the consensus for binding to the Grb2 SH2 domain (YXNX), and, accordingly, tyrosyl-phosphorylated Shp2 binds Grb2. But whereas Bennett et al. reported that Tyr-542 is the major phosphorylation and Grb2 association site (14Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (341) Google Scholar), two other studies concluded that Tyr-580 is the major Grb2 binding site (15Vogel W. Ullrich A. Cell Growth & Differ. 1996; 7: 1589-1597PubMed Google Scholar, 16Keegan K. Cooper J.A. Oncogene. 1996; 12: 1537-1544PubMed Google Scholar). The reason(s) for these discrepancies is (are) not clear. The effects of tyrosyl phosphorylation on Shp2 enzymatic activity and RTK signaling are also in dispute. The finding that Grb2 binds tyrosyl-phosphorylated Shp2, together with the requirement for Shp2 upstream of Ras in the ERK pathway (see above), suggested an “adapter model” of Shp2 action (14Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (341) Google Scholar, 17Li W. Nishimura R. Kashishian A. Batzer A.G. Kim W.J. Cooper J.A. Schlessinger J. Mol. Cell. Biol. 1994; 14: 509-517Crossref PubMed Google Scholar). In this model, tyrosyl-phosphorylated Shp2 contributes to Ras (and, ultimately, ERK) activation by recruiting Grb2/Sos complexes to the vicinity of the plasma membrane; hence, Shp2 functions as an adapter. Notably, it is unclear how the requirement for Shp2 catalytic activity can be reconciled with this model. Others have argued that tyrosyl phosphorylation of Shp2 stimulates its catalytic activity (12Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (488) Google Scholar, 18Lu W. Gong D. Bar-Sagi D. Cole P.A. Mol. Cell. 2001; 8: 759-769Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Early studies found a temporal correlation between Shp2 tyrosyl phosphorylation and increased Shp2 activity (12Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (488) Google Scholar). However, upon growth factor stimulation, Shp2 binds via its SH2 domains to RTKs and/or scaffolding adapters (e.g. Gab/Dos, insulin receptor substrate (IRS), and fibroblast growth factor receptor substrate (FRS) family members) at the same time that it becomes tyrosyl phosphorylated (11Feng G.S. Hui C.C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (582) Google Scholar, 12Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (488) Google Scholar, 13Lechleider R.J. Freeman Jr., R.M. Neel B.G. J. Biol. Chem. 1993; 268: 13434-13438Abstract Full Text PDF PubMed Google Scholar). Because engagement of the SH2 domains of Shp2 markedly enhances PTP activity (19Barford D. Neel B.G. Structure. 1998; 6: 249-254Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), these early studies could not distinguish between the effects of tyrosyl phosphorylation and SH2 domain engagement on Shp2 catalytic activity. More recently, protein ligation techniques were used to introduce a phosphonate at either Tyr-542 or Tyr-580 of Shp2 (18Lu W. Gong D. Bar-Sagi D. Cole P.A. Mol. Cell. 2001; 8: 759-769Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Phosphonate incorporation at Tyr-542 resulted in intramolecular binding to the N-terminal SH2, whereas phosphono-Tyr-580 binds to the C-terminal SH2. Moreover, both phosphonate derivatives were found to stimulate catalytic activity (to an extent similar to SH2 domain engagement) and display enhanced biological activity in a microinjection assay. These data argue for an “enzyme activation” model for Shp2 tyrosyl phosphorylation. Because Shp2 rapidly autodephosphorylates, however, a stable phosphonate might not have the same effect in cells as would a transiently phosphorylated tyrosyl residue. The function of Grb2 binding in the enzyme activation model is also unclear. Most importantly, there has been no clear demonstration that tyrosyl phosphorylation of Shp2 is required for its signaling functions. Several studies have investigated the effects of over-expressing tyrosyl phosphorylation site mutants of Shp2 on RTK signaling in vertebrate systems. Bennett et al. found no effect of such mutants on EGF and PDGF signaling in transiently transfected 293 cells (20Bennett A.M. Hausdorff S.F. O'Reilly A.M. Freeman R.M. Neel B.G. Mol. Cell. Biol. 1996; 16: 1189-1202Crossref PubMed Scopus (226) Google Scholar), nor did these mutants inhibit FGF-induced ERK activation, elongation movements, or mesoderm induction in Xenopus embryos (21O'Reilly A.M. Neel B.G. Mol. Cell. Biol. 1998; 18: 