SHP-1 dephosphorylates 3BP2 and potentially downregulates 3BP2-mediated T cell antigen receptor signaling Zhenbao Yu1, Meryem Maoui1, Zhizhuang J Zhao2, Yang Li1 and Shi-Hsiang Shen1,3 ´ Health Sector, Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA ´ Department of Medicine, McGill University, Montreal, Canada Keywords 3BP2; protein phosphatases; protein–protein interaction; SHP-1; T cell-receptor Correspondence Zhenbao Yu, Health Sector, Biotechnology Research Institute, National Research ´ ´ Council of Canada, Montreal, Quebec H4P 2R2, Canada Fax: +1 514 496 6319 Tel: +1 514 496 6377 E-mail: zhenbao.yu@nrc.ca (Received 14 December 2005, revised 28 February 2006, accepted 16 March 2006) doi:10.1111/j.1742-4658.2006.05233.x Src homology (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) is a critical inhibitory regulator in T cell-receptor (TCR) signaling However, the exact molecular mechanism underlying this is poorly defined, largely because the physiological substrates for SHP-1 in T cells remain elusive In this study, we showed that adaptor protein 3BP2 serves as a binding protein and a physiological substrate of SHP-1 3BP2 is phosphorylated on tyrosyl residue 448 in response to TCR activation, and the phosphorylation is required for T cell signalling, as indicated by transcriptional activation of nuclear factor activated in T cells (NFAT) Concurrently, phosphorylation of Tyr566 at the C-terminus of SHP-1 causes specific recruitment of 3BP2 to the phosphatase through the SH2 domain of the adaptor protein This leads to efficient dephosphorylation of 3BP2 and thereby termination of T cell signaling The study thus defines a novel function of the C-terminal segment of SHP-1 and reveals a new mechanism by which T cell signaling is regulated Protein tyrosine phosphorylation plays a critical role in various signal-transduction pathways in T lymphocytes [1] For example, ligation of the T cell antigen receptor (TCR) activates Src family protein tyrosine kinases (PTKs) such as Lck and Fyn, which in turn phosphorylate TCR n chain and CD3 e, d and c subunits within the immunoreceptor tyrosine-based activation motif (ITAM), resulting in the recruitment and activation of the ZAP70 and Syk PTKs [2,3] These activated PTKs further induce the tyrosine phosphorylation of multiple intracellular proteins, including the adapter proteins LAT [4] and SLP-76 [5] Phosphorylation of these adapter proteins creates docking sites for various Src-homology (SH2) domain-containing proteins such as PLCc, Grb2, Grap, Gads, Nck, Vav, c-CBL and Tec family tyrosine kinase Itk, leading to stimulation of downstream signaling pathways, and ultimately to T cell activation [6,7] Dephosphorylation of these tyrosine-phosphorylated proteins is a necessary counterpart for maintaining a balance between activation and quiescence of TCR signaling [8] SHP-1 is one such enzyme which can counterbalance PTK effects and terminate receptor-initiated signaling [9] SHP-1 is expressed primarily in hematopoietic cells and plays a critical role in the negative regulation of TCR signaling and T cell development Accordingly, thymocytes derived from motheaten (me) mice, which lack the expression of functional SHP-1, hyperproliferate in response to TCR stimulation [10–18] SHP-1 displays its negative function at diverse stages of TCR signaling For instance, SHP-1 constitutively associates with TCR and appears Abbreviations GST, glutathione S-transferase; IP, immunoprecipitation; ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; pTyr, phosphotyrosine; SH2, src homology 2; SHP, SH2 domain-containing PTPase; TCR, T cell antigen receptor FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2195 SHP-1 and 3BP2 in T cell receptor signaling Z Yu et al to dephosphorylate the TCR CD3e subunit and more distal signaling effectors following TCR activation [11] It has been also reported that SHP-1 is phosphorylated by activated Src family kinase Lck [19] in T cells and it, in turn, dephosphorylates and inactivates Lck and Fyn kinases [12,18,20] SHP-1 is also thought to regulate the activity of Syk family