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SHP-1 Associates with Both Platelet-derived Growth Factor Receptor and the p85 Subunit of Phosphatidylinositol 3-Kinase*

Open AccessPublished:February 06, 1998DOI:https://doi.org/10.1074/jbc.273.6.3687
      The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.
      Protein tyrosine phosphorylation is critical in many cellular processes including signal transductions, neoplastic transformation, and the control of the mitotic cycle. These cellular processes are regulated by the activities of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs).
      The abbreviations used are: PTP, protein tyrosine phosphatase; SH2, src homology 2; SHP, SH2 domain-containing PTP; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; EGF, epidermal growth factor; EpoR, erythropoietin receptor; GST, glutathione S-transferase; PI, phosphatidylinositol; Tyr(P), phosphotyrosine; SRE, serum-responsive element; PAGE, polyacrylamide gel electrophoresis; Luc, luciferase; PCR, polymerase chain reaction.
      1The abbreviations used are: PTP, protein tyrosine phosphatase; SH2, src homology 2; SHP, SH2 domain-containing PTP; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; EGF, epidermal growth factor; EpoR, erythropoietin receptor; GST, glutathione S-transferase; PI, phosphatidylinositol; Tyr(P), phosphotyrosine; SRE, serum-responsive element; PAGE, polyacrylamide gel electrophoresis; Luc, luciferase; PCR, polymerase chain reaction.
      One subfamily of cytoplasmic PTPs, referred to as SHP (
      • Adachi M.
      • Fisher E.H.
      • Ihle J.
      • Imai K.
      • Jirik F.
      • Neel B.
      • Pawson T.
      • Shen S.-H.
      • Thomas M.
      • Ullrich A.
      • Zhao Z.
      ), contains two SH2 domains at their N terminus. Three members of SHP have been identified. They are SHP-1, SHP-2, and Drosophila Csw. Csw (
      • Perkins L.A.
      • Larsen I.
      • Perrimon N.
      ), the likely homology of mammalian SHP-2, is essential in signaling from the Torso and Sevenless receptor tyrosine kinases (
      • Perkins L.A.
      • Larsen I.
      • Perrimon N.
      ,
      • Allard J.D.
      • Chang H.C.
      • Herbst R.
      • McNeill H.
      • Simon M.A.
      ). SHP-2 (previously also known as PTP2C, SH-PTP2, PTP1D, Syp, and SHPTP3) (
      • Adachi M.
      • Fisher E.H.
      • Ihle J.
      • Imai K.
      • Jirik F.
      • Neel B.
      • Pawson T.
      • Shen S.-H.
      • Thomas M.
      • Ullrich A.
      • Zhao Z.
      ) is widely expressed and involved in many signaling pathways mediated by multiple non-hematopoietic receptor protein tyrosine kinase and by hematopoietic receptors as well (reviewed in Ref.
      • Neel B.G.
      • Tonks N.K.
      ). Generally, SHP-2 plays a positive role in growth factor-stimulated signaling. However, SHP-2 also negatively regulates some cellular processes, such as PDGF receptor-mediated signaling and cell membrane ruffling (
      • Marengere L.E.M.
      • Waterhouse P.
      • Duncan G.S.
      • Mittrucker H.-W.
      • Feng G.-S.
      • Mak T.W.
      ,
      • Cossette L.J.
      • Hoglinger O.
      • Mou L.
      • Shen S.-H.
      ,
      • Saxton T.M.
      • Henkemeyer M.
      • Gasca S.
      • Shen R.
      • Rossi D.J.
      • Shalaby F.
      • Feng G.-S.
      • Pawson T.
      ) and EGF-dependent cell growth (
      • Reeves S.A.
      • Sinha B.
      • Baur I.
      • Reinhold D.
      • Harsh G.
      ). SHP-1 (previously known as PTP1C, SH-PTP1, HCP, PTPN6, or SHP) (
      • Adachi M.
      • Fisher E.H.
      • Ihle J.
      • Imai K.
      • Jirik F.
      • Neel B.
      • Pawson T.
      • Shen S.-H.
      • Thomas M.
      • Ullrich A.
      • Zhao Z.
      ) is predominantly expressed in hematopoietic cells, where it generally functions as a negative regulator (reviewed in Ref.
      • Neel B.G.
      • Tonks N.K.
      ), although with some exceptions (
      • Krautwald S.
      • Buscher D.
      • Kummer V.
      • Buder S.
      • Baccarini M.
      ). Recent studies, however, revealed that SHP-1 is also substantially expressed under the control of an alternative tissue-specific promoter in a variety of non-hematopoietic cells, especially in some malignant epithelial cells (
      • Shen S.-H.
