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Mutual Regulation of Protein-tyrosine Phosphatase 20 and Protein-tyrosine Kinase Tec Activities by Tyrosine Phosphorylation and Dephosphorylation

2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês

10.1074/jbc.m310401200

1083-351X

Naohito Aoki, Shuichi Ueno, Hiroyuki Mano, Sho Yamasaki, Masayuki Shiota, Hitoshi Miyazaki, Yumiko Yamaguchi-Aoki, Tsukasa Matsuda, Axel Ullrich,

Glycosylation and Glycoproteins Research

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism. PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism. Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation.Journal of Biological ChemistryVol. 285Issue 42PreviewVOLUME 279 (2004) PAGES 10765–10775 Full-Text PDF Open Access Protein-tyrosine phosphatases (PTPs) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; HSCF, hematopoietic stem cell fraction; PSTPIP, proline, serine, threonine phosphatase-interacting protein; PTK, protein-tyrosine kinase; BCR, B cell receptor; SH2, Src homology 2; SH3, Src homology 3; HA, hemagglutinin; GST, glutathione S-transferase; WT, wild type; ECL, enhanced chemiluminescence; PH, pleckstrin homology; TH, Tec homology; POV, pervanadate. are a large and structurally diverse family of enzymes that catalyze the dephosphorylation of tyrosine-phosphorylated proteins (1Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (603) Google Scholar, 2Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). Biochemical and kinetic studies have documented that Cys and an Asp residues in the catalytic domain are essential for the PTP activity. PTPs have been shown to participate as either positive or negative regulators of signaling pathways in a wide range of physiological processes, including cellular growth, differentiation, migration, and survival (1Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (603) Google Scholar, 2Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). Despite their important roles in such fundamental cellular processes, the mechanisms by which PTPs exert their effects are largely not understood. PTP20 (3Aoki N. Yamaguchi-Aoki Y. Ullrich A. J. Biol. Chem. 1996; 271: 29422-29426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), which is also known as hematopoietic stem cell fraction (HSCF) (4Cheng J. Daimaru L. Fennie C. Lasky L.A. Blood. 1996; 88: 1156-1167Crossref PubMed Google Scholar), PTP-K1 (5Huang K. Sommers C.L Grinberg A Kozak C.A. Love P.E. Oncogene. 1996; 13: 1567-1573PubMed Google Scholar), fetal liver phosphatase 1 (6Dosil M. Leibman N. Lemischka I.R. Blood. 1996; 88: 4510-4525Crossref PubMed Google Scholar), and brain-derived phosphatase 1 (7Kim Y.W. Wang H. Sures I. Lammers R. Martell K.J. Ullrich A. Oncogene. 1996; 13: 2275-2279PubMed Google Scholar), comprises the PEST family of PTPs together with PTP-PEST and PEP PTP and was originally isolated by screening a PC12 cDNA library. Overexpression of PTP20 in PC12 cells results in a more rapid and robust neurite outgrowth in response to nerve growth factor treatment, suggesting that PTP20 is involved in cytoskeletal reorganization (3Aoki N. Yamaguchi-Aoki Y. Ullrich A. J. Biol. Chem. 1996; 271: 29422-29426Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Mostly consistent with this observation, overexpression of a dominant negative mutant of fetal liver phosphatase 1 in K562 hematopoietic progenitor cells results in an inhibition of cell spreading and substrate adhesion in response to phorbol ester (6Dosil M. Leibman N. Lemischka I.R. Blood. 