Identification of Substrates of Human Protein-tyrosine Phosphatase PTPN22

Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3ϵ, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) ζ was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRζ, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRζ in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRζ when both were coexpressed in 293T cells, establishing TCRζ as a direct substrate of PTPN22. Stimulation of mature T cells activates a downstream signaling cascade involving temporally and spatially regulated phosphorylation and dephosphorylation events mediated by protein-tyrosine kinases and phosphatases, respectively. PTPN22 (Lyp), a non-receptor protein-tyrosine phosphatase, is expressed exclusively in cells of hematopoietic origin, notably in T cells where it represses signaling through the T cell receptor. We used substrate trapping coupled with mass spectrometry-based peptide identification in an unbiased approach to identify physiological substrates of PTPN22. Several potential substrates were identified in lysates from pervanadate-stimulated Jurkat cells using PTPN22-D195A/C227S, an optimized substrate trap mutant of PTPN22. These included three novel PTPN22 substrates (Vav, CD3ϵ, and valosin containing protein) and two known substrates of PEP, the mouse homolog of PTPN22 (Lck and Zap70). T cell antigen receptor (TCR) ζ was also identified as a potential substrate in Jurkat lysates by direct immunoblotting. In vitro experiments with purified recombinant proteins demonstrated that PTPN22-D195A/C227S interacted directly with activated Lck, Zap70, and TCRζ, confirming the initial substrate trap results. Native PTPN22 dephosphorylated Lck and Zap70 at their activating tyrosine residues Tyr-394 and Tyr-493, respectively, but not at the regulatory tyrosines Tyr-505 (Lck) or Tyr-319 (Zap70). Native PTPN22 also dephosphorylated TCRζ in vitro and in cells, and its substrate trap variant co-immunoprecipitated with TCRζ when both were coexpressed in 293T cells, establishing TCRζ as a direct substrate of PTPN22. T cell antigen receptor (TCR) 2The abbreviations used are: TCR, T cell antigen receptor; WT, wild type; DACS, D195A/C227S; ITAMs, immune tyrosine-based activation motifs; SH2, Src homology 2 domain; SH3, Src homology 3 domain; MES, 4-morpholineethanesulfonic acid; PTPN22cd, catalytic domain of PTPN22; PTP, protein-tyrosine phosphatase; HA, hemagglutinin; DTT, dithiothreitol; VCP, valosin containing protein; TCEP, tris(2-carboxyethyl)phosphine hydrochloride. signaling is important for the proliferation and differentiation of T cells. Upon binding of the peptide antigen presented by major histocompatibility complex, T cell antigen receptors initiate a cascade of signaling events mediated by protein-tyrosine kinases and adaptor proteins (1Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (540) Google Scholar, 2Cannons J.L. Schwartzberg P.L. Curr. Opin. Immunol. 2004; 16: 296-303Crossref PubMed Scopus (25) Google Scholar, 3Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar). TCR engagement brings the Src family kinase Lck into proximity with cytoplasmic domains of the invariant TCR subunits TCRζ, CD3ϵ, CD3γ, and CD3δ, each of which contains one or more immunoreceptor tyrosine-based activation motifs (ITAMs) that are phosphorylated at tyrosine residues within the conserved signature motif (YXXLX6-8YXXL) by Lck (4Pitcher L.A. van Oers N.S. Trends Immunol. 2003; 24: 554-560Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Phosphorylation of ITAMs creates high affinity binding sites for the tandem SH2 domains of the Syk family protein-tyrosine kinase Zap70 (1Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (540) Google Scholar, 2Cannons J.L. Schwartzberg P.L. Curr. Opin. Immunol. 