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• W2089908364 abstract "PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytosolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was co-immunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein. PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytosolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was co-immunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein. INTRODUCTIONExternal signals, such as growth factors, initiate their signals by binding to ligand-specific growth factor receptors on the cellular membrane (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4581) Google Scholar). Receptor autophosphorylation on multiple tyrosyl residues, as a result of ligand binding, provides high-affinity binding sites for specific cellular proteins containing SH21 1The abbreviations used are: SH2src homology 2PTPprotein- tyrosine phosphataseMAP kinasemitogen-activated protein kinaseEGFepidermal growth factorPDGFplatelet-derived growth factorMBPmyelin basic proteinPAGEpolyacrylamide gel electrophoresis. domains. These SH2 domain-phosphopeptide interactions recruit other signaling molecules to the receptors where they can be phosphorylated by the receptors and/or recruit still other proteins (2Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1429) Google Scholar). Among the SH2 domain-containing proteins recruited to the growth factor receptors is a protein-tyrosine phosphatase (PTP) designated as PTP2C (also termed as SH-PTP2, SH-PTP3, PTP1D, and Syp) (3Ahmad S. Banville D. Zhao Z. Fischer E.H. Shen S.-H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2197-2201Crossref PubMed Scopus (194) Google Scholar, 4Freeman R.M. Plutzky J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11239-11244Crossref PubMed Scopus (326) Google Scholar, 5Adachi M. Sekiya M. Miyachi T. Matsuno K. Hinoda Y. Imai K. Yachi A. FEBS Lett. 1992; 314: 335-339Crossref PubMed Scopus (83) Google Scholar, 6Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (490) Google Scholar, 7Feng G.-S. Hui C.-C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (583) Google Scholar). PTP2C is a widely expressed enzyme. It shares high homology with corkscrew, which is required for normal development of Drosophila by playing a positive role in the transduction of the torso signal acting in concert with D-Raf (8Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (329) Google Scholar). The parallel nature of the torso signal transduction pathway to MAP kinase activation in mammalian cells suggests that PTP2C may play a similar role in mammalian systems. Recent studies indeed suggest that PTP2C is involved in growth factor signaling and in MAP kinase activation. First, PTP2C binds to EGF and PDGF receptors (6Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (490) Google Scholar, 9Case R.D. Piccione E. Wolf G. Benett A.M. Lechleider R.J. Neel B.G. Shoelson S.E. J. Biol. Chem. 1994; 269: 10467-10474Abstract Full Text PDF PubMed Google Scholar, 10Kazlauskas A. Feng G.S. Pawson T. Valius M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6939-6943Crossref PubMed Scopus (188) Google Scholar) and insulin receptor substrate-1 (11Kuhné M.R. Pawson T. Lienhard G.E. Feng G.S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar). Second, phosphorylation of PTP2C at its C terminus couples GRB2 to PDGF receptor, providing a mechanism for PDGF-induced activation of Ras (12Li 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, 13Bennett 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 (344) Google Scholar). Third, overexpression of catalytically inactive forms of PTP2C blocks insulin-stimulated MAP kinase activation (14Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 15Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (347) Google Scholar, 16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar). Fourth, as a feedback reaction, PTP2C is phosphorylated and inhibited by MAP kinase (17Peraldi P. Zhao Z. Filloux C. Fischer E.H. Van Obberghen E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5002-5006Crossref PubMed Scopus (39) Google Scholar).MAP kinase activation via the Ras signaling pathway in mammalian cells has been well defined (18Avruch J. Zhang X.F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 19Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (897) Google Scholar). GRB2, an SH2 and SH3 domain-containing protein, works as an adaptor by binding directly or indirectly via another protein such as SHC to upstream autophosphorylated growth factor receptors or their substrates and to downstream nucleotide exchange factor mSOS, thus activating Ras. GTP-bound Ras facilitates the recruitment of Raf-1 to the plasma membrane, resulting in its activation followed by activation of MAP kinase kinase. MAP kinase kinase, in turn, activates MAP kinase which requires phosphorylation on both tyrosine and threonine residues.To investigate the role of PTP2C in growth factor signaling, we have expressed the native