1996; 88: 4510-4525Crossref PubMed Google Scholar). Recently, through yeast two-hybrid screening the proline, serine, threonine phosphatase-interacting protein (PSTPIP) and PSTPIP2 have been identified to be specific in vivo substrates for HSCF, because the phosphotyrosine (Tyr(P)) level of PSTPIP1 is significantly enhanced by coexpression of the catalytically inactive mutant (Cys to Ser) of PTP20 (8Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 9Wu Y. Dowbenko D. Lasky L.A. J. Biol. Chem. 1998; 273: 30487-30496Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). PSTPIP is tyrosine-phosphorylated both in BaF3 cells and in v-Src-transfected COS cells and is shown to be co-localized with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow (8Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar), strongly supporting the idea that PTP20/HSCF is a potential regulator of cytokinesis. PSTPIP also interacts with the C-terminal part of the cytosolic protein-tyrosine kinase (PTK) c-Abl, serves as a substrate for c-Abl, and can bridge interactions between c-Abl and PTP20 with the dephosphorylation of c-Abl by PTP20 (10Cong F. Spencer S. Cote J.F. Wu Y. Tremblay M.L. Lasky L.A. Goff S.P. Mol. Cell. 2000; 6: 1413-1423Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It has also been reported that PTP20 associates with the negative Src-family kinase regulator Csk via its Src homology 2 (SH2) domain and two putative sites of tyrosine phosphorylation of the phosphatase (11Wang B. Lemay S. Tsai S. Veillette A. Mol. Cell. Biol. 2001; 21: 1077-1088Crossref PubMed Scopus (51) Google Scholar). This association is thought to allow Csk and PTP20 to synergistically inhibit Src-family kinase activity by phosphorylating and dephosphorylating negative and positive regulatory tyrosine residues, respectively. Regarding post-translational regulation of the PEST family PTPs, it has been documented that phosphorylation of an N-terminal serine residue, which is well conserved in all members of the PEST PTP family, by protein kinase A results in the inhibition of its catalytic activity (12Garton A.J. Tonks N.K. EMBO J. 1994; 13: 3763-3771Crossref PubMed Scopus (106) Google Scholar). In addition to proline, serine, and threonine residues in the C-terminal PEST domain of PTP20, a large number of tyrosine residues exist in that region, suggesting the possibility that PTP20 is tyrosine-phosphorylated. Indeed, previous studies reveal that PTP20/HSCF becomes tyrosine-phosphorylated by constitutively active forms of Lck and v-Src kinases in transfected cells (8Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar, 11Wang B. Lemay S. Tsai S. Veillette A. Mol. Cell. Biol. 2001; 21: 1077-1088Crossref PubMed Scopus (51) Google Scholar) even though the physiological relevance of tyrosine phosphorylation on PTP20 remains unclear. In this study we addressed the question of PTP20 regulation with special emphasis on the relevance of tyrosine phosphorylation and its biological impact. Through co-expression with nonreceptor PTKs we found that Tec kinase strongly tyrosine-phosphorylated the catalytically inactive form of PTP20 and that Tec physically interacted with PTP20 in a tyrosine phosphorylation-dependent manner in transfected COS7 cells. Further analyses with a variety of mutants of PTP20 and Tec revealed that C-terminal tyrosine residues of PTP20 and the Tec SH2 domain were necessary in the regulation of respective state of phosphorylation. Ectopic expression of PTP20 in human immature Ramos B cells resulted in suppression of B-cell receptor-induced c-fos promoter activity. Moreover, we determined that tyrosine 281 of PTP20 played a role in the dephosphorylation activity of PTP20 against both Tec and PTP20 itself. Our findings suggest a negative feedback mechanism that mutually controls the tyrosine phosphorylation of Tec and PTP20 and regulates Tec activity and B cell receptor (BCR) signaling. Reagents—Antibodies to hemagglutinin (HA) epitope (Y-11), phosphotyrosine (PY99), glutathione S-transferase (GST) (Z-5), Src (SRC2), Lck (2102), JAK2 (M-126), JAK3 (C-21), Csk (C-20), and ZAP70 (LR) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to Tec, Itk, Btk, and Bmx were as described previously (13Mano H. Yamashita Y. Sato K. Yazaki Y. Hirai H. Blood. 