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• W2075121503 abstract "The protein-tyrosine phosphatase SHP-1 has been shown to be a negative regulator of multiple signaling pathways in hematopoietic cells. In this study, we demonstrate that SHP-1 dephosphorylates the lymphoid-specific Src family kinase Lck at Tyr-394 when both are transiently co-expressed in nonlymphoid cells. We also demonstrate that a GST-SHP-1 fusion protein specifically dephosphorylates Lck at Tyr-394 in vitro. Because phosphorylation of Tyr-394 activates Lck, the fact that SHP-1 specifically dephosphorylates this site suggests that SHP-1 is a negative regulator of Lck. The failure of SHP-1 to inactivate Lck may contribute to some of the lymphoid abnormalities observed in motheaten mice. The protein-tyrosine phosphatase SHP-1 has been shown to be a negative regulator of multiple signaling pathways in hematopoietic cells. In this study, we demonstrate that SHP-1 dephosphorylates the lymphoid-specific Src family kinase Lck at Tyr-394 when both are transiently co-expressed in nonlymphoid cells. We also demonstrate that a GST-SHP-1 fusion protein specifically dephosphorylates Lck at Tyr-394 in vitro. Because phosphorylation of Tyr-394 activates Lck, the fact that SHP-1 specifically dephosphorylates this site suggests that SHP-1 is a negative regulator of Lck. The failure of SHP-1 to inactivate Lck may contribute to some of the lymphoid abnormalities observed in motheaten mice. Src homology 2 glutathione S-transferase wild type Tyrosine phosphorylation is a critical control mechanism for growth, differentiation, cell cycle control, and cytoskeletal function. The signal transduction pathways involving tyrosine phosphorylation are regulated by the concerted action of protein-tyrosine kinases and protein-tyrosine phosphatases. Although much of the work has centered on the actions of the multitude of protein-tyrosine kinases, it is now apparent that the protein-tyrosine phosphatases play an equally diverse and important role. SHP-1 is a member of the SH21domain-containing family of nonmembrane protein-tyrosine phosphatases that is predominately expressed in hematopoietic cells. SHP-1 and the related phosphatases SHP-2 and csw in Drosophilaall share a similar structure of two tandem SH2 domains in the N terminus followed by the catalytic domain and a C-terminal tail (1Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (741) Google Scholar). In resting cells, the SHP-1 SH2 domains sterically inhibit the catalytic activity of SHP-1 through interactions with the catalytic domain (2Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar,3Martin A. Tsui H.W. Shulman M.J. Isenman D. Tsui F.W. J. Biol. Chem. 1999; 274: 21725-21734Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Following lymphocyte activation, the SH2 domains allow SHP-1 to bind to tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motifs (4D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar, 5Neel B.G. Curr. Opin. Immunol. 1997; 9: 405-420Crossref PubMed Scopus (140) Google Scholar). This binding serves to recruit SHP-1 to the membrane and relieve the steric inhibition, activating the phosphatase (2Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar). A naturally occurring SHP-1 deficiency exists in the motheaten mouse. These mice do not express any detectable SHP-1 protein and exhibit a panoply of hematopoietic defects, ranging from the overproliferation of macrophages and neutrophils to abnormal B- and T-cell development and hyper-responsiveness (6Shultz L.D. Rajan T.V. Greiner D.L. Trends Biotechnol. 1997; 15: 302-307Abstract Full Text PDF PubMed Scopus (147) Google Scholar). This suggests that SHP-1 plays an inhibitory role in hematopoietic cells. Consistent with this idea, SHP-1 has been shown to negatively regulate signaling downstream of the erythropoietin receptor, c-Kit, the granulocyte/macrophage colony-stimulating factor receptor, and the B- and T-cell antigen receptor (5Neel B.G. Curr. Opin. Immunol. 