kinase ZAP70 [21,22] Moreover, SHP-1 is known to interact with the adapter proteins Grb2 and SLP-76, although the physiological meaning is unclear [11,23,24] In this study, we identified 3BP2 as a novel SHP-1 substrate and binding protein 3BP2 was originally described as a PTK c-Abl SH3 domain-binding protein [25] It contains an N-terminal PH domain, a central proline-rich region that interacts with c-Abl, and a C-terminal SH2 domain Recently, the SH2 domain of 3BP2 has been shown to bind to the PTKs Syk and ZAP70 [26] As a result, overexpression of 3BP2 in T cells leads to increased nuclear factor activated T cell (NFAT-) and AP-1-dependent transcription [26,27] It has also been shown that 3BP2 plays a positive regulatory role in NK-cell-mediated cytotoxicity [28] and participates in the regulation of FccR1-mediated degranulation in basophilic cells [29] However, the mechanism by which 3BP2 exerts its positive effect on downstream signaling molecules remains elusive In this study, we demonstrate that the interplay of 3BP2 and SHP-1 has an important role in T cell signaling On the one hand, 3BP2 is phosphorylated on tyrosyl residue 448, and the tyrosine phosphorylation is critical for TCR signaling On the other hand, SHP-1 is phosphorylated on Tyr566 at the C-terminus and thereby recruits 3BP2 through SH2 domain interaction This leads to dephosphorylation of 3BP2 and termination of T cell signaling This study thus provides a novel mechanism by which 3BP2 and SHP-1 regulate T cell signaling Results 3BP2 interacts with SHP-1 in a yeast two-hybrid screen To demonstrate the molecular mechanism of SHP-1mediated regulation of TCR signaling, we searched for SHP-1-interacting proteins from human T cells using a modified yeast two-hybrid screen [30] The full-length SHP-1 with mutation of Cys455 to Ser (SHP-1 ⁄ C455S), which abolishes the protein tyrosine phosphatase (PTPase) catalytic activity but retains the binding ability to its substrates, was cloned into plasmid pBTM-116-Src [31] for two-hybrid screening 2196 Transformation of the plasmid in yeast results in the expression of Lex DNA-binding domain ⁄ SHP-1– C455S fusion protein and c-Src kinase Expression of c-Src allows the identification of tyrosine phosphorylation-dependent SHP-1-interacting proteins From 1.1 · 107 transformants with a human Jurkat T cell cDNA library, 124 were positive for both HIS3 and LacZ expression Sequence analyses of the 124 positive clones revealed that, among others, 11 independent clones of different lengths represented overlapping cDNAs of the SH3 domain-binding protein (3BP2) 3BP2 was originally characterized as an Abl SH3-interacting protein [25] It is composed of an N-terminal PH domain, a proline-rich region and a C-terminal SH2 domain Interestingly, all of the 3BP2 clones isolated in our two-hybrid screening contained at least the sequence encoding the entire SH2 domain, suggesting that the SH2 domain of 3BP2 is involved in mediating the SHP)1 ⁄ 3BP2 interaction Our additional studies demonstrated that the catalytic inactive Cys-to-Ser mutant of SHP-2, an enzyme structurally similar to SHP-1, was incapable of interacting with 3BP2 in the system (data not shown) This indicates a high specificity of the interaction between 3BP2 and SHP-1 3BP2 associates with SHP-1 in 293T cells when coexpressed with Lck To determine whether 3BP2 associates with SHP-1 in mammalian cells, we carried out a coimmunoprecipitation assay Because the interaction of 3BP2 with SHP-1 was first identified from a T cell cDNA library in a modified yeast two-hybrid system in which a Src family kinase c-Src was expressed, we cotransfected 293T cells with C-terminal myc-tagged 3BP2 (3BP2–myc), catalytically inactive SHP-1 (SHP-1 ⁄ C455S), and Src family kinases, Fyn and Lck, which are known to be involved in TCR signaling 3BP2–myc was immunoprecipitated with an anti-myc IgG As shown in Fig 1A, SHP1 ⁄ C455S was detected by western blot with the anti(SHP-1) IgG in the anti-myc immunoprecipitant from 293T cells cotransfected with wild-type Lck or catalytically activated Lck (Lck ⁄ Y505F) However, under the