1995; 85: 343-350Crossref PubMed Google Scholar). Antibody to PTP20 was prepared by immunizing rabbits with the N-terminal peptide of PTP20 (MSRQSDLVRSFLEQQEARDH), to which a cysteine residue was added to the C terminus, coupled to keyhole limpet hemocyanin (14Shiota M. Tanihiro T. Nakagawa Y. Aoki N. Ishida N. Miyazaki K. Ullrich A. Miyazaki H. Mol. Endocrinol. 2003; 17: 534-549Crossref PubMed Scopus (28) Google Scholar). Anti-human IgM antibody (Fab′)2 fragment was obtained from Southern Biotechnology Associates (Birmingham, AL). All other reagents were from Sigma unless otherwise noted. Plasmid Construction—The pSR-based expression vectors for Tec wild-type (WT), Tec kinase mutant, TecY187F, TecY518F, and Tec proteins lacking each subdomain were described previously (15Mano H. Yamashita Y. Miyazato A. Miura Y. Ozawa K. FASEB J. 1996; 10: 637-642Crossref PubMed Scopus (41) Google Scholar, 16Mao J. Xie W Yuan H. Simon M.I. Mano H. Wu D. EMBO J. 1998; 17: 5638-5646Crossref PubMed Scopus (86) Google Scholar). pEBG plasmids (17Mayer B.J. Hirai H. Sakai R. Curr. Biol. 1995; 5: 296-305Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) encoding each subdomain of Tec to express the GST-tagged proteins were previously described (18Yoshida K. Yamashita Y. Miyazato A. Ohya K. Kitanaka A. Ikeda U. Shimada K. Yamanaka T. Ozawa K. Mano H. J. Biol. Chem. 2000; 275: 24945-24952Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). HA epitope tagging to PTP20 at its N terminus and subsequently all the mutations (cysteine to serine, aspartic acid to alanine, and tyrosine to phenylalanine) in PTP20 were carried out by PCR-based strategy. To express GST-tagged PTP20 in mammalian cells, full-length PTP20 (amino acids 2–453), PTP catalytic domain (amino acids 2–308), and the C-terminal noncatalytic PEST domain (amino acids 271–453) were amplified by PCR and ligated into pEBG vector via the BamHI site. All the plasmids newly constructed were confirmed by sequencing. Expression plasmids for rat Csk and mouse JAK2 were generous gifts from Drs. M. Okada (Osaka University, Japan) and J. N. Ihle (St. Jude Children’s Research Hospital, Memphis, TN), respectively. Expression plasmids for mouse Src, Lck, Itk, Btk, Bmx, ZAP-70, and JAK3 were described elsewhere. Cell Culture and Transfection—COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (high glucose, Sigma) supplemented with 10% fetal calf serum. Ramos cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum. Upon transfection experiments COS7 cells were inoculated at a density of 4 × 105 cells/6-cm dish and grown overnight in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. Expression plasmids were transfected into the cells by the modified calcium phosphate precipitation method (19Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). After incubation under 3% CO2, 97% air for 18 h, the transfected cells were washed with phosphate-buffered saline twice and cultured in fresh Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum for another 24 h under humidified 5% CO2 and 95% air. Cell Lysis, Immunoprecipitation, GST Pull-down, and Western Blotting—The transfected cells were lysed with lysis buffer containing 50 mm Tris-HCl (pH 7.5), 5 mm EDTA, 150 mm NaCl, 10 mm sodium phosphate, 10 mm sodium fluoride, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml leupeptin. Lysates were directly subjected to immunoblotting, immunoprecipitation with the indicated antibodies plus protein G- or Protein A-Sepharose beads (Amersham Bioscience), or precipitation with GSH-Sepharose beads (Amersham Bioscience). Proteins in the immunoprecipitates and precipitates were further analyzed by immunoblotting with the indicated antibodies. The protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Bioscience) and light capture system (AE-6962, ATTO, Tokyo, Japan). c-fos Promoter Assay—Ramos cells (1 × 107/experiment) were subjected to electroporation with 2 μg of the pfos/luc reporter plasmid (20Yamashita Y. Watanabe S Miyazato A Ohya K. Ikeda U. Shimada K. Komatsu N. Hatake K. Miura Y. Ozawa K. Mano H. Blood. 