1997; 9: 405-420Crossref PubMed Scopus (140) Google Scholar, 7Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 8Kozlowski M. Larose L. Lee F. Le D.M. Rottapel R. Siminovitch K.A. Mol. Cell. Biol. 1998; 18: 2089-2099Crossref PubMed Scopus (178) Google Scholar, 9Jiao H. Berrada K. Yang W. Tabrizi M. Platanias L.C. Yi T. Mol. Cell. Biol. 1996; 16: 6985-6992Crossref PubMed Scopus (254) Google Scholar). The identification of physiological substrates of SHP-1 has been of considerable interest. In B cells, CD72 has been identified as anin vivo substrate of SHP-1 (10Wu Y. Nadler M.J. Brennan L.A. Gish G.D. Timms J.F. Fusaki N. Jongstra-Bilen J. Tada N. Pawson T. Wither J. Neel B.G. Hozumi N. Curr. Biol. 1998; 8: 1009-1017Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Additionally, SIRP-α and p62DOK have been identified as SHP-1 substrates in macrophages (11Timms J.F. Carlberg K. Gu H. Chen H. Kamatkar S. Nadler M.J. Rohrschneider L.R. Neel B.G. Mol. Cell. Biol. 1998; 18: 3838-3850Crossref PubMed Scopus (175) Google Scholar, 12Berg K.L. Siminovitch K.A. Stanley E.R. J. Biol. Chem. 1999; 274: 35855-35865Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In T cells, the adapter protein SLP-76 and the cytosolic tyrosine kinase ZAP-70 have been shown to be substrates of SHP-1 (13Binstadt B.A. Billadeau D.D. Jevremovic D. Williams B.L. Fang N. Yi T. Koretzky G.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1998; 273: 27518-27523Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 14Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (335) Google Scholar). We were interested in identifying additional substrates of SHP-1 in T cells. We initially focused our efforts on Lck because of the prolonged Lck catalytic activity observed in motheaten thymocytes (15Lorenz U. Ravichandran K.S. Burakoff S.J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9624-9629Crossref PubMed Scopus (157) Google Scholar) and because Lck is an upstream activator of ZAP-70 (16Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar). Lck is a lymphoid cell-specific member of the Src family of nonreceptor tyrosine kinases that is essential for both the development of T cells in the thymus and the response of mature T cells to signals arising from the T-cell antigen receptor (17Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Crossref PubMed Scopus (890) Google Scholar, 18Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (935) Google Scholar). Like all Src family kinases, Lck is activated and inhibited by tyrosine phosphorylation. Tyr-394 is the site of stimulatory phosphorylation, whereas Tyr-505 is the site of inhibitory phosphorylation. Here we have examined whether SHP-1 directly regulates Lck phosphorylation. The cDNAs encoding wild-type murine Lck (WT), F505 Lck, F394 Lck, and ΔSH2 Lck have been described previously (19Voronova A.F. Sefton B.M. Nature. 1986; 319: 682-685Crossref PubMed Scopus (162) Google Scholar, 20Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar). For the transient transfections used in this study, the Lck cDNAs were subcloned into the expression vector pCEP4 (Invitrogen). The SHP-1 cDNA was a kind gift from Dr. Matt Thomas (Washington University, St. Louis, MO). The catalytically inactive SHP-1 cDNAs (D419A and C453S) were constructed by polymerase chain reaction-mediated mutagenesis. For transient transfections, the SHP-1 cDNAs were subcloned in frame into the vector CS3+MT (a kind gift from Dr. Jon Cooper, Fred Hutchinson Cancer Research Center, Seattle, WA), which appends six copies of the Myc epitope tag to the N terminus. SHP-1 constructs lacking both SH2 domains (ΔΔSH2 and ΔΔSH2 D419A) were constructed by subcloning a restriction fragment encoding amino acids 203–595 in frame into the vector CS3+MT. The CD4 cDNA was a kind gift from Dr. Andrey Shaw (Washington University) and was subcloned into the vector pCMX (22Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1497) Google Scholar). The GST-SHP-1 fusion protein constructs were created by the polymerase chain reaction amplification of the nucleotides encoding amino acids 214–595 from either wild-type SHP-1 or catalytically inactive SHP-1 (C453S). The polymerase chain reaction product was subcloned in frame into the vector pGEX-2TK (Amersham Pharmacia Biotech). The fidelity of all polymerase chain reaction products was confirmed by automated DNA sequencing. Polyclonal rabbit anti-Lck antibodies and rabbit anti-phosphotyrosine antibodies have been described previously (23Hurley T.R. Sefton B.M. Oncogene. 1989; 4: 265-272PubMed Google Scholar, 24Kamps M.P. Sefton B.M. Oncogene Res. 1988; 3: 105-115PubMed Google Scholar). The mouse monoclonal anti-Myc antibody 9E10 (25Chan S. Gabra H. Hill F. Evan G. Sikora K. Mol. Cell. Probes. 