PTP2C and a catalytic inactive form of the enzyme in 293 human embryonic kidney cells. Our study demonstrated that expression of the mutant of PTP2C had a profound impact on protein tyrosine phosphorylation in the cells. Furthermore, while overexpression of the native enzyme increased EGF-stimulated MAP kinase activation, expression of the catalytically inactive mutant decreased such activation.EXPERIMENTAL PROCEDURESMaterialsHuman 293 cells were obtained from the American Type Culture Collection. Polyclonal anti-PTP2C (serum 1263) and anti-MAP kinase (serum 7884) were raised in rabbits against an SH2 domain-truncated form of PTP2C expressed in Escherichia coli (20Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar) and against a 22-amino acid peptide derived from the subdomain XI of MAP kinase (21Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar), respectively. Monoclonal ribophorin II antibody S.D.1 was kindly provided by Dr. David Meyer (UCLA). Epidermal growth factor (EGF) and monoclonal anti-phosphotyrosine and polyclonal sheep-anti-EGF receptor antibodies were purchased from Upstate Biotechnology Inc. Myelin basic protein (MBP) and [γ-32P]ATP were purchased from Sigma and Amersham, respectively.Buffer A: 50 mMβ-glycerophosphate (pH 7.3) and 2 mM EDTA. Buffer B: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM KCl, and 1.5 mM MgCl2. Both buffers were also supplemented with 5 mMβ-mercaptoethanol, 1 mM EGTA, 0.2 mM Na3VO4, 0.1 μM microcystin, 1.0 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 1 μM pepstatin A, and 1 μg/ml aprotinin.Construction and Expression of PTP2C in 293 CellsA catalytically inactive form of PTP2C, designated as PTP2C(C-S), was generated by substituting the highly conserved cysteine 459 with a serine in a polymerase chain reaction. The site of mutation was confirmed by DNA sequencing. The cDNA inserts corresponding to the native and the Cys-to-Ser mutant were constructed into the pRC/CMV vector (Invitrogen). Transfection was carried out according to the standard calcium phosphate co-precipitation technique. To obtain stable cell lines, cells were selected in medium containing 0.5 mg/ml geniticin (G418 sulfate, Life Technologies, Inc.), and single colonies expressing high level PTP2C or PTP2C(C-S) were obtained after 2-3 weeks. Wild type 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 μg/ml streptomycin, and 50 units/ml penicillin. The transfected cells were grown in the same medium supplemented with 0.5 mg/ml G418.Immunoprecipitation, Western Blotting, and PTP AssayConfluent 293 cells were lysed in Buffer A supplemented with 1% Triton X-100 and 0.1 M NaCl. The extract was cleared by centrifugation at full speed in a microcentrifuge. For immunoprecipitation with anti-PTP2C antibody, the extracts were preincubated with 50 μl of a 50% slurry of protein A-Sepharose (Pharmacia Biotech Inc.) for 2 h at 4°C. After a brief centrifugation, the supernatant was incubated with antibody prebound to protein A-Sepharose overnight. The beads were washed three times with the cold lysis buffer. For Western blot analyses, samples were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were probed with various primary antibodies and detected using the ECL system with horseradish peroxidase-conjugated secondary antibodies (Amersham) according to the manufacturer's protocol.For PTP activity assay, sodium vanadate was omitted from all the buffers used. Assays were performed with anti-PTP2C immunoprecipitates using 1 μM32P-labeled ENDpYINASL peptide (800 cpm/pmol) as the substrate at pH 5.0 as described (20Zhao Z. Larocque R. Ho W.T. Fischer E.H. Shen S.H. J. Biol. Chem. 1994; 269: 8780-8785Abstract Full Text PDF PubMed Google Scholar, 22Daum G. Solca F. Diltz C.D. Zhao Z. Cool D.E. Fischer E.H. Anal. Biochem. 1993; 211: 50-54Crossref PubMed Scopus (57) Google Scholar).Fractionation of Cell ExtractsCells were collected, washed with cold phosphate-buffered saline, and suspended in Buffer B. Lysis was performed by nitrogen cavitation for 20 min at 350 p.s.i. Nuclear pellets were removed by centrifugation at 800 × g for 20 min. The postnuclear extract was further centrifuged at 100,000 × g for 45 min to give a clear cytosolic supernatant and a pelleted membrane fraction. The latter pellet, washed once with Buffer B and then dissolved in Buffer B supplemented with 1% Triton X-100, was referred to as the membrane extract. Further fractionation of the membrane fraction was performed by employing a 17.5% Percoll density gradient. The postnuclear extract (1 ml) was loaded on the top of a 9-ml Percoll suspension made in buffer B. Sedimentation was performed for 1 h at 18,000 rpm with a Beckman 50 Ti rotor. A total of 20 fractions were collected from bottom to top.Stimulation of Cells and Assays for MAP Kinase and MAP Kinase KinaseCells (80-90% confluency) were starved at 0% serum for 24 h and then stimulated with EGF for various periods of time. The reactions were stopped by washing with ice-cold phosphate-buffered saline. Cells were