2004; 16: 296-303Crossref PubMed Scopus (25) Google Scholar), which is subsequently recruited to the TCR complex and activated by Lck (1Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (540) Google Scholar). Zap70, in turn, phosphorylates LAT (5Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 6Paz P.E. Wang S. Clarke H. Lu X. Stokoe D. Abo A. Biochem. J. 2001; 356: 461-471Crossref PubMed Scopus (133) Google Scholar) and SLP76 (7Wardenburg J.B. Fu C. Jackman J.K. Flotow H. Wilkinson S.E. Williams D.H. Johnson R. Kong G. Chan A.C. Findell P.R. J. Biol. Chem. 1996; 271: 19641-19644Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), key adaptor proteins in the TCR signaling pathway that relay the signal to downstream effectors and eventually lead to the activation of T cells. Phosphorylated Zap70 can also serve as a docking site for several components of the TCR signaling pathway including Lck itself (8Pelosi M. Di Bartolo V. Mounier V. Mege D. Pascussi J.M. Dufour E. Blondel A. Acuto O. J. Biol. Chem. 1999; 274: 14229-14237Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), Vav (9Katzav S. Sutherland M. Packham G. Yi T. Weiss A. J. Biol. Chem. 1994; 269: 32579-32585Abstract Full Text PDF PubMed Google Scholar), and Cbl (10Lupher Jr., M.L. Songyang Z. Shoelson S.E. Cantley L.C. Band H. J. Biol. Chem. 1997; 272: 33140-33144Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 11Fournel M. Davidson D. Weil R. Veillette A. J. Exp. Med. 1996; 183: 301-306Crossref PubMed Scopus (123) Google Scholar). Lck has two major phosphorylation sites at tyrosine 394 and tyrosine 505 (1Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (540) Google Scholar, 12Jullien P. Bougeret C. Camoin L. Bodeus M. Durand H. Disanto J.P. Fischer S. Benarous R. Eur. J. Biochem. 1994; 224: 589-596Crossref PubMed Scopus (20) Google Scholar, 13Flint N.A. Amrein K.E. Jascur T. Burn P. J. Cell. Biochem. 1994; 55: 389-397Crossref PubMed Scopus (12) Google Scholar). Tyrosine 394, which is within the activation loop of the kinase domain, is autophosphorylated upon T cell stimulation and phosphorylation at this site is required for maximal Lck kinase activity (1Palacios E.H. Weiss A. Oncogene. 2004; 23: 7990-8000Crossref PubMed Scopus (540) Google Scholar). Tyrosine 505, located at the carboxyl terminus of Lck, is phosphorylated by Csk (14Bergman 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, 15Gervais F.G. Chow L.M. Lee J.M. Branton P.E. Veillette A. Mol. Cell. Biol. 1993; 13: 7112-7121Crossref PubMed Google Scholar) to create an internal binding site to which the SH2 domain of Lck binds, thus inhibiting Lck activity (16Veillette A. Caron L. Fournel M. Pawson T. Oncogene. 1992; 7: 971-980PubMed Google Scholar). Zap70 is phosphorylated at multiple sites, some of which activate (tyrosines 319 and 493) and others that suppress (tyrosines 292 and 492) TCR signaling (10Lupher Jr., M.L. Songyang Z. Shoelson S.E. Cantley L.C. Band H. J. Biol. Chem. 1997; 272: 33140-33144Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Phosphorylation at tyrosine 493 by Lck augments the kinase activity of Zap70 (17Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18Chan 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), whereas phosphorylation at tyrosine 319 appears to generate a docking site for Lck but does not alter intrinsic Zap70 kinase activity (8Pelosi M. Di Bartolo V. Mounier V. Mege D. Pascussi J.M. Dufour E. Blondel A. Acuto O. J. Biol. Chem. 1999; 274: 14229-14237Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). T cell signaling is negatively regulated by the activity of protein-tyrosine phosphatases such as PTPH1, SHP1, and PEP that dephosphorylate components of the TCR signaling pathway (19Veillette A. Latour S. Davidson D. Annu. Rev. Immunol. 2002; 20: 669-707Crossref PubMed Scopus (211) Google Scholar, 20Mustelin T. Alonso A. Bottini N. Huynh H. Rahmouni S. Nika K. Louis-dit-Sully C. Tautz L. Togo S.H. Bruckner S. Mena-Duran A.V. al-Khouri A.M. Mol. Immunol. 