1998; 91: 1496-1507Crossref PubMed Google Scholar) plus 10 μg of expression plasmids for PTP20 or its mutants. Five hours after transfection cells were incubated for 5 h in the absence or presence of antibodies to human IgM (10 μg/ml). Luciferase activity was measured with the use of the dual luciferase assay system (Promega, Madison, WI). Tec Is a Potent Regulator of PTP20—Although PTP20 has been shown to be a substrate of v-Src (8Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar) and constitutively active Lck (11Wang B. Lemay S. Tsai S. Veillette A. Mol. Cell. Biol. 2001; 21: 1077-1088Crossref PubMed Scopus (51) Google Scholar), the physiological relevance of PTP20 tyrosine phosphorylation remains unknown. Northern blot analysis revealed that PTP20 was abundantly expressed in spleen, suggesting a role in the immune system (data not shown). Therefore, it was reasoned that other PTKs of immune cells might be involved in PTP20 regulation by tyrosine phosphorylation. To examine this possibility HA-tagged PTP20 was co-expressed with various cytosolic PTKs including Src and Lck in COS7 cells. We used a catalytically inactive form of PTP20 for this experiment because autodephosphorylation activity of PTP20 has been previously reported (8Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (153) Google Scholar). Cells were lysed, PTP20 was immunoprecipitated with anti-HA antibody, and the immune complexes were subjected to SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody. As shown in Fig. 1A, PTP20 was tyrosine-phosphorylated by Src and Lck and co-immunoprecipitated with proteins with 56 and 60 kDa, likely corresponding to Lck and Src, respectively. In the case of ectopic Lck expression, endogenous Src seemed to be included in the immune complex, as suggested by the presence of a 66-kDa phosphotyrosine-containing band. PTP20 was slightly tyrosine-phosphorylated by Csk and co-immunoprecipitated with a faintly tyrosine-phosphorylated 70-kDa band, which seemed unlikely to be Csk. JAK2 but not JAK3 also tyrosine-phosphorylated PTP20 and appeared to be co-immunoprecipitated with PTP20. Most notably, PTP20 was strongly tyrosine-phosphorylated by Tec and co-immunoprecipitated with a heavily tyrosine-phosphorylated protein of 74 kDa and other minor proteins of 120 and 35 kDa. Based on the molecular mass, the 74-kDa protein was likely to represent Tec. Itk, another member of Tec/Btk family, also tyrosine-phosphorylated PTP20 to a lesser extent and was co-immunoprecipitated, whereas related PTKs Btk and Bmx did not tyrosine phosphorylate PTP20 and were not co-immunoprecipitated. Because all the transfected PTKs were obviously expressed as compared with mock transfectant (Fig. 1, panel B), it was suggested that Tec tyrosine-phosphorylated PTP20 with the greatest efficiency. Tec Is a Potential Substrate of PTP20—To examine the relationship between PTP20 and Tec in more detail, Tec was co-transfected with WT or a catalytically inactive C/S form of PTP20 into COS7 cells, and either PTP20 or Tec was immunoprecipitated followed by immunoblotting with anti-phosphotyrosine antibody. When HA-PTP20 WT was expressed, no phosphorylated bands were visible in both anti-HA and anti-Tec immunoprecipitates, possibly due to dephosphorylation activity of PTP20 against both Tec and itself (Fig. 2). Two major bands with 74 and 50 kDa in the anti-HA and anti-Tec immune complexes were detected with