then collected and lysed in Buffer A by brief sonication. The extracts were cleared by centrifugation at 100,000 × g for 25 min. For protein kinase activity assay, samples were passed through a DEAE 52 minicolumn equilibrated with Buffer A. The column was washed with the same buffer and eluted with 0.3 M NaCl. The flow-through was collected for MAP kinase kinase assay and the elute for MAP kinase assay. The activity of MAP kinase kinase was determined by the ability of the enzyme to stimulate the MBP kinase activity of recombinant ERK2 in a coupled assay system as described (21Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar). MAP kinase was assayed with MBP as substrate in the presence of PKI peptide and calmidizolium (21Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar). Blanks were run without ERK2 and MBP for MAP kinase and the kinase kinase assays, respectively.To immunoprecipitate ERK1 and ERK2 with anti-MAP kinase antibody, cell extracts were brought to 0.5% SDS and then boiled for 10 min. After cooling down, samples were diluted 5-fold with the extraction buffer and incubated with anti-MAP kinase serum 7884 overnight. The cell extracts and immunoprecipitates were separated on 10% SDS gel. Western blotting was carried out as described above.RESULTSOverexpression of PTP2C and PTP2C(C-S) in 293 CellsConstructs containing PTP2C or the mutant PTP2C(C-S) and the pRC/CMV vector alone were used to transfect 293 cells. Clonal cell lines were isolated by G418 selection. Most of the clones selected an overexpressed substantial level of exogenous PTP2C or PTP2C(C-S) as compared with the vector control and the parental cells. Typically, about 6-8-fold overexpression was obtained as determined by Western blot with anti-PTP2C serum (Fig. 1) followed by densitometric analyses. The activity of PTP2C in the transfected cells was measured following immunoprecipitation of the cell extract with anti-PTP2C serum. To precipitate approximately equal portions of enzyme, extracts from cells overexpressing PTP2C or PTP2C(C-S) were diluted 6-fold with the extraction buffer while that from the vector control cells was undiluted. Upon incubation of an equal volume of cell extracts with anti-PTP2C serum and protein A-Sepharose, each immunoprecipitate gave about an equal amount of anti-PTP2C cross-reactivity in Western blotting analyses (data not shown). PTP activity in the immunoprecipitates was then assayed by employing the 32P-labeled peptide substrate ENDpYINASL in a liquid-solid phase reaction mixture (Table I). As expected, nearly equal amounts of activity were detected in the immunoprecipitates from the control cells and cells over-expressing PTP2C, corresponding to an approximately 6.5-fold total increase of PTP2C activity in the transfected cells. This suggests that the exogenously expressed PTP2C possessed the same activity as the endogenous enzyme. In contrast, the activity per mg of cell extract from the cells overexpressing PTP2C(C-S) was similar to that obtained from the control cells, indicating that the Cys-to-Ser mutant of PTP2C is totally inactive. Control experiments with preimmune serum showed essentially no PTP activity (data not shown).Table IActivity of PTP2C in 293 cell extracts Open table in a new tab Effects of the Altered PTP2C Expression on the Tyrosine Phosphorylation of Proteins in 293 CellsProtein tyrosine phosphorylation was detected by Western blotting analyses employing anti-phosphotyrosine antibody as shown in Fig. 1. Overexpression of the native PTP2C showed essentially no effect on protein tyrosine phosphorylation at the basal level. No significant effect was observed in serum-stimulated cells either (data not shown). However, overexpression of PTP2C(C-S) resulted in changes of tyrosine phosphorylation of proteins in the cells. In particular, a protein of approximately 43 kDa (designated as p43) was hyperphosphorylated on tyrosine. This protein ran as a rather broad band on SDS gel, probably due to differing degrees of phosphorylation on multiple sites. Note that tyrosine phosphorylation of another protein of approximately 100 kDa slightly increased also. Stimulation of the cells with serum did not cause significant change in the tyrosine phosphorylation of these proteins (data not shown).Increased tyrosine phosphorylation of proteins caused by expression of the catalytically inactive mutant of PTP2C has also been observed by others. In one study, hyperphosphorylation of a 120-kDa protein which associated with PTP2C was observed in insulin-stimulated NIH 3T3 cells (14Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar). In another study, increased tyrosine phosphorylation of the 125-kDa focal adhesion kinase was demonstrated in CHO cells, but the phosphorylation of this protein decreased upon insulin treatment of the cells (16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar). It is not known whether the 43-kDa and 100-kDa proteins we observed are related to the 120-kDa protein and focal adhesion kinase.Co-fractionation