161-177Crossref PubMed Scopus (96) Google Scholar). Csw also has a tyrosyl phosphorylation site that binds Grb2, and a mutant lacking this site rescues csw mutant embryos when expressed under heat shock promoter control (22Allard J.D. Herbst R. Carroll P.M. Simon M.A. J. Biol. Chem. 1998; 273: 13129-13135Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These results argue that the effects (if any) of tyrosyl phosphorylation on Shp2/Csw function can be bypassed, at least at sufficiently high levels of expression. Because all of the above studies used over-expression approaches, a requirement for Shp2 tyrosyl phosphorylation might have been obscured. To clarify the role of tyrosyl phosphorylation of Shp2, we used retroviral gene transduction to reconstitute 3T3-immortalized fibroblasts derived from Shp2 mutant mice (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar, 23Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (204) Google Scholar) with wild type (WT) Shp2 and Shp2 mutants lacking either or both tyrosyl phosphorylation sites. This approach allowed us to directly compare the signaling abilities of WT Shp2 and phosphorylation site mutants expressed at comparable levels. Our results show that tyrosyl phosphorylation is, in fact, required for normal levels and kinetics of ERK activation in some (FGF and PDGF) but not all (EGF) signaling pathways in fibroblasts. Our data also confirm our previous report that Tyr-542 is the major Grb2 binding site in tyrosylphosphorylated Shp2 and suggest that, in contrast to the predictions of the adapter model, Grb2 binding is not sufficient to mediate the signaling function of Shp2 in the Ras/ERK pathway. Cell Lines and Culture—3T3-immortalized fibroblasts (Ex3–/–cells) derived from a Shp2 exon 3-deficient mouse (23Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (204) Google Scholar) were maintained in Dulbecco's modified Eagles medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. cDNAs encoding WT human Shp2 (24Freeman Jr., R.M. Plutzky J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11239-11243Crossref PubMed Scopus (325) Google Scholar) and its Y542F, Y580F, and Y542,Y580F (14Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (341) Google Scholar) mutants were inserted into the retroviral vector pBABE-puro (25Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1886) Google Scholar). Additional details regarding the generation of these constructs are available from the corresponding author upon request. The resulting retroviral vectors were co-transfected with the Ecopack packaging vector (generously provided by Dr. D. G. Gilliland, Brigham and Women's Hospital, Boston, MA) into 293T cells (26Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). Twenty-four (24Freeman Jr., R.M. Plutzky J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11239-11243Crossref PubMed Scopus (325) Google Scholar) hours post-transfection, viral supernatants were collected and used to infect Shp2-mutant cells in the presence of Polybrene (4 μg/ml) for 4 h. Pools of infected cells were recovered following 1 week of puromycin (2 μg/ml) treatment and used for experiments. For growth factor stimulations, cells were starved for at least 16 h in serum-free Dulbecco's modified Eagle's medium and then exposed to EGF (20 ng/ml), IGF-1 (50 ng/ml), FGF (20 ng/ml), or PDGF (20 ng/ml). In some experiments, the Src-selective inhibitor SU6656 (Calbiochem) was added to a final concentration of 1 μm for 1 h before stimulation. Immunoprecipitations and Immunoblotting—Cells were washed three times with ice-cold phosphate-buffered saline and lysed in a buffer containing 1% (w/v) Triton X-100, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 10 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 0.5 μg/ml antipain, and 0.5 μg/ml pepstatin. Lysates were centrifuged at 10,000 × g at 4 °C for 30 min, and the protein concentration of clarified lysates was determined by BCA assay (Pierce). Immunoprecipitations and immunoblotting were performed as described previously (23Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (204) Google Scholar, 27Araki T. Yamada M. Ohnishi H. Sano S. Uetsuki T. Hatanaka H. J. Neurochem. 