      • Bastien L.
      • Posner B.I.
      • Chretien P.
      ,
      • Banville D.
      • Stocco R.
      • Shen S.-H.
      ,
      • Plutzky J.
      • Neel B.G.
      • Rosenberg R.D.
      ,
      • Vogel W.
      • Lammers R.
      • Huang J.
      • Ullrich A.
      ,
      • Uchida T.
      • Matozaki T.
      • Matsuda K.
      • Suzuki T.
      • Matozaki S.
      • Nakano O.
      • Wada K.
      • Konda Y.
      • Sakamoto C.
      • Kasuga M.
      ). In non-hematopoietic cells, the phosphatase activity of SHP-1 has been shown to positively regulate EGF- or serum-activated mitogenic signaling in 293 cells (
      • Su L.
      • Zhao Z.
      • Bouchard P.
      • Banville D.
      • Fischer E.H.
      • Krebs E.G.
      • Shen S.-H.
      ). The negative effect of SHP-1 on cytokine receptor-mediated signaling is exerted by dephosphorylation of the tyrosine-phosphorylated cytokine receptor itself or receptor-associated tyrosine-phosphorylated mediators (Refs.
      • Yi T.
      • Mui A.L.-F.
      • Krystal G.
      • Ihle J.N.
      and
      • Klingmuller U.
      • Lorenz U.
      • Cantley L.C.
      • Neel B.G.
      • Lodish H.F.
      and reviewed in Ref.
      • Neel B.G.
      • Tonks N.K.
      ). In other pathways, however, the mechanism(s) by which SHP-1 and SHP-2 positively or negatively regulate signaling is not fully understood. Recently, various methods for detecting protein interaction have identified potential targets for SHP-1 and SHP-2, including SHPS-1 (SIRPs), CD22, CTLA4, ZAP70, Killer inhibitory receptors, SHIP, STATs, the 97-kDa (100-kDa) and 135-kDa proteins (Ref.
      • Fujioka Y.
      • Matozaki T.
      • Noguchi T.
      • Iwamatsu A.
      • Yamao T.
      • Takahashi N.
      • Tsuda M.
      • Takada T.
      • Kasuga M.
      ,
      • Tailor P.
      • Jascur T.
      • Williams S.
      • Von Willebrand M.
      • Couture C.
      • Mustelin T.
      ,
      • Kharitonenkov A.
      • Chen Z.
      • Sures I.
      • Wang H.
      • Schilling J.
      • Ullrich A.
      ,
      • Manie S.N.
      • Astier A.
      • Haghayeghi N.
      • Canty T.
      • Druker B.J.
      • Hirai H.
      • Freedman A.S.
      ,
      • Liu L.
      • Damen J.E.
      • Ware M.D.
      • Krystal G.
      ,
      • Bone H.
      • Dechert U.
      • Jirik F.
      • Schrader J.W.
      • Welham M.J.
      ,
      • Carlberg K.
      • Rohrschneider L.R.
      ,
      • Gu H.
      • Griffin J.D.
      • Neel B.G.
      ,
      • Ram P.A.
      • Waxman D.J.
      and reviewed in Ref.
      • Neel B.G.
      • Tonks N.K.
      ). Identification of interacting proteins with these two SHPs can greatly facilitate the elucidation of the roles of these phosphatases in the signaling pathways. In this report, we demonstrate that SHP-1 directly associated with both the PDGF receptor and the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in non-hematopoietic MCF-7 and TRMP cells stimulated with PDGF and that its phosphatase activity negatively regulated PDGF receptor-mediated activation of the c-fospromoter.

      DISCUSSION

      As is the case with other receptor protein tyrosine kinases, upon binding of its ligand, the PDGF receptor is activated via dimerization, resulting in autophosphorylation of multiple tyrosine residues within its intracellular domain. The autophosphorylated PDGF receptor provides docking sites to recruit specific cellular SH2 domain-containing proteins, many of which have been identified, including the SH2 domain-containing PTP, SHP-2 (
      • Kazlauskas A.
      • Feng G.-S.
      • Pawson T.
      • Valius M.
      ,
      • Lechleider R.J.
      • Sugimoto S.
      • Bennett A.M.
      • Kashshian A.S.
      • Cooper J.A.
      • Shoelson S.E.
      • Walsh C.T.
      • Neel B.G.