anti-phosphotyrosine antibody only when the PTP20 C/S mutant was co-transfected with Tec. Reprobing with anti-Tec and anti-HA antibodies clearly revealed that the bands represent Tec and HA-PTP20. No phosphorylation of Tec was observed when Tec alone was introduced into COS7 cells, suggesting that the interaction between Tec and PTP20 was required for Tec phosphorylation and possibly activation. These results suggest that PTP20 is a substrate of Tec and that Tec is also a substrate of PTP20. Phosphotyrosine-dependent Interaction between PTP20 and Tec—Tec is composed of several distinct domains including pleckstrin homology (PH), Tec homology (TH), SH3, SH2, and kinase (KD) domains (Fig. 3, panel A). All of these domains are necessary for full function of Tec under physiological conditions (15Mano H. Yamashita Y. Miyazato A. Miura Y. Ozawa K. FASEB J. 1996; 10: 637-642Crossref PubMed Scopus (41) Google Scholar, 16Mao J. Xie W Yuan H. Simon M.I. Mano H. Wu D. EMBO J. 1998; 17: 5638-5646Crossref PubMed Scopus (86) Google Scholar). To examine which domains are involved in interaction with PTP20, Tec mutants each lacking one of the domains were co-transfected with the catalytically inactive form of PTP20 into COS7 cells. A kinase mutant as well as two mutants (Y187F and Y518F) where tyrosine residues were replaced by phenylalanines were also included. Cells were lysed, and PTP20 was immunoprecipitated followed by immunoblotting with anti-phosphotyrosine antibody. PTP20 was tyrosine-phosphorylated by the Y187F mutant as well as mutants lacking PH, TH, and SH3 domains to a similar extent as compared with Tec WT (Fig. 3, panel B). As expected, the Y518F mutant, which is missing the autophosphorylation site for Tec activation, and the inactive mutant of a kinase mutant could not tyrosine phosphorylate PTP20. Interestingly, the ΔPH mutant could tyrosine phosphorylate PTP20 but was not co-immunoprecipitated with PTP20. Most strikingly, the ΔSH2 mutant could not tyrosine phosphorylate PTP20 and was not co-immunoprecipitated with PTP20. When a membrane on which aliquots of total cell lysates were blotted was probed with anti-phosphotyrosine antibody, it was revealed that co-expression of the Tec ΔSH2 mutant and PTP20 resulted in no tyrosine phosphorylation on both molecules and that the Tec ΔPH mutant tyrosine-phosphorylated (Fig. 3, panel C). Tec SH2 domain-dependent interaction with PTP20 was further investigated by co-transfecting the PTP20 C/S mutant with plasmids encoding GST fusion proteins of Tec domains in the presence or absence of Tec into COS7 cells. Cell lysates were subjected to pull-down experiments with GSH-Sepharose beads. Precipitates were separated by SDS-PAGE followed by immunoblotting with the indicated antibodies. In the absence of full-length Tec co-expression, no substantial binding of PTP20 to any of the Tec domains was apparent (Fig. 3, panel D). In contrast, in the presence of full-length Tec, phosphorylated PTP20 bound to only the SH2 domain of Tec. Given that co-expression of Tec should result in marked tyrosine phosphorylation of PTP20 in COS7 cells, these data indicate that the PTP20-Tec interaction is mediated predominantly by the SH2 domain of Tec and phosphotyrosine residues of PTP20. Next, we tried to identify the binding site(s) for Tec in PTP20. Because the interaction of Tec with PTP20 was mediated by the Tec SH2 domain, potential tyrosine residues of phosphorylation were first taken into consideration. There are 13 tyrosine residues (Tyr-62, Tyr-68, Tyr-86, Tyr-101, Tyr-144, Tyr-192, Tyr-244, Tyr-281, Tyr-285, Tyr-303, Tyr-354, Tyr-381, Tyr-419) in the PTP20 sequence, and all the residues are perfectly conserved among human and mouse orthologs (Fig. 4). We focused our attention on the tyrosine residues Tyr-281, Tyr-285, Tyr-303, Tyr-354, Tyr-381, and Tyr-419 located