2004; 41: 687-700Crossref PubMed Scopus (80) Google Scholar). Overexpression of PEP, the mouse homolog of PTPN22, decreases TCR activation (21Gjorloff-Wingren A. Saxena M. Han S. Wang X. Alonso A. Renedo M. Oh P. Williams S. Schnitzer J. Mustelin T. Eur. J. Immunol. 2000; 30: 2412-2421Crossref PubMed Scopus (87) Google Scholar, 22Gjorloff-Wingren A. Saxena M. Williams S. Hammi D. Mustelin T. Eur. J. Immunol. 1999; 29: 3845-3854Crossref PubMed Scopus (161) Google Scholar); conversely, genetic ablation of PEP results in an increase in TCR signaling, notably in the effector/memory T cell population (23Hasegawa K. Martin F. Huang G. Tumas D. Diehl L. Chan A.C. Science. 2004; 303: 685-689Crossref PubMed Scopus (322) Google Scholar). PEP interacts with Csk, a negative regulator of Src family kinases, to repress TCR signaling in a synergistic manner (24Cloutier J.F. Veillette A. J. Exp. Med. 1999; 189: 111-121Crossref PubMed Scopus (359) Google Scholar). The physiological importance of PTPN22 in proper immune system regulation is further demonstrated by recent disease association studies linking a functional R620W protein variant, encoded by a C1858T single nucleotide polymorphism, to a significantly increased risk of autoimmune diseases including type 1 diabetes (25Bottini N. Musumeci L. Alonso A. Rahmouni S. Nika K. Rostamkhani M. MacMurray J. Meloni G.F. Lucarelli P. Pellecchia M. Eisenbarth G.S. Comings D. Mustelin T. Nat. Genet. 2004; 36: 337-338Crossref PubMed Scopus (1138) Google Scholar), rheumatoid arthritis (26Begovich A.B. Carlton V.E. Honigberg L.A. Schrodi S.J. Chokkalingam A.P. Alexander H.C. Ardlie K.G. Huang Q. Smith A.M. Spoerke J.M. Conn M.T. Chang M. Chang S.Y. Saiki R.K. Catanese J.J. Leong D.U. Garcia V.E. McAllister L.B. Jeffery D.A. Lee A.T. Batliwalla F. Remmers E. Criswell L.A. Seldin M.F. Kastner D.L. Amos C.I. Sninsky J.J. Gregersen P.K. Am. J. Hum. Genet. 2004; 75: 330-337Abstract Full Text Full Text PDF PubMed Scopus (1202) Google Scholar), and systemic lupus erythematosis (27Kyogoku C. Langefeld C.D. Ortmann W.A. Lee A. Selby S. Carlton V.E. Chang M. Ramos P. Baechler E.C. Batliwalla F.M. Novitzke J. Williams A.H. Gillett C. Rodine P. Graham R.R. Ardlie K.G. Gaffney P.M. Moser K.L. Petri M. Begovich A.B. Gregersen P.K. Behrens T.W. Am. J. Hum. Genet. 2004; 75: 504-507Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). The effects of the R620W variant on PTPN22 function have not been entirely defined but one consequence is to disrupt the interaction between Csk and PTPN22 (25Bottini N. Musumeci L. Alonso A. Rahmouni S. Nika K. Rostamkhani M. MacMurray J. Meloni G.F. Lucarelli P. Pellecchia M. Eisenbarth G.S. Comings D. Mustelin T. Nat. Genet. 2004; 36: 337-338Crossref PubMed Scopus (1138) Google Scholar, 26Begovich A.B. Carlton V.E. Honigberg L.A. Schrodi S.J. Chokkalingam A.P. Alexander H.C. Ardlie K.G. Huang Q. Smith A.M. Spoerke J.M. Conn M.T. Chang M. Chang S.Y. Saiki R.K. Catanese J.J. Leong D.U. Garcia V.E. McAllister L.B. Jeffery D.A. Lee A.T. Batliwalla F. Remmers E. Criswell L.A. Seldin M.F. Kastner D.L. Amos C.I. Sninsky J.J. Gregersen P.K. Am. J. Hum. Genet. 2004; 75: 330-337Abstract Full Text Full Text PDF PubMed Scopus (1202) Google Scholar). Despite the key role of PTPN22 in T cell signaling, there has to date been no systematic effort to identify its substrates in T cells. Reported substrates for PEP/PTPN22 include Zap70 and Src family kinases such as Fyn and Lck (22Gjorloff-Wingren A. Saxena M. Williams S. Hammi D. Mustelin T. Eur. J. Immunol. 1999; 29: 3845-3854Crossref PubMed Scopus (161) Google Scholar, 23Hasegawa K. Martin F. Huang G. Tumas D. Diehl L. Chan A.C. Science. 