and Co-immunoprecipitation of PTP2C(C-S) with p43Cytosolic and membrane fractions of postnuclear extracts were separated by high speed centrifugation. While almost all the PTP2C in the control cells and cells overexpressing the native PTP2C distributed in the cytosolic fraction, nearly 50% of the enzyme was associated with the membrane fraction in cells expressing PTP2C(C-S) (Fig. 2). PTP assays following immunoprecipitation of the membrane fraction with anti-PTP2C serum yielded essentially no activity, indicating that the anti-PTP2C cross-reactivity detected in the membrane fraction represents the mutant enzyme only. For all cases, almost no PTP2C was found in the nuclear fraction (data not shown). The hyperphosphorylated p43 was also found in the membrane fraction (Fig. 2). In addition, it was co-immunoprecipitated with PTP2C(C-S), which might indicate a direct association of these two proteins. Separation of the postnuclear extract from cells overexpressing PTP2C(C-S) on a Percoll density gradient was shown in Fig. 3. Nearly half of the anti-PTP2C cross-reactivity was detected on the top of the gradient (fractions 16-20), which presumably represent the cytosolic fraction. Most of the rest was found in the middle of the gradient (fractions 6-12) where p43 was also detected. The distribution pattern of p43 and associated PTP2C(C-S) followed that of the EGF receptor, a plasma membrane marker, but differed from that exhibited by the endoplasmic reticulum maker ribophorin II (23Crimaudo C.M. Hortsch H. Gausepohl H. Meyer D.I. EMBO J. 1987; 6: 75-82Crossref PubMed Scopus (65) Google Scholar). The data indicate that PTP2C(C-S) and the hyperphosphorylated p43 were partitioned in the plasma membrane fraction. Based on the results, one can postulate that PTP2C(C-S) caused the hyperphosphorylation of p43 by preventing its dephosphorylation by the endogenous native enzyme and that tyrosine-phosphorylated p43, in turn, serves as an anchor for PTP2C(C-S) on the plasma membrane.Figure 2:Distribution of PTP2C, PTP2C(C-S), and the hyperphosphorylated p43 in cell fractions. Lanes V, N, and M represent 293 cells transfected with the vector alone, native PTP2C, and mutant PTP2C(C-S), respectively. Cytosolic (lane 1) and membrane (lane 2) fractions were separated by centrifugation at 100,000 × g. Cell extracts and their immunoprecipitates with anti-PTP2C were subject to SDS-PAGE and Western blotting analyses as described in Fig. 1. The arrow indicates the co-precipitated, hyperphosphorylated p43.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Fractionation of cell extracts from 293 cells expressing PTP2C(C-S). Postnuclear extracts were fractionated using a Percoll density gradient as described under “Experimental Procedures.” Fractions were subject to SDS-PAGE and Western blotting analyses with anti-EGF receptor, anti-PTP2C, anti-ribophorin II, and anti-phosphotyrosine (for p43). The number for each fraction is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of the Altered PTP2C Expression on MAP Kinase ActivationTo examine the role of PTP2C in the MAP kinase activation pathway, 293 cells with altered PTP2C expression were stimulated with EGF. Fig. 4 illustrates the time courses of MAP kinase and MAP kinase kinase activation. The activity profile of both kinases followed a typical biphasic pattern with peak activity occurring at 5 min. Altered PTP2C expression did not affect the time course of activation significantly. However, there was a small but significant impact on its magnitude. Overexpression of the native PTP2C increased the peak of EGF-stimulated MAP kinase kinase activity by about 30% while the expression of the catalytically inactive mutant decreased it by approximately 50% (Fig. 4A). Similar effects were observed for the activity of MAP kinase (Fig. 4B). These results were reproduced in other clonal cell lines selected.Figure 4:Activation of MAP kinase kinase (A) and MAP kinase (B) in 293 cells transfected with vector alone (■–■), PTP2C (▲- - -▲), and PTP2C(C-S) (●- - -●). Serum-starved cells were stimulated with 20 ng/ml EGF for the indicated time. MAP kinase activity was determined by incubating 5-μg samples with MBP and [γ-32P]ATP (1000 cpm/pmol) for 10 min. MAP kinase kinase was analyzed by incubating 2-μg samples with ERK2 and cold ATP for 10 min followed by the addition of MBP and 32P-labeled ATP and further incubating for 10 min. See “Experimental Procedures” for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effects of the altered PTP2C expression were also apparent in MAP kinase gel shift assays and Western blot analyzed with anti-phosphotyrosine (Fig. 5). Mobility shifts of MAP kinase (ERK1 and ERK2) on SDS-gel reflect the phosphorylations on the enzyme, which are required for its activation. As shown in Fig. 5A, 5- and 10-min stimulation of cells with EGF resulted in a significant portion of both ERK1 and ERK2 shifted in cells with the vector control and the native PTP2C, while much less was observed in the cells expressing the