same conditions, SHP-1 ⁄ C455S could not be coimmunoprecipitated with 3BP2–myc from 293T cells cotransfected with catalytically activated Fyn (Fyn ⁄ Y531F) or the plain control vector (Fig 1A) In reciprocal experiments involving immunoprecipitating SHP-1 ⁄ C445S with anti-(SHP-1) IgG, 3BP2–myc was detected in the immunoprecipitant from cells transfected with catalytically activated Lck but not in the immunoprecipitant from the cells transfected with wild-type Lck FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) SHP-1 and 3BP2 in T cell receptor signaling IgG anti-Lck 82 Western blot: anti-myc WC L first IP no Abs + + anti-Lck + + + + no Abs + + second IP: anti-myc IgG + + + + + + IgG + + precipitant anti-Lck - SHP-1 3BP2-myc unbound no Abs kinase B Fy n-Y Lc 531F k Lc k-Y 50 5F A Fy n-Y 53 Lc 1F k Lc k-Y 50 5F Z Yu et al 10 3BP2-myc 82 62 62 47 47 82 Western blot: anti-SHP-1 Western blot: anti-Lck Lck SHP-1 62 10 82 47 3BP2 + + + + - + + + + + + - Fy n-Y 53 1F Lc k Lc k-Y 50 5F - SHP-1 3BP2-myc 62 IP: anti-SHP-1 Fy n-Y 53 1F Lc k Lc k-Y 50 5F kinase Fy n-Y 53 1F Lc k Lc k-Y 50 5F IP: anti-myc + + + + + + + + + + + + Western blot: anti-myc 47 + + 10 82 160 SHP-1 82 3BP2-myc 62 Western blot: anti-pTyr 62 Western blot: anti-SHP-1 SHP-1 47 47 Whole cell lysates IP: anti-myc IP: anti-SHP-1 Fig 3BP2 associates with SHP-1 when coexpressed with Lck in 293T cells (A) 293T cells were transfected with 3BP2–myc, SHP1 ⁄ C445S and Src family of kinases Fyn or Lck as indicated The cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine IgG for 48 h after transfection and then lysed without any treatment Whole-cell lysates were subjected to immunoprecipitation and western blot analysis with anti-myc IgG, anti-SHP-1 IgG or anti-phosphotyrosine IgG Molecular mass (kDa) is indicated to the left of the gel (B) 293T cells were transfected with 3BP2–myc, SHP-1 ⁄ C445S and autoactivated Lck (Lck ⁄ Y505F) Forty-eight hours after transfection the cells were lysed and the whole-cell lysates were subjected to immunoprecipitation with anti-Lck IgG, IgG or without antibody as control The unbound proteins after the first immunoprecipitation were subjected to immunoprecipitation with anti-myc IgG The proteins collected in each step were analyzed by western blot as indicated (Fig 1A, right) although SHP-1 could be coimmunoprecipitated with 3BP2–myc by anti-myc IgG from the wild-type Lck-transfected cell This indicates that low amounts of associated proteins could not be detected in some coimminoprecipitation experiments 3BP2, SHP-1, and Lck all contain SH2 domains and potential tyrosine phosphorylation sites that can mediate protein–protein interactions To examine whether the association of 3BP2 with SHP-1 could be mediated by Lck, we cotransfected these three proteins into 293T cells and carried out a two-step immunoprecipitation experiment Whole-cell lysates were first subjected to immunoprecipitation with anti-Lck IgG or control antibody (mouse IgG or protein A–Sepharose 4B beads alone) and the unbound proteins were then subjected to immunoprecipitation with anti-myc IgG If the interaction of 3BP2 with SHP-1 is mediated by Lck, removal of Lck from whole-cell lysates by anti-Lck IgG immunoprecipitation should reduce the amount of SHP-1 coprecipitated with 3BP2–myc However, as shown in Fig 1B, although Lck was essentially depleted from whole-cell lysates by anti-Lck IgG (Fig 1B, upper, lane 4), coimmunoprecipitation of SHP-1 with 3BP2–myc was not affected (Fig 1B, lower, lanes 8–10) Moreover, neither 3BP2–myc (Fig 1B, middle, lane 7) nor SHP-1 (Fig 1B, lower, lane 7) was detected in the anti-Lck immunoprecipitates Parallel reciprocal experiments showed that Lck was not coimmunoprecipitated with 3BP2–myc by anti-myc IgG either (Fig 1B, upper, lanes and 9) Taken together, these results indicate that the association of 3BP2 with SHP-1 is not mediated by Lck, although its kinase activity is required for the interaction in 293 cells FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2197 SHP-1 and 3BP2 in T cell receptor signaling Z Yu et al 3BP2 interacts with SHP-1 through