1987; 1: 73-82Crossref PubMed Scopus (50) Google Scholar) and the mouse monoclonal anti-phosphotyrosine antibody 4G10 (26Druker B.J. Mamon H.J. Roberts T.M. N. Engl. J. Med. 1989; 321: 1383-1391Crossref PubMed Scopus (245) Google Scholar) were a kind gift from Jill Meisenhelder and Dr. Tony Hunter (The Salk Institute, La Jolla, CA). The mouse monoclonal anti-CD4 antibody OKT4 (27Reinherz E.L. Kung P.C. Goldstein G. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4061-4065Crossref PubMed Scopus (920) Google Scholar) was a kind gift from Dr. Bob Hyman (The Salk Institute). 293, a human embryonic kidney cell line (28Harrison T. Graham F. Williams J. Virology. 1977; 77: 319-329Crossref PubMed Scopus (175) Google Scholar), was grown in Dulbecco's modified Eagle's medium (Cellgro, Mediatech) supplemented with 10% calf serum (Hyclone). 293 cells were seeded onto 5-cm gelatin-coated Petri dishes and were transfected using a calcium phosphate transfection system (Life Technologies, Inc.) as per the manufacturer's protocol. Cells were washed once with Tris-buffered saline and lysed in ice-cold radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 100 kallikrein-inactivating units/ml aprotinin, and 1 mm Na3VO4). Lysates were clarified by centrifugation at 35,000 × gfor 30 min. Lysates were subjected to immunoprecipitation using either a rabbit anti-Lck antibody or a monoclonal anti-CD4 antibody (OKT4). Immune complexes were collected on Pansorbin cells (Calbiochem), washed three times in radioimmune precipitation buffer, and used for subsequent analysis. Immunoprecipitated Lck and total cellular lysates were resolved by SDS-polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (Millipore). Western blotting was carried out with rabbit anti-Lck antibodies, anti-phosphotyrosine antibodies, or anti-Myc antibodies as described previously (23Hurley T.R. Sefton B.M. Oncogene. 1989; 4: 265-272PubMed Google Scholar, 24Kamps M.P. Sefton B.M. Oncogene Res. 1988; 3: 105-115PubMed Google Scholar). Bound antibodies were detected either by enhanced chemiluminescence or by 125I-protein A (ICN) and a PhosphorImager (Molecular Dynamics) as indicated. 293 cells were transiently transfected with the DNA constructs as indicated. At 24–48 h post-transfection, cells were washed twice in Tris-buffered saline. Cells were biosynthetically labeled in 2 ml of phosphate-free Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal calf serum and32Pi(H332PO4, ICN, 0.5 mCi/ml) for 2 h at 37 °C. Cells were washed with Tris-buffered saline and lysed in radioimmune precipitation buffer, and 32P-labeled Lck was isolated by immunoprecipitation as described.32P-labeled Lck was resolved by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and excised from the membrane following identification by autoradiography. The excised membrane containing the labeled Lck was then digested withl-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin as described previously (29Luo K. Hurley T.R. Sefton B.M. Oncogene. 1990; 5: 921-923PubMed Google Scholar). Two-dimensional tryptic peptide mapping was carried out on cellulose thin layer chromatography plates (EM Science) by electrophoresis at pH 8.9 in the first dimension followed by ascending chromatography in phosphochromatography buffer in the second dimension as described previously (30Hunter T. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1311-1315Crossref PubMed Scopus (1560) Google Scholar). The labeled peptides were visualized using a PhosphorImager. 293 cells were transiently co-transfected with cDNAs encoding WT Lck or F505 Lck and CD4. At 24 h post-transfection, the CD4-Lck complex was immunoprecipitated as described above with OKT4. The CD4-Lck immunoprecipitates were washed once in TN (50 mm Tris-HCl, pH 7.4, and 150 mm NaCl) and once in phosphatase assay buffer (100 mm HEPES, pH 7.4, 150 mm NaCl, 10 mm dithiothreitol, and 5 mm EDTA). Immunoprecipitated CD4-Lck was resuspended in phosphatase assay buffer and incubated with 5 μg of soluble, purified GST-SHP-1 fusion protein in a final reaction volume of 20 μl for 1 h at 4 °C unless otherwise indicated. To terminate the reaction, the immunoprecipitates were washed three times with radioimmune precipitation buffer and resuspended in SDS-polyacrylamide gel electrophoresis sample buffer. For the two-dimensional tryptic peptide analysis of the in vitro dephosphorylated products, the above procedure was employed except that the transfected 293 cells were labeled biosynthetically with 32Pi for 2 h prior to immunoprecipitation. Peptide