Specific Dephosphorylation of the Lck Tyrosine Protein Kinase at Tyr-394 by the SHP-1 Protein-tyrosine Phosphatase

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). was with rabbit anti-Lck anti-phosphotyrosine 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). antibodies were either by or by A and a as 293 cells were transiently transfected with the DNA constructs as cells were washed in Tris-buffered Cells were in 2 of Dulbecco's modified Eagle's medium supplemented with 10% calf serum for 2 at Cells were washed with Tris-buffered saline and lysed in radioimmune precipitation buffer, and Lck was by immunoprecipitation as Lck was resolved by SDS-polyacrylamide gel transferred to a and from the membrane identification by The membrane the Lck was as described previously K. T.R. Sefton B.M. Oncogene. 1990; Google Scholar). was on by electrophoresis at pH in the followed by in buffer in the as described previously T. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 77: PubMed Scopus Google Scholar). The were using a 293 cells were transiently with cDNAs encoding Lck or F505 Lck and the was as described with The were washed once in (50 mm Tris-HCl, pH 7.4, and 150 mm and once in phosphatase buffer mm pH 7.4, 150 mm NaCl, mm and mm Immunoprecipitated was in phosphatase buffer and with of GST-SHP-1 fusion protein in a reaction of for 1 at the the were washed three times with radioimmune precipitation buffer and in SDS-polyacrylamide gel electrophoresis For the of the in the was that the transfected 293 cells were with for 2 to were using GST-SHP-1 and a of the in which The was by Jill Meisenhelder using an Inc.) and The was in SHP-1 buffer (50 mm pH 2 mm EDTA, and 2 mm were in a of in and phosphate was by R. I. J. 1994; PubMed Scopus Google Scholar). The of phosphate the reaction was from a using sodium phosphate buffer, pH whether the phosphorylation of Lck on tyrosine was by we co-expressed Lck and SHP-1 by transient transfection in 293 cells. We Lck from cells and the of Lck tyrosine phosphorylation by using anti-phosphotyrosine antibodies In all of our we used a of SHP-1 lacking both SH2 domains to SHP-1 activity of the SH2 domains has been shown to catalytic activity by the steric 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, 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 catalytic activity of ΔΔSH2 SHP-1 and the of the activity of ΔΔSH2 SHP-1 in not Lck is tyrosine-phosphorylated in 293 cells when expressed of Lck are observed when the protein is in 293 cells. This is to of phosphorylation, which is in by activated protein kinases R. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar). wild-type SHP-1 was co-expressed with Lck, the tyrosine phosphorylation of Lck was as using a of catalytically inactive SHP-1 not the tyrosine phosphorylation of Lck that the catalytic activity of SHP-1 was for the of Lck The of Lck protein was in the from cells Lck and SHP-1 with from cells Lck with the of the Lck was for the Lck protein in the The expression of wild-type and catalytically inactive SHP-1 was shown to be by of the cell lysates using anti-Myc antibodies of Lck phosphorylation in 293 cells Lck was 293 cells Lck and SHP-1 and by on Lck from cells wild-type Lck Lck from cells wild-type Lck and ΔΔSH2 Lck from cells Lck and ΔΔSH2 are with the of electrophoresis and in the of are the of the in Tyr-505 to that of Tyr-394 as shown are of three Lck is in 293 cells, it is tyrosine-phosphorylated at both Tyr-394 and Tyr-505 T.R. Sefton B.