the SH2 domain of 3BP2 Interaction of the SH2 domain of 3BP2 with SHP-1 is mediated by phosphorylation of SHP-1 at Tyr566 Because both 3BP2 and SHP-1 contain SH2 domains, the interaction of 3BP2 with SHP-1 might be through either binding of the SH2 domains of SHP-1 to tyrosine-phosphorylated 3BP2 or that of the SH2 domain of 3BP2 to phosphorylated SHP-1 We thus constructed a glutathione S-transferase (GST) fusion protein of the SHP-1 SH2 domains (GST–SHP-1–2SH2) and also of the 3BP2 SH2 domain (GST)3BP2–SH2), and carried out GST pull-down experiments to determine which of these two possibilities accounts for the observed association As shown in Fig 2A, SHP1 ⁄ C455S was precipitated by GST)3BP2–SH2 In the same condition, SHP-2 could not be pulled down by GST)3BP2–SH2 Western blot with anti-phophotyrosine IgG indicates that both SHP-1 and SHP-2 were phosphorylated These results suggest that 3BP2 specifically interacts with SHP-1 but not SHP-2 In contrast, 3BP2–myc could not be pulled down by GST–SHP-1– 2SH2 (Fig 2B) Note that, under the same conditions, GST–SHP-1–2SH2 was able to pull down S2V, a siglec family receptor previously identified as an SHP-1-binding protein [32], suggesting that the GST–SHP-1–2SH2 fusion protein was properly folded These results suggest that 3BP2 interacts with SHP-1 through the SH2 domain of 3BP2 and, presumably, tyrosine-phosphorylated SHP)1 We next determined which tyrosine residue(s) of SHP1 is (are) involved in the interaction using tyrosine-tophenylalanine mutants SHP-1 contains a C-terminal noncatalytic tail that bears three potential phosphorylated tyrosine residues (Tyr538, Tyr543 and Tyr566) We mutated each of them and cotransfected the resulting mutants with 3BP2–myc and Lck into 293T cells As shown in Fig 3B, although both SHP-1 ⁄ Y538F and SHP-1 ⁄ Y543F were detected in the anti-myc immunoprecipitants, SHP-1 ⁄ Y566 was not detectable, suggesting that 3BP2 binds to the phosphorylated Tyr566 of SHP-1 Interestingly, we found that both wild-type and catalytically inactive SHP-1 (SHP1 ⁄ C455S) could be coprecipitated by 3BP2–myc with anti-myc IgG (Fig 2B and data not shown), indicating that Tyr566, the 3BP2-binding site of SHP-1 was not dephosphorylated by SHP-1 in this condition However, western blot analyses of the anti-SHP-1 immunoprecipitants with anti-phosphotyrosine IgG showed that the tyrosine phosphorylation level of SHP-1 ⁄ Y566F mutant is much lower than that of wild-type SHP-1, SHP-1 ⁄ Y538F, SHP-1 ⁄ Y543F and SHP-1 ⁄ C455S ⁄ Y566F (Fig 3F), suggesting that Tyr566 is the major phosphorylation site of SHP-1 in this condition The anti-phosphotyrosine western blot - pervanadate A Western blot: anti-SHP-1 SHP-1 + SHP-1 IgG Western blot: anti-pTyr IP :anti-SHP-1 Western blot: anti-SHP-2 H2 P2 -S GS T IgG IP :anti-SHP-2 S2 V-m yc 3B P2 -m yc +S 3B 2V P2 -m -m yc yc S2 V-m yc 3B P2 -m yc +S 2V -m yc 3B P2 -m yc B SHP-2 Western blot: anti-pTyr GS T-3 B WC L SHP-2 Western blot: anti-myc S2V-myc 3BP2-myc WCL 2198 GST-SHP-1-2SH2 Fig SHP-1 associates with 3BP2 through the SH2 domain of 3BP2 293T cells were transfected with SHP-1 ⁄ C455S, SHP2 ⁄ C459S, 3BP2–myc and 3BP2–myc plus S2V-myc, respectively Forty-eight hours after transfection, the cells were treated with 0.5 mM pervanadate for 30 The whole-cell lysates were incubated with GST, GST)3BP2–SH2 or GST–SHP-1–2SH2 bound on glutathione Sepharose and subjected to immunoprecipitation as indicated The proteins precipitated were analyzed by western blot with anti-SHP-1, anti-SHP-2, anti-phosphotyrosine or anti-myc IgG FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) Z Yu et al SHP-1 and 3BP2 in T cell receptor signaling 5S + + + + + + + + - - + + + + + + + SHP-1 Lck 3BP2-myc + WT C4 55 S SDS treatment - - + + + - - + + + + + + + + + + + - - WT C4 55 S 56 6F G WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S/Y + + + + + + + + WT WT WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S/Y 56 6F WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S /Y + + + + + + + + WT - + + + + + + + SHP-1 Lck 3BP2-myc - + + + + + + + C WT C4 B 66 F A - + + + + + + + 