dephosphorylation reactions were carried out using purified GST-SHP-1 and a Lck-derived phosphopeptide consisting of the sequence CIEDNEpYTAREGA in which pY represents phosphorylated Tyr-394. The Lck-derived phosphopeptide was synthesized by Jill Meisenhelder using an ABI 432A peptide synthesizer (Applied Biosystems Inc.) and standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) synthesis methods. The peptide was resuspended in SHP-1 assay buffer (50 mm NaOAc-HOAc, pH 5.0, 2 mm EDTA, and 2 mm dithiothreitol). Reactions were carried out in a volume of 20 μl in 96-well microtiter plates, and phosphate release was measured by malachite green assay (31Harder K.W. Owen P. Wong L.K. Aebersold R. Clark-Lewis I. Jirik F.R. Biochem. J. 1994; 298: 395-401Crossref PubMed Scopus (184) Google Scholar). The amount of phosphate released during the reaction was calculated from a standard curve generated using sodium phosphate buffer, pH 7.0. To examine whether the phosphorylation of Lck on tyrosine was affected by SHP-1, we co-expressed Lck and SHP-1 by transient transfection in 293 cells. We immunoprecipitated Lck from these cells and analyzed the level of Lck tyrosine phosphorylation by Western blotting using anti-phosphotyrosine antibodies (Fig.1). In all of our experiments, we used a mutant of SHP-1 lacking both SH2 domains to augment SHP-1 activity (ΔΔSH2). Deletion of the SH2 domains has been shown to increase catalytic activity by relieving the steric hindrance of the catalytic site by the SH2 domain (2Pei D. Lorenz U. Klingmuller U. Neel B.G. Walsh C.T. Biochemistry. 1994; 33: 15483-15493Crossref PubMed Scopus (186) Google Scholar, 3Martin A. Tsui H.W. Shulman M.J. Isenman D. Tsui F.W. J. Biol. Chem. 1999; 274: 21725-21734Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We confirmed the increased catalytic activity of ΔΔSH2 SHP-1 and the absence of the activity of ΔΔSH2 D419A SHP-1 in vitro (data not shown). Lck is tyrosine-phosphorylated in 293 cells when expressed alone (Fig.1 A,lane 1). Multiple forms of Lck are observed when the protein is overexpressed in 293 cells. This heterogeneity is attributable to differing degrees of serine phosphorylation, which is catalyzed in part by activated mitogen-activated protein kinases (32Watts J.D. Sanghera J.S. Pelech S.L. Aebersold R. J. Biol. Chem. 1993; 268: 23275-23282Abstract Full Text PDF PubMed Google Scholar). When wild-type SHP-1 was co-expressed with Lck, the tyrosine phosphorylation of Lck was reduced ∼1.8-fold as quantified using a PhosphorImager (Fig. Fig.1 A, lane 2). Co-expression of catalytically inactive SHP-1 (D419A) did not reduce the tyrosine phosphorylation of Lck ( Fig.1 A,lane 3), indicating that the catalytic activity of SHP-1 was required for the reduction of Lck phosphotyrosine levels. The level of Lck protein was slightly higher in the immunoprecipitates from cells co-expressing Lck and SHP-1 compared with immunoprecipitates from cells expressing Lck alone ( Fig.1 B,lanes 2andFig.3 compared with lane 1). Therefore, the quantitation of the Lck phosphotyrosine levels was normalized for the Lck protein level present in the immunoprecipitates. The expression of wild-type and catalytically inactive SHP-1 was shown to be equivalent by Western blotting of the cell lysates using anti-Myc antibodies ( Fig.1C,lanes 2and3).Figure 3Analysis of Lck phosphorylation in 293 cells co-expressing SHP-1. Lck was immunoprecipitated from32P-labeled 293 cells co-expressing Lck and SHP-1 and analyzed by two-dimensional tryptic peptide mapping on thin layer cellulose plates. A, Lck from cells expressing wild-type Lck alone; B, Lck from cells co-expressing wild-type Lck and ΔΔSH2 SHP-1; C, Lck from cells co-expressing Lck and ΔΔSH2 D419A SHP-1. Origins are marked with arrowheads. Arrows indicate the directions of electrophoresis and chromatography. Y505, peptide containing phosphorylated Tyr-505; Y394, peptide containing phosphorylated Tyr-394. Values in the lower left-hand corner of eachpanel are the ratio of the PhosphorImager units present in Tyr-505 to that of Tyr-394 (set as 1). Data shown are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When Lck is overexpressed in 293 cells, it is tyrosine-phosphorylated at both Tyr-394 and Tyr-505 (33Hartley D.A. Hurley T.R. Hardwick J.S. Lund T.C. Medveczky P.G. Sefton B.M. J. Biol. Chem. 1999; 274: 20056-20059Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, the reduction of total Lck