in the C-terminal PEST domain of PTP20, and 6 residues were individually mutated. First, the mutants were tested for the extent of tyrosine phosphorylation by Tec in transfected COS7 cells. Total cell lysates were subjected to anti-phosphotyrosine blotting. Fig. 5, panel A, demonstrates that the PTP20 mutants (Y281F, Y303F, Y354F, Y381F) in which Tyr-281, Tyr-303, Tyr-354, and Tyr-381 were individually mutated exhibited dramatic reduction in tyrosine phosphorylation levels, whereas no apparent reduction for Y285F and Y419F was observed. Combinational mutation of Tyr-281, Tyr-303, Tyr-354, and Tyr-381 totally abolished tyrosine phosphorylation of PTP20. In keeping with these data, anti-phosphotyrosine blotting also demonstrated that tyrosine phosphorylation of Tec was concomitantly reduced. This observation was further extended by GST pull-down experiments using the Tec SH2 domain. COS7 cells were then transfected with PTP20 YF variants together with Tec and Tec-Tec SH2, as outlined in Fig. 3, panel C. Mutation of either Tyr-281, Tyr-303, Tyr-354, or Tyr-381 of PTP20 resulted in reduced binding capacity of PTP20 to the Tec SH2 domain, and again, such binding was completely abrogated by substituting all the tyrosine residues (Fig. 5, panel B). Together these data clearly indicate that four tyrosine residues in the C-terminal non-catalytic region of PTP20 are involved in not only binding to the Tec SH2 domain but also in the phosphorylation and subsequent activation of Tec. We asked whether the C-terminal non-catalytic region of PTP20 was enough for phosphorylation and activation of Tec. To this end, PTP20 deletion mutants lacking either an N-terminal catalytic or a C-terminal non-catalytic segment were made, but the resultant constructs could not be expressed in COS7 cells, although comparable amounts of transcripts were detected (data not shown). To solve this problem, the N-terminal PTP domain and the C-terminal PEST domain were inserted into pEBG vector and were expressed as GST fusion proteins in COS7 cells. These pEBG plasmids encoding the PTP domain and full length of PTP20 C/S mutant and the PEST domain of PTP20 were co-transfected into COS7 together with Tec. Anti-phosphotyrosine blotting documented that Tec was highly tyrosine-phosphorylated with the full-length but not the PTP domain of the PTP20 C/S mutant (Fig. 6, panel A), supporting previous data shown in Fig. 5, where the C-terminal part of PTP20 was essential for tyrosine phosphorylation of Tec. Interestingly, the presence of the PEST domain of PTP20 caused tyrosine phosphorylation of PTP20, but the extent was lower than in the presence of the full-length PTP20 C/S mutant. Equivalent expression of each construct was confirmed by Western blotting with anti-Tec and anti-GST antibodies. To further examine the involvement of the PEST domain, lysates were precipitated with GSH-Sepharose beads followed by immunoblotting with anti-phosphotyrosine antibody. A phosphorylated 74-kDa band, which was shown to be Tec by immunoblotting, was co-precipitated with full-length PTP20 C/S mutant, whereas the PTP domain alone could not capture Tec (Fig. 6, panel B). A faint tyrosine-phosphorylated band with the same mobility of 74 kDa that co-precipitated with the PEST domain appeared to be Tec but could not be detected by our anti-Tec antibody presumably due to sensitivity. These results suggest that the PEST domain of PTP20 is necessary but not sufficient for not only hyperphosphorylation and activation of, but also association with Tec. Negative Regulatory Roles of PTP20 in BCR Signaling—All the experiments documented above were conducted in transfected COS7 cells. To demonstrate a physiological relevance of the PTP20-Tec interaction, evidence of such an association in non-transfected cells was required. To this end we selected human Ramos immature B cells, because it has been reported that they express relatively high amounts of endogenous Tec (21Kitanaka A. Mano H. Conley M.E. Campana D. Blood. 