      ). In our immunoprecipitation experiments, we found that SHP-1, structurally related to SHP-2, also associated with the PDGF receptor in response to ligand stimulation in the non-hematopoietic MCF-7 and TRMP cell lines. Notably, the p85 subunit of PI 3-kinase was also co-precipitated with SHP-1. To address the nature of these interactions, we took the advantage of available TRMP cell lines transfected with mutant PDGFR Y740F and PDGFR Y751F which are unable to bind to p85. We demonstrated that although no p85 was associated with the mutant PDGF receptor, SHP-1 was still co-precipitated with the mutant PDGF receptor, suggesting that SHP-1 associates with the PDGF receptor without p85 mediation. Additionally, in the cells expressing only the mutant PDGF receptor, SHP-1 could associate with p85 without the binding of p85 to the PDGF receptor. These results suggest that SHP-1 independently associates with both the PDGF receptor and p85. The in vitro binding experiments further showed that SHP-1 associated with the PDGF receptor and p85 through its SH2 domains, whereas peptide competition experiments subsequently specified that the N-terminal domain of SHP-1 was responsible for both of these interactions. The binding sites for SHP-1 on both the PDGF receptor and p85, however, are presently unknown. We attempted to identify the binding sites by two approaches, phosphopeptide competition and using PDGF receptor mutants for co-immunoprecipitation. Experiments with phosphopeptide competition in co-precipitation (Fig. 4 A) likely excluded the possibility that SHP-1 bound to Tyr1009, a binding site for the structurally related SHP-2 (
      • Kazlauskas A.
      • Feng G.-S.
      • Pawson T.
      • Valius M.
      ,
      • Lechleider R.J.
      • Sugimoto S.
      • Bennett A.M.
      • Kashshian A.S.
      • Cooper J.A.
      • Shoelson S.E.
      • Walsh C.T.
      • Neel B.G.
      ). The experiments also excluded the binding of SHP-1 to Tyr771, a GTPase activating protein-binding site (
      • Kashishian A.
      • Kazlauskas A.
      • Cooper J.A.
      ,
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ). We have used PDGF receptor mutants PDGFR Y740F, Y751F, Y771F, Y1009F, and Y1021F in co-immunoprecipitation with SHP-1. All these mutations were unable to interfere with the co-precipitation of the receptors with SHP-1 (Fig. 2 B),
      Z. Yu, M. L. Jaramillo, D. Banville, and S.-H. Shen, unpublished results.
      suggesting that the SH2 domain of SHP-1 does not bind to these phosphorylation sites. Similarly, the SH2 domain of SHP-1 does not appear to bind to the reported phosphorylation sites of Tyr368, Tyr580, and Tyr607 in p85 (
      • Hayashi H.
      • Nishioka Y.
      • Kamohara S.
      • Kanai F.
      • Ishii K.
      • Fukui Y.
      • Shibasaki F.
      • Takenawa T.
      • Kido H.
      • Katsunuma N.
      • Ebina Y.
      ) as the corresponding tyrosine-phosphorylated peptides failed to block the association of SHP-1 with p85 in co-immunoprecipitation (Fig. 4 B).
      The function of SHP-1 in PDGF receptor signaling was studied by assessing the effect of wild type SHP-1 and its dominant negative mutants on PDGF-induced c-fos promoter activation. In TRMP cells, the overexpression of wild type SHP-1 markedly inhibited the response of the c-fos promoter to PDGF stimulation, whereas the expression of biochemically dominant negative mutants increased the response. These results suggest that SHP-1 can negatively regulate PDGF receptor-mediated signaling at least in SRE-regulated transcription in TRMP cells. However, in other cell lines, particularly CCL39 cell, it was reported that overexpression of SHP-1 did not affect PDGF-induced DNA synthesis (
      • Rivard N.
      • McKenzie F.R.
      • Brondello J.-M.
      • Pouyssegur J.
      ). It appears that, depending on the cell type, its compartmentalization, and its targeting molecules, SHP-1 could play different roles in growth factor-activated pathways, such as a negative role observed here in the PDGF-induced c-fos promoter activation, a positive role reported in EGF-stimulated pathway (
      • Su L.
      • Zhao Z.
      • Bouchard P.
      • Banville D.
      • Fischer E.H.
      • Krebs E.G.
      • Shen S.-H.
      ), or no apparent effect on PDGF-stimulated DNA synthesis (
      • Rivard N.
      • McKenzie F.R.
      • Brondello J.-M.
      • Pouyssegur J.
      ) and on EGF-stimulated mitogen-activated protein kinase activation and Elk-1 transaction (
      • Bennett A.M.
      • Hausdorff S.F.
      • O'Reilly A.M.
      • Freeman Jr., R.M.
      • Neel B.G.
      ).