tyrosine phosphorylation induced by SHP-1 could be a result of the reduced phosphorylation of Tyr-394, Tyr-505, or both. To distinguish between these possibilities, we examined the effect of SHP-1 on the phosphorylation of two mutants of Lck, F505 Lck and F394 Lck. These mutants are tyrosine-phosphorylated only at Tyr-394 or Tyr-505, respectively. We co-expressed these mutants of Lck in 293 cells with either catalytically active ΔΔSH2 SHP-1 or catalytically inactive ΔΔSH2 D419A SHP-1 by transient transfection and analyzed Lck tyrosine phosphorylation by Western blotting using anti-phosphotyrosine antibodies (Fig. 2). As with wild-type Lck, both F505 Lck and F394 Lck were tyrosine-phosphorylated in 293 cells (Fig. 2 A, lanes 1 and 4). The tyrosine phosphorylation of F505 Lck was reduced by co-expression of catalytically active SHP-1 (Fig.2 A, lane 2). Catalytically inactive SHP-1 did not affect the tyrosine phosphorylation of F505 Lck (Fig. 2 A,lane 3). In contrast, the tyrosine phosphorylation of F394 Lck was not affected by co-expression with catalytically active SHP-1 (Fig. 2 A, lane 5), suggesting that expression of SHP-1 does not alter the phosphorylation of Tyr-505. To confirm that the dephosphorylation of wild-type Lck by SHP-1 occurred at Tyr-394, we analyzed Lck phosphorylation by two-dimensional tryptic peptide analysis. To do this, we co-expressed wild-type Lck with either catalytically active SHP-1 or catalytically inactive SHP-1 and labeled the co-transfected cells biosynthetically with32Pi. Lck was isolated by immunoprecipitation, and its phosphorylation was analyzed by two-dimensional tryptic peptide analysis (Fig. 3). In cells expressing Lck alone, both Tyr-505 and Tyr-394 were phosphorylated with 1.4-fold more phosphate present at Tyr-505 than at Tyr-394 (Fig.3 A). The expression of catalytically active SHP-1 reduced the amount of phosphate at Tyr-394, increasing the ratio of phosphate in the Tyr-505-containing peptide to that of the Tyr-394-containing peptide to 5.6-fold (Fig. 3 B). Co-expression of catalytically inactive SHP-1 did not change the relative phosphorylation state of Tyr-394 or Tyr-505 (Fig. 3 C). These results demonstrate that SHP-1 causes the dephosphorylation of Lck at Tyr-394. The crystal structure of the inactive forms of Hck and c-Src, two other Src family kinases, shows that the SH2 domain binds intramolecularly to a conserved phosphorylated tyrosine residue in the extreme C terminus of the molecule, keeping the kinase in an inactive conformation (34Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar, 35Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar). Because Lck is activated either by mutation of this conserved tyrosine (Tyr-505) or by mutation of the SH2 domain (20Amrein K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, 36Veillette A. Caron L. Fournel M. Pawson T. Oncogene. 1992; 7: 971-980PubMed Google Scholar), it is reasonable to infer that the SH2 domain of Lck interacts intramolecularly with the C-terminal tyrosine residue in the same manner as Hck or c-Src. It is a possibility that SHP-1 can dephosphorylate either Tyr-394 or Tyr-505, but the Lck SH2 domain interferes with the dephosphorylation of Tyr-505 by SHP-1, thus giving rise to the apparent preference of SHP-1 to dephosphorylate Tyr-394. Alternatively, the apparent preferential dephosphorylation of Tyr-394 could be specified by the amino acid sequences surrounding Tyr-394 and Tyr-505. To examine whether the Lck SH2 domain protected against dephosphorylation of Tyr-505, we co-expressed a mutant of Lck that lacks the SH2 domain (ΔSH2 Lck) with catalytically activated SHP-1 by transient transfection and analyzed Lck phosphorylation by two-dimensional tryptic peptide analysis (Fig.4). In cells expressing ΔSH2 Lck alone, both Tyr-505 and Tyr-394 were phosphorylated, although there was 0.57-fold less phosphate present at Tyr-505 than at Tyr-394 (Fig.4 A), the opposite of what we observed with wild-type Lck. The change in the ratio of phosphate between Tyr-394 and Tyr-505 in ΔSH2 Lck could be attributable to increased dephosphorylation of Tyr-505 by endogenous tyrosine phosphatases because this site is no longer protected by the SH2 domain, or it could be attributable to increased Lck phosphorylation at Tyr-394. Co-expression of catalytically active SHP-1 with ΔSH2 Lck reduced the amount of phosphate at Tyr-505, further decreasing the ratio of phosphate in the Tyr-505-containing