2000; 74: 659-668Crossref PubMed Scopus (27) Google Scholar). The monoclonal anti-phosphotyrosine antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY.), and monoclonal anti-Shp2 and anti-Grb2 antibodies were from Transduction Laboratories (Lexington, KY). Polyclonal anti-Shp2 and anti-MAPK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-phospho-MAPK and phospho-SHP-2 (Tyr-542) and (Tyr-580) antibodies were from Cell Signaling Technology (Beverly, Mass.). All immuno-reagents were used at the concentrations recommended by their manufacturers. MAPK Assays—Cells were lysed in 20 mm Tris HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm Na3VO4, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride and clarified by centrifugation at 10,000 × g for 30 min. Clarified lysates were incubated overnight at 4 °C with 2 μg of polyclonal anti-ERK2 antibodies (Santa Cruz Biotechnology). Protein A-Sepharose (30 μl) was added, and the mixture was rotated at 4 °C for 30–60 min. Immune complex kinase assays using myelin basic protein as substrate were performed as described previously (28Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (245) Google Scholar). Incorporated 32P was visualized by autoradiography and quantified by using NIH image 1.62. Data were evaluated by factorial analysis of variance and Newman-Keuls multiple range test. To clarify the physiological function of tyrosyl phosphorylation of Shp2, we reconstituted Ex3–/–fibroblasts with WT Shp2, mutants of either (Y542F or Y580F) or both (Y542F,Y580F) tyrosyl phosphorylation sites, or the parental retrovirus as a control (Fig. 1A). All experiments were performed with pools of retrovirally infected cells to average out possible inter-clonal variation. Immunoblot analysis using antibodies directed against the Shp2 C terminus revealed that the levels of expression of the reconstituted WT and mutant Shp2 proteins were comparable in each pool (Fig. 1B), thus enabling direct comparison of their signaling properties. An immunoreactive species smaller than WT Shp2 was also detected in each pool, including cells expressing the parental retrovirus alone. As described previously (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar), this truncated Shp2 species arises as a consequence of splicing around the neomycin resistance cassette that was used to replace exon 3. Although the N-terminally truncated protein has markedly increased PTP activity, because of its missing N-terminal SH2 domain it fails to target appropriately and, in all settings tested thus far, behaves as an Shp2 hypomorph (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar, 10Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (184) Google Scholar, 23Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (204) Google Scholar, 29Qu C.K. Shi Z.Q. Shen R. Tsai F.Y. Orkin S.H. Feng G.S. Mol. Cell. Biol. 1997; 17: 5499-5507Crossref PubMed Scopus (148) Google Scholar, 30Qu C.K. Yu W.M. Azzarelli B. Cooper S. Broxmeyer H.E. Feng G.S. Mol. Cell. Biol. 1998; 18: 6075-6082Crossref PubMed Scopus (106) Google Scholar, 31Oh E.S. Gu H. Saxton T.M. Timms J.F. Hausdorff S. Frevert E.U. Kahn B.B. Pawson T. Neel B.G. Thomas S.M. Mol. Cell. Biol. 1999; 19: 3205-3215Crossref PubMed Scopus (193) Google Scholar, 32Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Because of the key role of Shp2 in RTK-evoked ERK activation, we compared the response of WT and mutant Shp2-reconstituted cells to several growth factors. In WT cells, FGF and PDGF stimulation resulted in strong tyrosyl phosphorylation of Shp2. In contrast, IGF-1 and EGF failed to evoke detectable Shp2 tyrosyl phosphorylation (Fig. 1C). Why stimulation with some but not all RTKs leads to tyrosyl phosphorylation of Shp2 is not known, although this difference may be more apparent than real. When over-expressed in heterologous cells, the insulin receptor can evoke detectable Shp2 tyrosyl phosphorylation (33Stein-Gerlach M. Kharitonenkov A. Vogel W. Ali S. Ullrich A. J. Biol. Chem. 1995; 270: 24635-24637Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and EGF-stimulated tyrosyl phosphorylation of Shp2 also has been reported in some cell systems (13Lechleider R.J. Freeman Jr., R.M. Neel B.G. J. Biol. Chem. 1993; 268: 13434-13438Abstract Full Text PDF PubMed Google Scholar, 15Vogel W. Ullrich A. Cell Growth & Differ. 