1998; 91: 940-948Crossref PubMed Google Scholar). As shown above, interaction of PTP20 with Tec is mediated by tyrosine phosphorylation of PTP20, and PTP20 has autodephosphorylation activity, implying that it would be difficult to detect a phosphotyrosine-dependent interaction of PTP20 with other molecules including Tec endogenously. To overcome this experimental difficulty, protein-tyrosine phosphorylation was induced in Ramos cells by treatment with pervanadate (POV). Cells were starved for 16 h in serum-free medium and then either left unstimulated or treated with 0.1 mm POV for 30 min and lysed. Cell lysates were immunoprecipitated with either anti-phosphotyrosine antibody or anti-Tec antibody. Our PTP20-specific antibody could not be used due to its inability in immunoprecipitation experiments. In anti-phosphotyrosine immunoprecipitates, specific bands with 74 and 50 kDa corresponding to human Tec and PTP20 were detected only upon POV treatment (Fig. 7). A tyrosine-phosphorylated band with 50 kDa in the anti-Tec immunoprecipitates was readily detected by the anti-PTP20 antibody but only when cells received POV pretreatment (Fig. 7). These results indicate that endogenous Tec and PTP20 interact with each other in a phosphotyrosine-dependent manner in Ramos B cells. Although upstream regulators such as cytokine receptors, lymphocyte surface antigens, G protein-coupled receptors, receptor type PTKs, or integrins for Tec in blood cells including Ramos B cells have been relatively well investigated (13Mano H. Yamashita Y. Sato K. Yazaki Y. Hirai H. Blood. 1995; 85: 343-350Crossref PubMed Google Scholar, 20Yamashita Y. Watanabe S Miyazato A Ohya K. Ikeda U. Shimada K. Komatsu N. Hatake K. Miura Y. Ozawa K. Mano H. Blood. 1998; 91: 1496-1507Crossref PubMed Google Scholar, 22Machide M. Mano H. Todokoro K. Oncogene. 1995; 11: 619-625PubMed Google Scholar, 23Matsuda T. Takahashi-Tezuka M. Fukada T. Okuyama Y. Fujitani Y. Tsukada S. Mano H. Hirai H. Witte O.N. Hirano T. Blood. 1995; 85: 627-633Crossref PubMed Google Scholar, 24Miyazato A. Yamashita Y. Hatake K. Miura Y. Ozawa K. Mano H. Cell Growth Differ. 1996; 7: 1135-1139PubMed Google Scholar, 25Tang B. Mano H. Yi T. Ihle J.N. Mol. Cell. Biol. 1994; 14: 8432-8437Crossref PubMed Scopus (88) Google Scholar, 26Yamashita Y. Miyazato A. Shimizu R. Komatsu N. Miura Y. Ozawa K. Mano H. Exp. Hematol. 1997; 25: 211-216PubMed Google Scholar), only limited information regarding downstream regulators of Tec has been available so far. If the data obtained in transfected COS7 cells are true, PTP20 would be thought to play a negative regulatory role in Tec-mediated signaling. To examine this, either the PTP20 WT, the inactive C/S mutant, or another form of catalytically inactive mutant D/A was transiently co-transfected with the pfos/luc reporter plasmid into Ramos cells, because the promoter of the c-fos proto-oncogene is activated in response to BCR cross-linking in the cells. Cells were either left unstimulated or treated with anti-human IgM F(ab′)2 fragments for 5 h. Cell lysates were assayed for luciferase activity. BCR cross-linking induced a marked activation of the c-fos promoter (Fig. 8). Expression of PTP20 WT totally inhibited BCR-induced activation of the c-fos promoter as well as its basal activity, whereas only about 20% inhibition of the promoter activation was observed in the co-expression of catalytically inactive forms of PTP20, strongly indicating that PTP20 is a negative regulator of BCR-Tec-c-fos signaling. Tyrosine Phosphorylation of PTP20 by Tec Modulates Its Catalytic Activity against Tec as Well as Itself—We demonstrated that specific tyrosine residues Tyr-281, Tyr-