2004; 303: 685-689Crossref PubMed Scopus (322) Google Scholar, 24Cloutier J.F. Veillette A. J. Exp. Med. 1999; 189: 111-121Crossref PubMed Scopus (359) Google Scholar). It is likely that other substrates exist, e.g. Csk-binding phosphoproteins. In this study, we developed an unbiased approach to identify substrates of PTPN22 using substrate trapping methods combined with protein identification by mass spectrometry. Using this approach, we identified both known and potentially novel substrates of PTPN22, including Lck, Zap70, VCP, Vav, CD3ϵ, and TCRζ. We also provide biochemical evidence for direct interactions between PTPN22 and activated Lck and Zap70, and characterize the specificity of tyrosine dephosphorylation in these two substrates. Antibody Reagents, Cell Lines, and Cell Culture—Human Jurkat and 293T cell lines were purchased from ATCC and cultured in RPMI 1640, 10% fetal bovine serum or Dulbecco's modified Eagle's medium, 10% fetal bovine serum, respectively. Sf9 insect cells were obtained from Invitrogen and cultured in ExCel420. HA.7 and HA.11 antibodies to the hemagglutinin affinity tag were obtained from Sigma and Covance, respectively; anti-(His)5 antibody was purchased from Qiagen. An antibody to phosphotyrosine 416 of Src that cross-reacts with phosphotyrosine 394 of Lck was purchased from Cell Signaling, as were antibodies to LCK phosphotyrosine 505, Zap70, Vav, Zap70 phosphotyrosine 319, and Zap70 phosphotyrosine 493. Anti-TCRζ (6B10.2), anti-phospho-TCRζ, and anti-CD3ϵ antibodies were from Santa Cruz Biotechnology. 4G10 anti-phosphotyrosine antibody was from Upstate Biotechnology. Expression Plasmids and Transient Transfections—A cDNA encoding the catalytic domain (cd) of human PTPN22 (amino acids 1 to 300) was cloned from a human leukocyte cDNA library (Clontech) into the Escherichia coli expression vector pET104.1, with a His6 tag at the COOH terminus to facilitate purification. The vector provides a biotin conjugation site at the NH2 terminus of the protein, enabling in vivo biotinylation of PTPN22cd by endogenous E. coli biotin ligases. Substrate trap mutants of PTPN22cd, including D195A, C227S, D195A/C227S, and D195A/Q274A were constructed using the GeneTailor mutagenesis system (Invitrogen). The full-length PTPN22 cDNA was cloned into the BamHI/NotI sites of pIRES-GFP (Qbiogene), with an HA tag added at the NH2 terminus to obtain the mammalian expression construct, pCMV5-HAPTPN22. For expression in 293T cells, the full-length Lck, TCRζ, and Zap70 cDNAs were cloned from the human leukocyte cDNA library into pCDNA3.1 (Invitrogen). A Y505F mutant of Lck was constructed by site-directed mutagenesis as described above for the substrate trap mutants. For expression of TCRζ in E. coli, a cDNA encoding the entire cytoplasmic domain (amino acids 52-164) of human TCRζ (cyto-TCRζ) was cloned into the BamHI/XhoI sites of pET21a, incorporating a His6 tag at the protein COOH terminus. For baculovirus expression in Sf9 cells, Lck and Zap70 were cloned into pFastbac1, with a His6 tag at the NH2 or COOH terminus, respectively. The cloning, expression, and purification of LckΔ224 (residues 225-509) was as described (28Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (423) Google Scholar) except tyrosine 505 was mutated to phenylalanine. Transfection of DNA into 293T cells was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Expression and Purification of Recombinant Proteins—Sf9 cells were infected with baculovirus containing Lck or Zap70 at a multiplicity of infection of 2. Cells were harvested 48 h after infection and lysed with lysis buffer A (20 mm Tris·Cl, pH 7.6, 200 mm NaCl, 20 mm imidazole, 1% Triton X-100, 1× Complete protease inhibitor mixture (Roche), 1 mm TCEP). The cell lysate was loaded on a nickel-nitrilotriacetic acid column (Qiagen), washed extensively with wash buffer A (20 mm Tris·Cl, pH 7.6, 300 mm NaCl, 20 mm imidazole, 0.25 mm TCEP), and the proteins were eluted with elution buffer A (20 mm Tris·Cl, pH 7.6, 300 mm NaCl, 250 mm imidazole, 0.25 mm TCEP). The recovered proteins were then loaded on a Superdex 200 (Amersham Biosciences) column pre-equilibrated with gel filtration buffer (20 mm Tris·Cl, pH 7.6, 150 mm NaCl, 