peptide to that of the Tyr-394-containing peptide to 0.26-fold (Fig. 4 B). Co-expression of catalytically inactive SHP-1 with ΔSH2 Lck did not change the relative phosphorylation state of Tyr-394 or Tyr-505 (Fig.4 C). These results suggest that the specificity of SHP-1 for Tyr-394 is attributable in part to the Lck SH2 domain sterically hindering the access of SHP-1 to Tyr-505. The results of the co-expression experiments demonstrated that the expression of SHP-1 reduced the phosphorylation of Lck specifically at Tyr-394 in vivo. However, these experiments did not distinguish between direct dephosphorylation of Lck by SHP-1 or indirect induction of Lck dephosphorylation. To determine whether SHP-1 could dephosphorylate Lck directly, we expressed and purified recombinant GST fusion proteins consisting of the catalytic domain and the C terminus of either wild-type SHP-1 or a catalytically inactive SHP-1 (C453S). These purified GST-SHP-1 fusion proteins did not contain the tandem SH2 domains and are therefore fully active. To obtain a suitable substrate, we co-expressed CD4 and either F505 Lck or wild-type Lck in 293 cells and immunoprecipitated the CD4-Lck complex with anti-CD4 antibodies. We then incubated the immunoprecipitates with the recombinant GST-SHP-1 fusion proteins and analyzed Lck phosphorylation by Western blotting with anti-phosphotyrosine antibodies (Fig.5). We isolated the CD4-Lck complex by immunoprecipitation of CD4 because our Lck antiserum contains anti-GST reactivity, and we wanted to avoid binding the GST-SHP-1 fusion protein to the immunoprecipitate during the in vitrodephosphorylation reaction. In addition, immunoprecipitation of the CD4-Lck complex by anti-CD4 antibodies eliminates the potential of interference because of the antibody directly bound to Lck. Wild-type GST-SHP-1 dephosphorylated both F505 Lck (Fig. 5 A, comparelane 2 with lane 1) and wild-type Lck (Fig.5 A, compare lane 5 with lane 4). The extent of dephosphorylation was considerably greater with F505 Lck. This suggested that SHP-1 directly dephosphorylated Lck at Tyr-394in vitro. In contrast, the catalytically inactive GST-SHP-1 did not dephosphorylate either F505 Lck or wild-type Lck (Fig.5 A, lanes 3 and 6). We used SHP-1 at a concentration of 3.5 μm in the above experiment. To determine how much SHP-1 was required for efficient dephosphorylation of Lck in vitro, we incubated CD4-F505 Lck immunoprecipitates with varying amounts of purified GST-SHP-1 and analyzed Lck phosphorylation by Western blotting with anti-phosphotyrosine antibodies. Greater than 75% dephosphorylation could be achieved using GST-SHP-1 at a concentration of 70 nm (Fig. 5 C, lane 5), and 50% dephosphorylation was observed using a GST-SHP-1 at a concentration of 7 nm (Fig. 5 C, lane 7). We also determined the kinetics of dephosphorylation of a Lck-derived peptide containing phosphorylated Tyr-394 (data not shown). After establishing reaction conditions that allowed linear reaction rates as a function of both incubation time and phosphatase concentration, we measured dephosphorylation of the Lck peptide as a function of peptide concentration. The Lck peptide exhibited an averageKm of 118 ± 40 μm in four independent experiments. To examine the apparent specificity for Tyr-394 in vitro in another way, we co-expressed CD4 and wild-type Lck in 293 cells, labeled the cells biosynthetically with 32Pi, and isolated the CD4-Lck complex by immunoprecipitation. We then incubated the immunoprecipitates with active and inactive GST-SHP-1 catalytic domain fusion proteins in vitro and analyzed dephosphorylation of Lck by two-dimensional tryptic peptide analysis (Fig.6). The amount of phosphate at Tyr-505 and Tyr-394 in the substrate alone was approximately equal (Fig.6 A). Wild-type GST-SHP-1 dephosphorylated Tyr-394 preferentially, yielding a product that contained 10-fold more phosphate at Tyr-505 than at Tyr-394 (Fig. 6 B). As expected, catalytically inactive GST-SHP-1 or GST did not affect either Tyr-394 or Tyr-505 phosphorylation (Fig. 6, C and D). In this experiment we chose to analyze Lck phosphorylation at the reaction end point instead of analyzing initial rates of dephosphorylation. Therefore, it is important to note that the difference in dephosphorylation of Tyr-394 versus Tyr-505 could actually be greater than the difference that we observed. We examined here whether the Src family kinase Lck was a substrate of the SHP-1 