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the of total Lck tyrosine phosphorylation by SHP-1 be a of the phosphorylation of or we examined the of SHP-1 on the phosphorylation of two of Lck, F505 Lck and F394 Lck. These are tyrosine-phosphorylated at Tyr-394 or We co-expressed of Lck in 293 cells with either catalytically ΔΔSH2 SHP-1 or catalytically inactive ΔΔSH2 SHP-1 by transient transfection and Lck tyrosine phosphorylation by using anti-phosphotyrosine antibodies with wild-type Lck, both F505 Lck and F394 Lck were tyrosine-phosphorylated in 293 cells 2 1 and The tyrosine phosphorylation of F505 Lck was by of catalytically SHP-1 inactive SHP-1 not the tyrosine phosphorylation of F505 Lck 2 In the tyrosine phosphorylation of F394 Lck was not by with catalytically SHP-1 2 that expression of SHP-1 not the phosphorylation of that the of wild-type Lck by SHP-1 at we Lck phosphorylation by analysis. do we co-expressed wild-type Lck with either catalytically SHP-1 or catalytically inactive SHP-1 and the cells Lck was by and phosphorylation was by In cells Lck both Tyr-505 and Tyr-394 were with phosphate at Tyr-505 at Tyr-394 The expression of catalytically SHP-1 the of phosphate at the of phosphate in the to that of the to of catalytically inactive SHP-1 not the phosphorylation of Tyr-394 or Tyr-505 These demonstrate that SHP-1 the of Lck at The structure of the inactive of and two Src family kinases, that the SH2 domain to a tyrosine in the terminus of the the kinase in an inactive F. I. J. Nature. 1997; PubMed Scopus Google Scholar, W. M.J. Nature. 1997; PubMed Scopus Google Scholar). Because Lck is activated either by of this tyrosine or by of the SH2 domain K.E. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4247-4251Crossref PubMed Scopus (164) Google Scholar, A. L. M. Pawson T. Oncogene. 1992; Google Scholar), it is to that the SH2 domain of Lck with the C-terminal tyrosine in the as or is a that SHP-1 either Tyr-394 or the Lck SH2 domain with the of Tyr-505 by to the apparent of SHP-1 to the apparent of Tyr-394 be by the amino Tyr-394 and whether the Lck SH2 domain of we co-expressed a of Lck that the SH2 domain with catalytically activated SHP-1 by transient transfection and Lck phosphorylation by In cells ΔSH2 Lck both Tyr-505 and Tyr-394 were was phosphate at Tyr-505 at Tyr-394 the of we observed with wild-type Lck. The in the of phosphate Tyr-394 and Tyr-505 in ΔSH2 Lck be to of Tyr-505 by tyrosine phosphatases because this site is by the SH2 or it be to Lck phosphorylation at of catalytically SHP-1 with ΔSH2 Lck the of phosphate at the of phosphate in the to that of the to of catalytically inactive SHP-1 with ΔSH2 Lck not the phosphorylation of Tyr-394 or Tyr-505 These that the of SHP-1 for Tyr-394 is in to the Lck SH2 domain sterically the of SHP-1 to The of the that the expression of SHP-1 the phosphorylation of Lck specifically at Tyr-394 in not of Lck by SHP-1 or of Lck whether SHP-1 Lck we expressed and fusion of the catalytic domain and the terminus of either wild-type SHP-1 or a catalytically inactive SHP-1 (C453S). These GST-SHP-1 fusion not the tandem SH2 domains and are a we co-expressed CD4 and either F505 Lck or wild-type Lck in 293 cells and the with anti-CD4 We the with the GST-SHP-1 fusion and Lck phosphorylation by with anti-phosphotyrosine antibodies We the by immunoprecipitation of CD4 because our Lck and we to binding the GST-SHP-1 fusion protein to the the in In immunoprecipitation of the by anti-CD4 antibodies the of because of the antibody directly to Lck. GST-SHP-1 both F505 Lck 2 with and wild-type Lck with The of was with F505 Lck. This that SHP-1 directly Lck at vitro. In the catalytically inactive GST-SHP-1 not either F505 Lck or wild-type Lck and We used SHP-1 at a of in the much SHP-1 was for of Lck in we Lck with of GST-SHP-1 and Lck phosphorylation by with anti-phosphotyrosine be using GST-SHP-1 at a of and was observed using a GST-SHP-1 at a of We also the of of a Tyr-394 not reaction that reaction as a of both and phosphatase we of the Lck as a of The Lck an of in the apparent for Tyr-394 in in we co-expressed CD4 and wild-type Lck in 293 cells, the cells with and the by We the with and inactive GST-SHP-1 catalytic domain fusion in and of Lck by The of phosphate at Tyr-505 and Tyr-394 in the substrate was GST-SHP-1 Tyr-394 a product that phosphate at Tyr-505 at Tyr-394 catalytically inactive GST-SHP-1 or not either Tyr-394 or Tyr-505 phosphorylation and In this we to Lck phosphorylation at the reaction of of it is important to that the in of Tyr-394 Tyr-505 be the that we We examined whether the Src family kinase Lck was a substrate of the SHP-1 protein-tyrosine demonstrate that Lck is directly and specifically by SHP-1 at a tyrosine in the M.L. K.