1996; 7: 1589-1597PubMed Google Scholar). Conceivably, RTK-evoked tyrosyl phosphorylation of Shp2 occurs only when surface expression of the receptor exceeds a certain minimal level. We have not quantified EGF receptor, PDGF receptor, IGF-1 receptor, or FGF receptor surface expression in WT or Shp2 mutant-reconstituted cells. However, we did observe higher and/or more sustained growth factor-evoked ERK activation in cells exposed to saturating levels of FGF or PDGF (which stimulate Shp2 tyrosyl phosphorylation) compared with those treated with saturating levels of EGF or IGF (Fig. 1D). Previous studies have shown that increasing RTK expression levels can prolong the kinetics of ERK activation (34Dikic I. Schlessinger J. Lax I. Curr. Biol. 1994; 4: 702-708Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 35Duckworth B.C. Cantley L.C. J. Biol. Chem. 1997; 272: 27665-27670Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Although several other explanations remain possible, these data along with reports of EGF- or insulin/IGF1-evoked Shp2 tyrosyl phosphorylation in other cell systems are consistent with the possibility that Shp2 phosphorylation depends on the strength of the RTK signal delivered. Alternatively, the ability of a particular growth factor to evoke Shp2 tyrosyl phosphorylation may depend on the proximity of Shp2 to the activated RTK. Notably, the PDGFR has a direct binding site for Shp2 (36Lechleider R.J. Sugimoto S. Bennett A.M. Kashishian A.S. Cooper J.A. Shoelson S.E. Walsh C.T. Neel B.G. J. Biol. Chem. 1993; 268: 21478-21481Abstract Full Text PDF PubMed Google Scholar, 37Kazlauskas A. Feng G.S. Pawson T. Valius M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6939-6943Crossref PubMed Scopus (187) Google Scholar), whereas Shp2 is recruited to the FGF receptor signaling complex via FRS-2, which binds constitutively to the FGF receptor (38Kouhara H. Hadari Y.R. Spivak-Kroizman T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 39Hadari Y.R. Kouhara H. Lax I. Schlessinger J. Mol. Cell. Biol. 1998; 18: 3966-3973Crossref PubMed Scopus (268) Google Scholar). In contrast, Shp2 is recruited to the EGF receptor via Gab1 (40Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (594) Google Scholar), and the insulin/IGF-1 receptors are recruited primarily via members of the IRS family (41Kuhne M.R. Pawson T. Lienhard G.E. Feng G.S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar), which associate less tightly with the respective activated RTKs. In fibroblasts, Shp2 is required for sustained ERK activation in response to EGF, IGF-1, FGF, and PDGF (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar, 10Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (184) Google Scholar, 23Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (204) Google Scholar, 32Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). To assess the effects of Shp2 tyrosyl phosphorylation, we monitored ERK activation by phospho-specific antibody immunoblotting in cells expressing WT Shp2 or the phosphorylation site mutants (Fig. 2). As expected, restoring WT Shp2 expression increased ERK activation in response to all growth factors, particularly at later time points, compared with vector control-infected cells (Figs. 2, B–F). Consistent with the absence of tyrosyl phosphorylation of Shp2 in response to EGF stimulation, we found no difference in EGF-evoked ERK activation between WT, Y542F-, or Y580F-expressing cells (Fig. 2A). Mutation of both Tyr-542 and Tyr-580 also had no effect on EGF-evoked ERK activation (data not shown). In contrast, ERK activity induced by FGF (Fig. 2B) or PDGF (Fig. 2C) was decreased in Y542F and Y580F mutant cells compared with WT cells, although it remained higher than that in control (vector alone) cells. ERK activation was decreased even further in cells expressing the double phosphorylation site mutant (Y542F,Y580F) than in single mutant (Y542F or Y580F)-expressing cells, although the Y542F,Y580F mutant retained some ability to restore ERK activation compared with vector control cells (Figs. 2, D and E). Direct assessment of ERK activity by immune complex kinase assays confirmed that, compared with WT-expressing cells, ERK activation in response to FGF or PDGF was impaired in cells expressing phosphorylation site mutants of Shp2 (Fig. 2F). Taken together, these data indicate that tyrosyl phosphorylation of Shp2 enhances its ability to restore normal ERK activation to Shp2-mutant cells, although mutants of Shp2 that are unable to become tyrosyl phosphorylated retain some function. Next, we examined the effect of each Tyr → Phe mutation on total tyrosyl phosphorylation of Shp2 and its association with Grb2 in growth