mutant PTP2C. Although the gel shifts were not perfectly correlated with the activity as has been reported (21Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar), the data indicate that the decreased MAP kinase activity in cells with PTP2C(C-S) was due to reduced phosphorylation. Further supporting this were the results from Western blot analyses of the immunoprecipitated MAP kinase (ERK1 and ERK2) with anti-phosphotyrosine antibody. EGF stimulated transient tyrosine phosphorylation of ERK1 and ERK2. Compared with the cells containing the vector alone, a higher level of phosphotyrosine was observed for the cells overexpressing the native PTP2C, while a lower level was obtained with cells expressing the mutant PTP2C. At basal level, neither significant gel shift nor tyrosine phosphorylation was detected, suggesting that the basal MBP kinase activity at zero time (see Fig. 4B) was probably due to protein kinases other than MAP kinase. Additional experiments indicated that the altered PTP2C expression had essentially no effect on the dose response of 293 cells to EGF and autophosphorylation of the EGF receptor upon stimulation (data not shown).Figure 5:Gel mobility shift (A) and tyrosine phosphorylation of MAP kinase (B). Serum-starved 293 cells transfected with the vector control, native PTP2C, and mutant PTP2C(C-S) were stimulated with 20 ng/ml EGF for the indicated time. Cell extracts (A) or anti-MAP kinase immunoprecipitates (B) were separated on 10% SDS gel, transferred to polyvinylidene difluoride membrane, and blotted with polyclonal anti-MAP kinase antibody (A) or anti-phosphotyrosine monoclonal antibody (B). Positions of ERK1 and ERK2 are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn this study, we found that overexpression of PTP2C slightly increased EGF-induced activation of MAP kinase while overexpression of the catalytically inactive mutant of PTP2C modestly decreased such activation. The remaining activation observed in the latter case could be attributed to endogenous PTP2C in the cells and/or a parallel activation pathway independent of PTP2C. In any case, the data suggest that the phosphatase activity of PTP2C is at least partly required for MAP kinase activation. These results are consistent with the recent reports on the positive role of corkscrew in Drosophila torso signaling (8Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (329) Google Scholar) and the blockage of insulin-induced MAP kinase activation by catalytically inactive forms of Syp and SH-PTP2 in 3T3 and CHO cells (14Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 15Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (347) Google Scholar, 16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar). The precise mechanism by which PTP2C regulates the MAP kinase activation pathway is unknown. Binding of PTP2C to growth factor receptors, including the EGF receptor, has been well documented (6Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (490) Google Scholar, 7Feng G.-S. Hui C.-C. Pawson T. Science. 1993; 259: 1607-1611Crossref PubMed Scopus (583) Google Scholar), but it is not known whether PTP2C can specifically dephosphorylate the autophosphorylated receptors. If it does, overexpression of PTP2C might reduce phosphorylation of the receptor and thus down-regulate MAP kinase activation. Conversely, expression of the inactive mutant of PTP2C should stabilize such phosphorylation and thus potentiate the signaling pathway. Opposite effects were observed in this study, with the native enzyme increasing the signal and the mutant decreasing the signaling. Therefore, it is unlikely that the PTP2C acts at the receptor level but instead probably functions downstream of the receptor. Recent studies demonstrated that PTP2C can couple GRB2 to the PDGF receptor by binding to the PDGF receptor via its SH2 domains and to GRB2 via its tyrosine-phosphorylated C-terminal segment. The GRB2-SOS complex would activate Ras and finally turn on the MAP kinase pathway (12Li 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, 13Bennett 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 (344) Google Scholar). However, according to this model, one should expect that the Cys-to-Ser mutation of PTP2C, which diminishes auto-dephosphorylation of the enzyme and thus increases its phosphorylation (24Bouchard P. Zhao Z. Banville D. Dumas F. Fischer E.H. Shen S.-H. J. Biol. Chem. 1994; 269: 19585-19589Abstract Full Text PDF PubMed Google Scholar), should enhance MAP kinase activation. Therefore, the present results suggest a more complicated role for PTP2C than serving as an adaptor for GRB2 binding. PTP2C might play a more important role in the aforementioned signaling pathway by dep" @default.
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• W2089908364 title "Altered Expression of Protein-tyrosine Phosphatase 2C in 293 Cells Affects Protein Tyrosine Phosphorylation and Mitogen-activated Protein Kinase Activation" @default.
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