1 mm DTT). The purity of purified Lck and Zap70 was greater than 95% as assessed by SDS-PAGE (data not shown). Purified Lck and Zap70 were used immediately after preparation because a substantial loss of activity was observed in samples that were subjected to freeze-thaw cycles. The cytoplasmic domain of human TCRζ was expressed in E. coli BL21(DE3) cells. Cells were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 37°C for 3 h. Cyto-TCRζ was purified to >90% homogeneity by affinity and gel filtration chromatography exactly as described for Lck and Zap70. The wild type and substrate trap mutants of PTPN22cd were expressed in E. coli BL21(DE3) cells. Cells were grown to an A600 = 0.7 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside at 20 °C for 16 h. The harvested cell pellet was lysed in lysis buffer B (20 mm Tris·Cl, pH 7.6, 1 m NaCl, 20 mm imidazole, 1% Triton X-100, 1× Complete protease inhibitor mixture, 1 mm TCEP). Cell lysates were loaded on a nickel-nitrilotriacetic acid column and proteins were eluted as described above for Lck and Zap70. The pooled protein fractions were dialyzed against MES buffer (20 mm MES, pH 6.0, 50 mm NaCl, 1 mm EDTA, 2 mm DTT) at 4 °C overnight, and loaded on a Source S-15 cation exchange column pre-equilibrated with MES buffer. PTPN22cd was eluted with a 50°-250 mm linear NaCl gradient over 25 column volumes. The pooled fractions containing PTPN22cd were concentrated and loaded on a Superdex 200 column pre-equilibrated with Hepes buffer (25 mm Hepes, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm DTT). After gel filtration, the purity of PTPN22cd was greater than 95%. All of the substrate-trapping mutants of PTPN22cd were purified in a similar manner to >95% homogeneity. Substrate Trapping—Substrate trapping was performed as described (29Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (686) Google Scholar), with minor modifications. Briefly, 1 × 109 Jurkat cells were treated with 100 μm pervanadate for 30 min and collected by centrifugation. The cell pellet was lysed with 10 ml of lysis buffer C (25 mm Hepes, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1× Complete protease inhibitor mixture, 1 mm EDTA), treated with 5 mm iodoacetic acid on ice for 5 min, neutralized by addition of 10 mm DTT, and centrifuged to remove debris. The purified biotinylated PTPN22cd (WT and substrate trap mutants) were coupled to streptavidin beads following the supplier's protocol (Pierce). 100 mg of Jurkat lysate was incubated with 1 mg of protein equivalent of PTPN22cd-coated beads at 4 °C for 1 h. The beads were pelleted and washed 3 times for 5 min with lysis buffer C supplemented with 1 mm DTT. Bound proteins were eluted with 1× SDS sample buffer at room temperature for 1 h. The eluted proteins were then boiled, aliquots were analyzed by a 4°-20% gradient of SDS-PAGE, and stained with SilverSnap (Pierce), a reagent that is compatible with subsequent mass spectrometric peptide identification. The vanadate competition experiment was performed as described (30Palka H.L. Park M. Tonks N.K. J. Biol. Chem. 2003; 278: 5728-5735Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Mass Spectrometry Identification—Protein bands excised from a silver-stained gel were digested with trypsin (Promega) using an in-gel digestion robot (Investigator ProGest, Genomic Solutions, Ann Arbor, MI). The eluted tryptic peptides were concentrated to 2 μl using a SpeedVac and reconstituted to 10 μl in 1% formic acid (Fluka). 