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.F. T. Hunter T. Sefton B.M. Mol. Cell. Biol. 4: PubMed Scopus Google Scholar). Because phosphorylation of Tyr-394 activates Lck N. Veillette A. Mol. Cell. Biol. 1990; 10: 5197-5206Crossref PubMed Scopus (111) Google Scholar, Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), SHP-1 is an of Lck In to the phosphorylation of phosphorylation of Tyr-505 in Lck by the tyrosine protein kinase Science. 1991; PubMed Scopus Google Scholar, S. M. A. H. Nature. 1991; PubMed Scopus Google Scholar) of a inactive by binding of the SH2 domain to the terminus F. I. J. Nature. 1997; PubMed Scopus Google Scholar, M. T. J. K.E. M. K. EMBO J. 1992; PubMed Scopus Google Scholar, M. Weiss A. EMBO J. PubMed Scopus Google Scholar). We not any of Tyr-505 in wild-type Lck or in F394 Lck by of an SH2 domain of binding to and this Tyr-505 from in demonstrate that of the SH2 domain Tyr-505 to by of the of SHP-1 for Tyr-394 from the of Tyr-505 by the Lck SH2 The tyrosine phosphatase has been shown to either Tyr-394 or Tyr-505 in Lck T.R. Johnson Hyman R. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar, M. J. R. D. Yang P.C. N. Pingel S. M. D.R. EMBO J. PubMed Scopus Google Scholar). demonstrate that SHP-1 is to the the Lck SH2 domain and This that in a physiological SHP-1 on Lck at it is a that SHP-1 on Lck at it the of the Lck SH2 domain by Because we of Lck in vivo at Tyr-394 when SHP-1 was or when Lck was with a of SHP-1 in it be that our do not that Tyr-394 is a substrate of we were to demonstrate of Lck when it was with as a as In the of that we for the is not to the and of two to two of SHP-1 in the physiological substrate SIRP-α that have been confirmed by J. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This is with the that the Tyr-394 of Lck is a physiological substrate of of SHP-1 is to of T cells and T-cell (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, G. I. Siminovitch K.A. J. Med. 1996; PubMed Scopus Google Scholar, J. D. Yang Y. Siminovitch K.A. J. Immunol. 1999; Google Scholar, Neel B.G. Lorenz U. Immunol. 1999; PubMed Scopus Google Scholar, Matthews R.J. J. Immunol. 1999; Google Scholar, D.R. Williams S. T. Thomas M.L. J. Immunol. 1999; Google Scholar). This suggests that the of tyrosine-phosphorylated substrates by SHP-1 plays a role in the of T cell and in wild-type mice. 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 that ZAP-70 may be a of Because ZAP-70 phosphorylation and activity play an important role in T cell and A.C. M. Weiss A. Science. 1994; PubMed Scopus Google Scholar, D. J. Chan A.C. T.J. Weiss A. Science. 1994; PubMed Scopus Google Scholar), a of ZAP-70 to SHP-1 deficiency contribute to the of T cells in motheaten mice. demonstrate that Lck is also a of SHP-1 and is inhibited by Because Lck activates ZAP-70 by phosphorylation at 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 that the of SHP-1 on ZAP-70 phosphorylation and activity are it is equally that SHP-1 on both Lck and ZAP-70 to inhibit our that the failure of SHP-1 to Lck at Tyr-394 may contribute to the of motheaten mice. our are 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 that SHP-1 directly Lck activity in We the Matt Thomas Jon Cooper, Andrey Tony Jill and Bob Hyman for kind of We also and for critical of the