factor-stimulated cells. Interestingly, the truncated mutant Shp2 expressed in Ex3–/–fibroblasts (and all of its derivative lines) retained the ability to undergo tyrosyl phosphorylation in response to PDGF, although to a significantly lesser extent than in WT Shp2-expressing cells (Fig. 3A). These data suggest that, in contrast with a previous report on EGFR signaling (10Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (184) Google Scholar), truncated Shp2 can be recruited to the vicinity of the PDGFR. Thus, the Ex3–/–protein may have some residual signaling potential and, consistent with this notion, we have found that unlike Ex3–/–embryos, which die at embryonic days 8.5–10, totally null Shp2 embryos succumb before embryonic day 6.5. 2W. Yang and B. G. Neel, unpublished data. Surprisingly, however, total tyrosyl phosphorylation of Shp2 in cells expressing either Y542F or Y580F, as measured by immunoblotting with monoclonal anti-phosphotyrosine antibody 4G10 (Fig. 3A) or several other anti-phosphotyrosine antibodies (data not shown), appeared to be reduced to levels seen in cells expressing only the parental retrovirus (Fig. 3A). Similar results were obtained following stimulation with FGF (Fig. 3B). Even more surprising, although the Y542F mutant lost virtually all Grb2-binding ability, PDGF-evoked Grb2 binding to Shp2 in Y580F-expressing cells was restored to WT levels despite the apparent lack of any Shp2 tyrosyl phosphorylation of this mutant (Fig. 3A). Likewise, the Y580F mutant retained about half the Grb2 binding ability of WT Shp2 in FGF-stimulated cells despite lacking any obvious tyrosyl phosphorylation as detected by anti-phosphotyrosine immunoblotting (Fig. 3B). These results appeared to suggest dissociation between Shp2 tyrosyl phosphorylation (as measured by total anti-phosphotyrosine immunoblotting) and Grb2 binding. On the other hand, they also suggested that Tyr-542 was required for most of the ability of Grb2 to bind to Shp2 following PDGF or FGF stimulation. These are at least two potential explanations for this apparent paradox. Both tyrosines might be necessary for efficient phosphorylation of Shp2. Alternatively, the anti-phosphotyrosine antibodies used for immunoblotting might not recognize mono-phosphorylated Tyr-542 or Tyr-580 effectively. To distinguish between these possibilities, we used recently developed phospho-specific antibodies that recognize Tyr-542 or Tyr-580 (Figs. 3, C and D). Immunoblotting with Tyr(P)-542-specific antibodies revealed that, following PDGF stimulation, Tyr-542 was phosphorylated to comparable extents in WT and Y580F cells. In contrast, no phosphorylation was detected in Y542F-expressing cells, confirming the specificity of these antibodies for Tyr(P)-542 (Fig. 3C, left panel). Immunoblotting with anti-Tyr(P)-580 revealed no phosphorylation in Y580F cells, as expected. Surprisingly, however, Y542F-expressing cells exhibited an ∼50% decrease in Tyr-580 phosphorylation (Fig. 3C, right panel). These data suggest that Tyr-542 must be phosphorylated to promote efficient Tyr-580 phosphorylation in response to PDGF stimulation. Following PDGF stimulation, the Y542F and Y580F mutants also exhibited increased mobility on SDS-PAGE compared with WT Shp2. Whether this reflects the direct effects of tyrosyl phosphorylation on the mobility of Shp2 in SDS-PAGE or a more indirect consequence of tyrosyl phosphorylation, e.g. on seryl/threonyl phosphorylation of Shp2, remains to be determined. The tyrosyl phosphorylation site mutations had a somewhat different effect on FGF signaling. Whereas mutation of Tyr-580 had no effect on Tyr-542 phosphorylation in PDGF signaling, FGF-evoked phosphorylation of Tyr-542 was reduced by ∼50% in Y580F-expressing cells; note that Y580F and WT Shp2 are expressed at comparable levels in these cells (Fig. 1B and 3D). As in PDGF signaling, however, Tyr-542 phosphorylation also was required for efficient phosphorylation of Tyr-580 (Fig. 3D). Taken together, these data suggest a model in which, in the absence of growth factor stimulation, the C terminus of Shp2 is in a “closed” conformation wherein the tyrosyl phosphorylation sites, Tyr-580 in particular, are relatively inaccessible. Following PDGF stimulation, Tyr-542 probably becomes phosphorylated first and then promotes “opening” of the closed form, permitting efficient phosphorylation of Tyr-580. Consistent with our previous work, but in contrast to the conclusions of others (15Vogel W. Ullrich A. Cell Growth & Differ. 