5 μl of the tryptic peptides were analyzed by light chromatography/mass spectrometry using an Ultimate HPLC system (Dionex/LC Packings, Sunnyvale, CA) with a reversed phase C18 column (Dionex) and injected into a mass spectrometer (Qstar Pulsar; Applied Biosystems, Foster City, CA). Protein identification was done by a data base search using the Mascot search engine from Matrix Sciences (London, United Kingdom). In Vitro Phosphorylation and Dephosphorylation Assays—Purified Lck was autoactivated in kinase buffer (20 mm Tris·Cl, pH 7.6, 10 mm MgCl2, 1 mm MnCl2, 150 mm NaCl) containing 100 μm ATP at 37 °C for 10 min. Purified Zap70 and cyto-TCRζ were phosphorylated in vitro by Lck (20:1 Zap70:Lck molar ratio; 60:1 TCRζ:Lck molar ratio) at 37 °C for 10 min. After Lck treatment, the reaction mixtures were dialyzed against Hepes buffer at 4 °C overnight. For dephosphorylation studies, 1 μg of activated Lck, Zap70, or cyto-TCRζ was incubated at 37 °C for 30 min in Hepes buffer with 10, 20, 40, and 80 ng of wild type PTPN22cd or 80 ng of the C227S active site mutant. To assay the activity of PTPN22cd, 1 μg of recombinant protein was incubated with 200 μl of 10 mm p-nitrophenyl phosphate in HAC buffer (50 mm acetic acid, pH 5.0, 2 mm DTT) at room temperature for 5 min. The reaction was stopped with the addition of 50 μl of 1 n NaOH. The optical density at 405 nm was read in a 96-well plate reader (SpectraMax; Molecular Devices). Wild type PTPN22 is active against p-nitrophenyl phosphate, but the C227S mutant has no detectable activity with this substrate (data not shown). In Vitro Binding Assays—200 ng of non-activated or activated Lck, Zap70, or TCRζ were incubated with 5 μg of purified PTPN22cd (wild type and substrate trap mutants) coupled to 10 μl of streptavidin beads in Hepes buffer at 4 °C for 1 h. The bound proteins were washed with the same buffer 3 times, and the beads were boiled in 1× SDS sample buffer for 3 min. 50 ng of input proteins (25% of total input) and 100% of eluted proteins were analyzed by SDS-PAGE and proteins captured by PTPN22 were quantitated by densitometry (ImageQuant). Immunoprecipitation and in Vivo Dephosphorylation Assays—For co-immunoprecipitation of TCRζ with PTPN22, 8 μg of pCDNA3.1-Lck (Y505F), 8 μg of pCNDA3.1-TCRζ, and 8 μg of pCMV5-HAPTPN22 (wild type or DACS double mutant) were co-transfected into 1 × 107 293T cells. 24 h after transfection, cells were harvested and subjected to immunoprecipitation with anti-HA.7 antibody. For in vivo dephosphorylation of Lck by PTPN22, 2 μg of pCDNA3.1-Lck and pCMV5-HAPTPN22 (wild type or DACS double mutant) were co-transfected into 1.5 × 106 293T cells. For in vivo dephosphorylation of Zap70 or TCRζ, 1.3 μg of pCDNA3.1-Zap70 or pCNDA3.1-TCRζ plus 1.3 μg each of pCDNA3.1-Lck (Y505F) and pCMV5-HAPTPN22 (wild type or DACS mutant) were co-transfected into 1.5 × 106 293T cells. 24 h after transfection, the cells were washed once with PBS and lysed with the addition of 300 μlof1× SDS sample buffer. After boiling for 5 min, 10 μl of lysate was analyzed by SDS-PAGE and Western blotting. Identification of Potential Substrates of PTPN22—PTPN22 contains two distinct functional domains, an NH2-terminal catalytic domain and a COOH-terminal proline-rich domain. The COOH-terminal domain participates in additional protein-protein interactions that likely contribute to protein localization or scaffolding functions but do not necessarily provide substrates for PTPN22 phosphatase activity. To reduce the complexity of protein-protein interactions and facilitate the identification of potential direct substrates of PTPN22, we produced and evaluated substrate trap variants of the PTPN22 catalytic domain (PTPN22cd). Mutations at key residues important for catalytic function have been employed as substrate traps for several tyrosine phosphatases, either singly or in combination (29Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (686) Google Scholar, 30Palka H.L. Park M. Tonks N.K. J. Biol. Chem. 2003; 278: 5728-5735Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 31Tonks N.K. FEBS Lett. 2003; 546: 140-148Crossref PubMed Scopus (319) Google Scholar, 32Xie L. Zhang Y.L. Zhang Z.Y. Biochemistry. 