82 120 3BP2-myc 80 60 SHP-1 WB: anti-myc 62 47 40 IP: anti-myc IP: anti-myc WB: anti-myc IP: anti-SHP-1 WB: anti-SHP-1 82 WB: anti-SHP-1 WB: anti-pTyr 62 WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S/Y 56 6F WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S/Y 56 6F WT 66 F F ++ + + + + + + 120 3BP2-myc 80 SHP-1 Lck 3BP2-myc S IP: anti-myc WT C4 55 WCL C4 55 S + + + + + + + + 47 WT + + + + + + + + WT -++ + + + + + WT - + + + + + + + SHP-1 Lck 3BP2-myc - + + + + + + + E /Y5 WT C4 55 Y5 S 38 Y5 F 43 Y5 F 66 C4 F 55 S D - - + + + - - + + + + + + + + + + + SHP-1 60 82 40 62 whole cell lysates IP: anti-myc 47 IP: anti-SHP-1 Western blot: anti-pTyr WCL WB: anti-Lck WCL WB: anti-SHP-1 Fig SHP-1 associates with 3BP2 through the phosphorylated tyrosine residue 566 of SHP-1 (A–F) 293T cells were cotransfected with 3BP2–myc, Lck ⁄ Y505F and SHP-1 or its mutants Forty-eight hours after transfection, the cells were lysed and whole-cell lysates were subjected to immunoprecipitation and western blot with the indicated antibodies (G) 293T cells were cotransfected with 3BP2 myc, Lck ⁄ Y505F and SHP-1 or its catalytically inactive mutant (SHP-1 ⁄ C455S) Forty-eight hours after transfection, the cells were lysed The whole-cell lysates were equally divided into two portions One portion of the lysates was treated with 1% SDS at 100 °C for 10 The other portion was left untreated The lysates were then diluted 10 times with lysis buffer and subjected to immunoprecipitation and western blot as described analysis of the anti-myc immunoprecipitants showed that 3BP2 was phosphorylated and dephosphorylated by wild-type SHP-1, and the SHP-1 ⁄ Y538F and SHP1 ⁄ Y543F mutants (Fig 3E), but not by the catalytically inactivated mutants (C455S and C455S ⁄ Y566F) 3BP2 was also partially dephosphorylated by SHP1 ⁄ Y566F mutant (Fig 3E,D, lane 7) although it did not associate with this mutant (Fig 3B, lane 7), suggesting that SHP-1 may be also able to directly dephospharylate 3BP2 without association of the two proteins through the SH2 domain–phosphotyrosine interaction in the condition with the overexpression of the two proteins To exclude the possibility that the major tyrosinephosphorylated protein in the anti-myc precipitants is not 3BP2–myc but another protein of similar molecular mass that might be comimmunoprecipitated with 3BP2–myc, we treated the whole-cell lysates by adding SDS to 1% and heating the samples at 100 °C for 10 before immunoprecipitation This should disrupt protein–protein interactions Treated samples were then diluted 10 times with lysis buffer and subjected to immunoprecipitation Such treatment is expected to eliminate the coimmunoprecipitation of any 3BP2-binding proteins from 3BP2 with anti-myc IgG As shown in Fig 3G, the tyrosine-phosphorylated protein with the same molecular mass as 3BP2– myc was detected in the anti-myc precipitants from the SDS-treated samples as well as in those from nontreated samples This result further confirms that 3BP2 was tyrosine phosphorylated More significantly, the phosphorylated 3BP2 was nearly completely dephosphorylated by wild-type SHP-1, but not by its catalytically inactive mutant SHP-1 ⁄ C455S (Fig 3E), suggesting that 3BP2 is a potential substrate for SHP-1 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2199 SHP-1 and 3BP2 in T cell receptor signaling Z Yu et al promoter-driven Renilla luciferase and 3BP2 or its mutants, and then stimulated the cells with PMA plus ionomycin Firefly luciferase activity was normalized by Renilla luciferase activity As the results show that 3BP2 and its mutants did not affect the T cell response to the PMA ⁄ ionomycin stimulation (data not shown), we normalized the transfection efficiencies determined by the stimulation with PMA plus ionomycin As shown in Fig 5, although mutation on other tyrosine residues of 3BP2 did not exert appreciable effects on 3BP2-mediated NFAT activation, the Y448F mutation reduced the effect of 3BP2 on NFAT activation These results suggest that phosphorylation of 3BP2 on Tyr448 plays an important role for its function in TCR signaling Tyr448 is