1996; 7: 1589-1597PubMed Google Scholar, 16Keegan K. Cooper J.A. Oncogene. 1996; 12: 1537-1544PubMed Google Scholar), Tyr-542 must also be the major Grb2 binding site in response to PDGF, because Grb2 binding is essentially normal in Y580F-expressing cells, whereas it is absent in cells expressing Y542F. Tyr-542 phosphorylation probably occurs first in response to FGF stimulation as well; but here, Tyr-580 must also undergo phosphorylation for WT levels of Tyr-542 phosphorylation to be attained. Further studies will be required to determine whether Tyr-580 phosphorylation is required for effective phosphorylation of Tyr-542 by its respective kinase or to stabilize it from dephosphorylation, most likely auto-dephosphorylation (33Stein-Gerlach M. Kharitonenkov A. Vogel W. Ali S. Ullrich A. J. Biol. Chem. 1995; 270: 24635-24637Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It also is not clear whether tyrosyl phosphorylation per se evokes the conformational changes proposed above or whether it acts more indirectly, e.g. by promoting seryl/threonyl phosphorylation and/or binding of other proteins. Notably, Shp2 has a proline-rich region between Tyr-542 and Tyr-580 (Fig. 1A); conceivably, phosphorylation of either (or both) of these residues could regulate binding of an SH3 domain-containing protein. Regardless, Tyr-542 is probably the major Grb2 binding site in response to FGF signaling as well, because in Y580F-expressing cells Grb2 binding to Shp2 is reduced in parallel to the reduction in Tyr-542 phosphorylation (Figs. 3, B and D). One potential explanation for the apparently ordered phosphorylation of Tyr-542 and Tyr-580 is that these sites could be the targets of different PTKs. For example, Tyr-542 might be phosphorylated by the RTK itself, whereas Tyr-580 would be phosphorylated by a downstream PTK. Src family kinases (SFKs) are attractive candidates for such downstream PTKs, because they are activated in a variety of RTK signaling pathways (42Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3456) Google Scholar, 43Abram C.L. Courtneidge S.A. Exp. Cell Res. 2000; 254: 1-13Crossref PubMed Scopus (339) Google Scholar). Moreover, Shp2 is tyrosyl phosphorylated in viral Src-transformed cells and also is a good Src substrate in vitro (11Feng G.S. Hui C.C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (582) Google Scholar, 44Hakak Y. Hsu Y.S. Martin G.S. Oncogene. 2000; 19: 3164-3171Crossref PubMed Scopus (66) Google Scholar). To test whether SFKs phosphorylate Shp2 downstream of growth factor receptors, we treated WT Shp2-expressing cells with the Src selective inhibitor SU6656 (45Blake R.A. Broome M.A. Liu X. Wu J. Gishizky M. Sun L. Courtneidge S.A. Mol. Cell. Biol. 2000; 20: 9018-9027Crossref PubMed Scopus (528) Google Scholar). However, there was no effect of this inhibitor on either Tyr-542 or Tyr580 phosphorylation in FGF- or PDGF-stimulated cells (Fig. 4A). As an independent test of the role of SFKs, we compared growth factor-evoked Shp2 phosphorylation in cells lacking Src, Fyn, and Yes (SYF cells) and SYF cells reconstituted with Src (46Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (644) Google Scholar). Consistent with the inhibitor studies, there was no difference in phosphorylation of either Tyr-542 or Tyr-580 in SYF and SYF Src cells (Fig. 4B). These data indicate that SFKs are not required for phosphorylation of either Tyr-542 or Tyr-580 in response to FGF or PDGF and suggest that each is a direct target of the FGFR and PDGFR, respectively. We cannot exclude the possible involvement of another downstream PTK in the phosphorylation of either Tyr-542 or Tyr-580. Notably, however, two other types of downstream PTKs, the Abl and Tec family PTKs, typically require SFKs to become activated (47Plattner R. Kadlec L. DeMali K.A. Kazlauskas A. Pendergast A.M. Genes Dev. 1999; 13: 2400-2411Crossref PubMed Scopus (367) Google Scholar, 48Rawlings D.J. Scharenberg A.M. Park H. Wahl M.I. Lin S. Kato R.M. Fluckiger A.C. Witte O.N. Kinet J.P. Science. 1996; 271: 822-825Crossref PubMed Scopus (371) Google Scholar, 49Miller A.T. Berg L.J. Curr. Opin. Immunol. 