      At present, the mechanism(s) by which SHP-1 positively or negatively regulates various growth factor-activated pathways is largely unknown. In our co-immunoprecipitation experiments, we observed that overexpression of wild-type SHP-1 substantially reduced the intensity of tyrosine phosphorylation on the PDGF receptor, whereas expression of the catalytically inactive mutant SHP-1 C455S dramatically increased the tyrosine phosphorylation of the receptor and p85. This result may suggest that SHP-1 targets the autophosphorylated PDGF receptor as its substrate and dephosphorylates the receptor on certain tyrosine residues, thus inhibiting the ligand-stimulated signaling, a mechanism similar to that found in cytokine receptor-mediated signalings where SHP1 negatively regulates these signal transduction pathways (
      • Yi T.
      • Mui A.L.-F.
      • Krystal G.
      • Ihle J.N.
      ,
      • Klingmuller U.
      • Lorenz U.
      • Cantley L.C.
      • Neel B.G.
      • Lodish H.F.
      ). The intensity of tyrosine-phosphorylated p85 was also greatly increased in cells expressing catalytically inactive mutant SHP1 C455S. It is likely that SHP1 also targets p85 as a substrate. It is unknown whether dephosphorylation of p85 contributes the negative effect of SHP1 on the PDGF receptor relaying signaling.
      Interestingly, SHP-2 can also positively or negatively regulate mitogen-stimulated pathways. The positive function of SHP-2 was reported in EGF, insulin, and PDGF-activated signal transductions (
      • Rivard N.
      • McKenzie F.R.
      • Brondello J.-M.
      • Pouyssegur J.
      ,
      • Bennett A.M.
      • Hausdorff S.F.
      • O'Reilly A.M.
      • Freeman Jr., R.M.
      • Neel B.G.
      ,
      • Valius M.
      • Kazlauskas A.
      ,
      • Xiao S.
      • Rose D.W.
      • Sasaoka T.
      • Maegawa H.
      • Burke Jr., T.R.
      • Roller P.P.
      • Shoelson S.E.
      • Olefsky J.M.
      ,
      • Bennett A.M.
      • Tang T.L.
      • Sugimoto S.
      • Walsh C.T.
      • Neel B.G.
      ,
      • Tauchi T.
      • Feng G.S.
      • Marshall M.S.
      • Shen R.
      • Mantel C.
      • Pawson T.
      • Broxmeyer H.E.
      ,
      • Zhao Z.
      • Tan Z.
      • Wright J.H.
      • Diltz C.D.
      • Shen S.-H.
      • Krebs E.G.
      • Fischer E.H.
      ,
      • Milarski K.L.
      • Saltiel A.R.
      ,
      • Noguchi T.
      • Matozaki T.
      • Horita K.
      • Fujioka Y.
      • Kasuga M.
      ,
      • Yamauchi K.
      • Milarski K.L.
      • Saltiel A.R.
      • Pessin J.E.
      ). Likewise, it was also reported that SHP-2 had a negative role or was not required in PDGF receptor signaling (
      • Saxton T.M.
      • Henkemeyer M.
      • Gasca S.
      • Shen R.
      • Rossi D.J.
      • Shalaby F.
      • Feng G.-S.
      • Pawson T.
      ,
      • Bennett A.M.
      • Hausdorff S.F.
      • O'Reilly A.M.
      • Freeman Jr., R.M.
      • Neel B.G.
      ,
      • Valius M.
      • Secrist J.P.
      • Kazlauskas A.
      ,
      • Klinghoffer R.A.
      • Duckworth B.
      • Valius M.
      • Cantley L.
      • Kazlauskas A.
      ,
      • DeMali K.A.
      • Whiteford C.C.
      • Ulug E., T.
      • Kazlauskas A.
      ). We also found that the dominant negative mutants of SHP-2 dramatically suppressed the EGF-stimulated signal pathway (
      • Zhao Z.
      • Tan Z.
      • Wright J.H.
      • Diltz C.D.
      • Shen S.-H.
      • Krebs E.G.
      • Fischer E.H.
      ),2 but it had no effect on the PDGF-activated mitogenesis in TRMP cells.2 The seemingly conflicting reports on the function of SHP-1 and SHP-2 in growth factor receptor protein tyrosine kinase signaling are probably due to redundancy and convergence of multiple signals from the growth factor receptors and the existence of multiple substrates that may be differentially expressed and/or differentially regulated by these PTPs. Thus, both SHP-1 and SHP-2 may have diversified functions in complicated and multiple signaling pathways.

      Acknowledgments

      We thank Normand Jolicoeur and Patrice Bouchard for technical assistance, Dr. Jacek Slon for peptide synthesis, Dr. Traian Sulea for assistance of designing SHP-1 deletion mutant by molecular modeling, and Dr. Darrell Mousseau for useful comments on the manuscript.

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