2002; 41: 4032-4039Crossref PubMed Scopus (81) Google Scholar). We tested D195A and C227S (equivalent to D181A and C215S in PTP1B, respectively; Ref. 29Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (686) Google Scholar) as single mutants as well as the double mutants D195A/C227S and D195A/Q274A for their ability to trap tyrosine-phosphorylated proteins from Jurkat cell lysates. The D195A/C227S mutant of PTPN22cd proved superior to the other substrate trap mutants (data not shown) and was then used to pull down potential substrates of PTPN22. Whole cell lysates from Jurkat cells treated with the phosphatase inhibitor pervanadate to stabilize tyrosine-phosphorylated proteins were subjected to affinity purification with the wild type and substrate-trapping DACS mutant of PTPN22cd (Fig. 1). In the absence of pervanadate treatment, neither the wild type protein nor the DACS mutant pulled down significant amounts of proteins (Fig. 1A, lanes 1 and 2). Following pervanadate treatment, several proteins including p270, p100, p90, p70, p56, p40, and p23 were trapped by the DACS mutant but not by the wild type protein (Fig. 1A, lanes 3 and 4). Bands corresponding to the proteins pulled down by the DACS protein were excised from the gel, digested in situ, and peptide fragments were identified by mass spectrometry. The p270 and p40 proteins were identified as spectrin and actin, respectively. Because these two abundant proteins are routinely identified in other mass spectrometry-based peptide identification experiments, we believe they represent nonspecific interactions of no biological significance. The p100 protein was identified as valosin containing protein (VCP), a member of the AAA (ATPases associated with multiple cellular activities) family of ATPases known to be phosphorylated on tyrosine residues following T cell receptor activation (33Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar). The p90 protein was identified as Vav, a guanine nucleotide exchange factor also implicated in T cell signaling (34Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar). The p70, p56, and p23 proteins were identified as Zap70, Lck, and CD3ϵ, respectively, all proteins known to participate in T cell signaling. For each of these proteins, at least two (Vav, Lck), and as many as 71 (VCP) distinct peptides were identified by mass spectrometry (see examples in Fig. 1, B-D). The substrate trap protein identifications made by mass spectrometry were confirmed in repeat pull down experiments followed by immunoblotting with antibodies against the corresponding proteins. In agreement with the mass spectrometry results, the DACS mutant of PTPN22cd pulled down Zap70, Lck, Vav, and CD3ϵ from pervanadate-treated Jurkat cell lysates, whereas wild type PTPN22cd did not (Fig. 1E). Although immunoblotting was not performed for VCP, the abundance of peptides identified from this protein and the fact that it was not routinely observed in other mass spectrometry-based peptide identification experiments (data not shown) is consistent with VCP also being a potential substrate for PTPN22. Because CD3ϵ is a component of the TCR that is phosphorylated at an ITAM, we asked whether TCRζ, another ITAM-containing TCR component, might also be a potential substrate for PTPN22. Indeed, the DACS mutant of PTPN22cd specifically pulled down TCRζ from pervandate-treated Jurkat cell lysates (Fig. 1E, lanes 9 and 10). PTPN22 Interacts Directly with Activated Lck and Zap70—Proteins identified from cell lysates by substrate trapping could represent either direct substrates of PTPN22 or non-substrate proteins trapped indirectly as part of a multiprotein complex. To confirm direct PTPN22cd-substrate interactions, we repeated the in vitro substrate trap experiments with purified recombinant proteins. Native or DACS mutant PTPN22cd were coupled to streptavidin beads and used to trap purified LCK or ZAP70. Lck purified from insect Sf9 cells is phosphorylated at tyrosine 505 but not at tyrosine 394. Following autoactivation in the presence of ATP and Mg2+, tyr