2002; 14: 331-340Crossref PubMed Scopus (73) Google Scholar). Our results also argue strongly against the Grb2 adapter model for positive signaling by Shp2. Although mutation of Tyr-580 results in marked diminution of the ability of Shp2 to restore normal ERK activation in response to PDGFR stimulation (Figs. 2, C and D), Y580F Shp2 retains essentially WT Grb2 binding in PDGF-stimulated cells (Fig. 3A). Likewise, Y580F retains ∼50% Grb2 binding in FGF-stimulated cells. Because heterozygotic Shp2-deficient fibroblasts express about half the level of Shp2 as WT cells yet activate ERK normally in response to FGF (8Saxton T.M. Henkemeyer M. Gasca S. Shen R. Rossi D.J. Shalaby F. Feng G.S. Pawson T. EMBO J. 1997; 16: 2352-2364Crossref PubMed Scopus (399) Google Scholar),2 the level of Grb2 binding in Y580F cells should have been sufficient to restore Shp2 function if the adapter model was correct. Instead, our results support alternative explanations for the role of tyrosyl phosphorylation of Shp2. Cole and co-workers have provided compelling evidence that tyrosyl phosphorylation of Shp2 can result in increased Shp2 activity (18Lu W. Gong D. Bar-Sagi D. Cole P.A. Mol. Cell. 2001; 8: 759-769Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and increased Shp2 activity might well explain our results. But, in addition to becoming tyrosyl-phosphorylated, Shp2 binds via its SH2 domains to the PDGFR and Gab1 following PDGF stimulation and to Gab1 and FRS2 in response to FGF. Engagement of the N-terminal SH2 domain of Shp2 fully activates the enzyme; therefore, it is unclear how/why tyrosyl phosphorylation would be required to promote additional Shp2 activation. Conceivably, a significant amount of tyrosyl-phosphorylated Shp2 transmits signals while unbound to the above phosphotyrosyl proteins. However, a second alternative is suggested by the role of tyrosyl phosphorylation of the receptor protein-tyrosine phosphatase RPTPα. ∼20% of RPTPα is phosphorylated constitutively on Tyr-789 and, like Shp2, tyrosyl-phosphorylated RPTPα binds Grb2 (50den Hertog J. Tracy S. Hunter T. EMBO J. 1994; 13: 3020-3032Crossref PubMed Scopus (159) Google Scholar, 51Su J. Batzer A. Sap J. J. Biol. Chem. 1994; 269: 18731-18734Abstract Full Text PDF PubMed Google Scholar). Recent studies show that tyrosyl phosphorylation of RPTPα is essential for its major function, the dephosphorylation of inhibitory C-terminal phosphotyrosines of Src family PTKs (SFKs) (52Zheng X.M. Shalloway D. EMBO J. 2001; 20: 6037-6049Crossref PubMed Scopus (57) Google Scholar, 53Zheng X.M. Resnick R.J. Shalloway D. EMBO J. 2000; 19: 964-978Crossref PubMed Scopus (206) Google Scholar). Inactive SFKs assume a closed state with their C-terminal phosphotyrosines bound intramolecularly to their respective SH2 domains. Phosphorylated Tyr-789 of RPTPα competes with SFK C-terminal tyrosyl residues for binding to SFK SH2 domains, thereby “prying open” the closed form and facilitating dephosphorylation of SFK C-terminal tyrosyl residues by the RPTPα catalytic domain. Grb2 competes with SFK C-terminal phosphotyrosines for binding to RPTPα Tyr(P)-789; thus, Grb2 functions as an inhibitor of the biological activity of RPTPα. By analogy, tyrosyl-phosphorylated Shp2 might engage the SH2 domain of a potential substrate(s), thereby increasing its accessibility to the PTP domain. If so, then key substrates for Shp2 might be identified by virtue of their ability to bind to phosphotyrosyl peptides derived from the sequences surrounding Tyr-542 and/or Tyr-580. In summary, our studies reveal new complexity in Shp2 regulation and function. We have shown that phosphorylation of the two Shp2 C-terminal tyrosines most likely occurs in an ordered fashion, with Tyr-542 phosphorylation preceding phosphorylation of Tyr-580. Moreover, our results show unambiguously that Tyr-542 is the major Grb2 binding site in Shp2, at least in fibroblasts stimulated with PDGF or FGF. Most importantly, although it is well established that the PTP activity of Shp2 is essential for its positive signaling functions, we have demonstrated for the first time that tyrosyl phosphorylation has an important but more auxiliary role, potentiating the actions of Shp2 in some, but not all, RTK signaling pathways. Further studies will be required to clarify just how tyrosyl phosphorylation regulates Shp2 function." @default.
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• W2000343242 title "Tyrosyl Phosphorylation of Shp2 Is Required for Normal ERK Activation in Response to Some, but Not All, Growth Factors" @default.
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