protein-tyrosine phosphatase. Our results demonstrate that Lck is dephosphorylated directly and specifically by SHP-1 at Tyr-394, a conserved tyrosine in the activation loop (37Casnellie J.E. Harrison M.L. Hellstrom K.E. Krebs E.G. J. Biol. Chem. 1982; 257: 13877-13879Abstract Full Text PDF PubMed Google Scholar, 38Voronova A.F. Buss J.E. Patschinsky T. Hunter T. Sefton B.M. Mol. Cell. Biol. 1984; 4: 2705-2713Crossref PubMed Scopus (75) Google Scholar). Because phosphorylation of Tyr-394 activates Lck (21Abraham N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, 39Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar), SHP-1 is therefore an inhibitor of Lck activity. In contrast to the phosphorylation of Tyr-394, phosphorylation of Tyr-505 in Lck by the ubiquitous tyrosine protein kinase Csk (40Thomas J.E. Soriano P. Brugge J.S. Science. 1991; 254: 568-571Crossref PubMed Scopus (85) Google Scholar, 41Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar) induces formation of a biologically inactive conformation by allowing intramolecular binding of the SH2 domain to the phosphorylated C terminus (34Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar, 42Bergman M. Mustelin T. Oetken C. Partanen J. Flint N.A. Amrein K.E. Autero M. Burn P. Alitalo K. EMBO J. 1992; 11: 2919-2924Crossref PubMed Scopus (274) Google Scholar, 43Sieh M. Bolen J.B. Weiss A. EMBO J. 1993; 12: 315-321Crossref PubMed Scopus (187) Google Scholar). We did not observe any dephosphorylation of Tyr-505 in wild-type Lck or in F394 Lck by SHP-1. Both of these proteins contain an intact SH2 domain capable of intramolecular binding to phosphorylated Tyr-505, and this could protect Tyr-505 from dephosphorylation. Our results in Fig. 4 demonstrate that deletion of the SH2 domain renders Tyr-505 susceptible to dephosphorylation by SHP-1. Some of the specificity of SHP-1 for phosphorylated Tyr-394 therefore comes from the protection of phosphorylated Tyr-505 by the Lck SH2 domain. The tyrosine phosphatase CD45 has been shown to dephosphorylate either Tyr-394 or Tyr-505 in Lck (44Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Crossref PubMed Scopus (413) Google Scholar, 45Baker M. Gamble J. Tooze R. Higgins D. Yang F.T. O'Brien P.C. Coleman N. Pingel S. Turner M. Alexander D.R. EMBO J. 2000; 19: 4644-4654Crossref PubMed Scopus (49) Google Scholar). Our results demonstrate that unlike CD45, SHP-1 is unable to displace the intramolecular interaction between the Lck SH2 domain and phosphorylated Tyr-505. This would suggest that in a physiological context, SHP-1 would only act on Lck phosphorylated at Tyr-394. However, it still is a possibility that SHP-1 could act on Lck phosphorylated at Tyr-505, although it presumably would require the prior displacement of the Lck SH2 domain by another molecule. Because we demonstrated dephosphorylation of Lck in vivo at Tyr-394 when SHP-1 was overexpressed or when Lck was incubated with a high concentration of SHP-1 in vitro (3.5 μm), it could be argued that our data do not provide evidence that Tyr-394 is a high affinity substrate of SHP-1. However, we were able to demonstrate significant dephosphorylation of Lck when it was incubated with as low a concentration as 7 nm SHP-1. In addition, the Km value of 118 ± 40 μm that we obtained for the Tyr-394-containing phosphopeptide is not dissimilar to the Km values (72 and 80 μm) of two peptides corresponding to two sites of SHP-1 dephosphorylation in the physiological substrate SIRP-α that have been confirmed by structural studies (46Yang J. Cheng Z. Niu T. Liang X. Zhao Z.J. Zhou G.W. J. Biol. Chem. 2000; 275: 4066-4071Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This is consistent with the hypothesis that the Tyr-394 of Lck is a physiological substrate of SHP-1. Loss of SHP-1 is known to cause hyperactivity of T cells and lower T-cell selection thresholds (15Lorenz U. Ravichandran K.S. Burakoff S.J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9624-9629Crossref PubMed Scopus (157) Google Scholar, 47Pani G. Fischer K.D. Mlinaric-Rascan I. Siminovitch K.A. J. Exp. Med. 1996; 184: 839-852Crossref PubMed Scopus (180) Google Scholar, 48Zhang J. Somani A.K. Yuen D. Yang Y. Love P.E. Siminovitch K.A. J. Immunol. 1999; 163: 3012-3021PubMed Google Scholar, 49Carter J.D. Neel B.G. Lorenz U. Int. Immunol. 1999; 11: 1999-2014Crossref PubMed Scopus (71) Google Scholar, 50Johnson K.G. LeRoy F.G. Borysiewicz L.K. Matthews R.J. J. Immunol. 1999; 162: 3802-3813PubMed Google Scholar, 51Plas D.R. Williams C.B. Kersh G.J. White L.S. White J.M. Paust S. Ulyanova T. Allen P.M. Thomas M.L. J. Immunol. 1999; 162: 5680-5684PubMed Google Scholar). This suggests that the dephosphorylation of tyrosine-phosphorylated substrates by SHP-1 plays a role in the regulation of T cell activation and selection in wild-type mice. Plas et al. (14Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (335) Google Scholar) have provided evidence that ZAP-70 may be a target of SHP-1. Because ZAP-70 phosphorylation and activity play an important role in T cell activation and maturation (52Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. Parslow T.G. Weiss A. Science. 1994; 264: 1599-1601Crossref PubMed Scopus (436) Google Scholar, 53Elder M.E. Lin D. Clever J. Chan A.C. Hope T.J. Weiss A. Parslow T.G. Science. 1994; 264: 1596-1599Crossref PubMed Scopus (433) Google Scholar), a loss of ZAP-70 dephosphorylation attributable to SHP-1 deficiency could well contribute to the hyperactivation of T cells in motheaten mice. Our data demonstrate that Lck is also a direct target of SHP-1 and is inhibited by SHP-1. Because Lck activates ZAP-70 by phosphorylation at Tyr-493 in ZAP-70 (16Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (325) Google Scholar), our results could suggest that the effects of SHP-1 on ZAP-70 phosphorylation and activity are indirect. However, it is equally possible that SHP-1 could act on both Lck and ZAP-70 to inhibit signaling. Nevertheless, our results suggest that the failure of SHP-1 to dephosphorylate Lck at Tyr-394 may contribute to the phenotype of motheaten mice. Indeed, our data are consistent with the prolonged activity of Lck in observed in motheaten thymocytes (15Lorenz U. Ravichandran K.S. Burakoff S.J. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9624-9629Crossref PubMed Scopus (157) Google Scholar) and suggest strongly that SHP-1 directly inhibits Lck activity in vivo. We thank the late Matt Thomas (1953–1999), Jon Cooper, Andrey Shaw, Tony Hunter, Jill Meisenhelder, and Bob Hyman for kind gifts of reagents. We also thank Roberta Schulte and Kambiz Amdjadi for critical reading of the manuscript." @default.
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• W2075121503 title "Specific Dephosphorylation of the Lck Tyrosine Protein Kinase at Tyr-394 by the SHP-1 Protein-tyrosine Phosphatase" @default.
• W2075121503 cites W1505892283 @default.
• W2075121503 cites W1524455540 @default.
• W2075121503 cites W1582379379 @default.
• W2075121503 cites W1583957430 @default.
• W2075121503 cites W1778557300 @default.
• W2075121503 cites W1870135658 @default.
• W2075121503 cites W1964203957 @default.
• W2075121503 cites W1964774404 @default.
• W2075121503 cites W1969318766 @default.
• W2075121503 cites W1979769420 @default.
• W2075121503 cites W1991683855 @default.
• W2075121503 cites W2011147958 @default.
• W2075121503 cites W2011467252 @default.
• W2075121503 cites W2015376574 @default.
• W2075121503 cites W2016553217 @default.
• W2075121503 cites W2016954161 @default.
• W2075121503 cites W2018826402 @default.
• W2075121503 cites W2019670504 @default.
• W2075121503 cites W2021451944 @default.
• W2075121503 cites W2026738139 @default.
• W2075121503 cites W2028669354 @default.
• W2075121503 cites W2034152669 @default.
• W2075121503 cites W2035562653 @default.
• W2075121503 cites W2041375044 @default.
• W2075121503 cites W2053465303 @default.
• W2075121503 cites W2055444005 @default.
• W2075121503 cites W2057723407 @default.
• W2075121503 cites W2065289702 @default.
• W2075121503 cites W2066560193 @default.
• W2075121503 cites W2067198866 @default.
• W2075121503 cites W2077173353 @default.
• W2075121503 cites W2085530715 @default.
• W2075121503 cites W2088215519 @default.
• W2075121503 cites W2088948208 @default.
• W2075121503 cites W2090309067 @default.
• W2075121503 cites W2097071478 @default.
• W2075121503 cites W2100869656 @default.
• W2075121503 cites W2109202813 @default.
• W2075121503 cites W2136770665 @default.
• W2075121503 cites W2143899077 @default.
• W2075121503 cites W2149827890 @default.
• W2075121503 cites W2156320225 @default.
• W2075121503 cites W2160584487 @default.
• W2075121503 cites W2170218211 @default.
• W2075121503 cites W4313343519 @default.
• W2075121503 cites W4313376981 @default.
• W2075121503 cites W87002874 @default.
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