the major phosphorylated residue of 3BP2 in response to TCR engagement and is critical for 3BP2 function in TCR signaling 3BP2 is a positive regulator of TCR signaling and is phosphorylated on tyrosine residues in response to TCR engagement [26] To further study the function of 3BP2 phosphorylation in the regulation of TCR signaling, we determined which tyrosine residue(s) of 3BP2 can be phosphorylated Four potential phosphorylation sites, namely Tyr174, Tyr183, Tyr448 and Tyr485, were predicted (http://kinasephos.mbc nctu.edu.tw) We mutated these tyrosine residues to phenylalanine and transfected these mutants into Jurkat cells The tyrosine-phosphorylation status of these mutants was examined following stimulation with antiCD3 IgG OKT3 As shown in Fig 4, although none of the mutations Y174F, Y183F and Y485F exerted any evident effect on the tyrosine phosphorylation of 3BP2, mutation Y448F almost completely abolished tyrosine phosphorylation, suggesting that Tyr448 is the major tyrosine-phosphorylated residue in response to TCR activation We next determined the effects of these 3BP2 mutants on NFAT activation To so, we cotransfected 3BP2 or its mutants with NFAT-luciferase (firefly) reporter into Jurkat cells and stimulated the cells with anti-CD3 IgG, and PMA plus ionomycin, respectively To determine whether the expression of 3BP2 and its mutants affects stimulation of the expression of NFAT-driven luciferase by PMA ⁄ ionomycin, we transfected the Jurkat cells with NFATluciferase vector, pRL-TK vector which expresses TK 3BP2 WT OKT3 82 IP: anti-myc WB: anti-pTyr 2' 3BP2/Y174F 10' 2' 10' SHP-1 dephosphorylates 3BP2 in TCR signaling and negatively regulates 3BP2-induced NFAT activation Because tyrosine phosphorylations of SHP-1 at the C-terminal residues can activate its phosphatase activity [33] and 3BP2 interacts with phosphorylated SHP-1, it is expected that the tyrosine-phosphorylated 3BP2 is a potential substrate for activated SHP-1 during their interaction The substrate characteristic of 3BP2 for SHP-1 was primarily demonstrated in 293T cells where the tyrosine-phosphorylated 3BP2 was largely dephosphorylated by wild-type SHP-1, but not by catalytically inactive mutant SHP-1 ⁄ C455S (Fig 3E) To further investigate the substrate nature of 3BP2 for SHP-1 during T cell signaling, we cotransfected 3BP2–myc with wild-type SHP-1, its catalyti- 3BP2/Y183F 2' 10' 3BP2/Y448F 10' 3BP2/Y485F 2' 10' 3BP2-myc 62 82 IP: anti-myc WB: anti-myc 3BP2-myc 62 Fig Identification of Tyr448 as the major phosphorylated residue of 3BP2 in response to TCR engagement Jurkat T cells were transfected with 3BP2 or its mutants as indicated Forty-eight hours after transfection, the cells (5 · 107) were stimulated with anti-CD3 IgG (OKT3) at 37 °C for 0, or 10 as described in Experimental procedures Whole-cell lysates were immunoprecipitated with anti-myc IgG and the precipitated proteins were subjected to western blot analysis with anti-phosphotyrosine (pTyr) and anti-Myc IgG, respectively 2200 FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) - + - + - + SH P1/Y 56 6F SH P1/C SH P1/W T Ve c OKT3 82 45 5S SHP-1 and 3BP2 in T cell receptor signaling tor Z Yu et al - + 3BP2-myc WB: anti-pTyr 62 82 Fig Effect of the mutation of tyrosine residues on 3BP2-induced NFAT activation Jurkat T cells were cotransfected with NFAT-luciferase reporter and 3BP2–myc or its mutants as indicated Twenty hours after transfection, the cells were incubated with either no addition, anti-CD3 IgG (OKT3) or PMA plus ionomycin at 37 °C for h as described in Experimental procedures Luciferase activity in cell extracts was assayed and the data were normalized by the maximal response obtained in the presence of PMA plus ionomycin The results shown are means ± SE from three independent assays performed in two separate experiments cally inactive mutant SHP-1 ⁄ C455S, and mutant SHP1 ⁄ Y566F, which is not capable of associating with 3BP2, into human Jurkat T cells, respectively The tyrosine-phosphorylation status of 3BP2 was examined following anti-CD3 IgG stimulation in the transfected cells As shown in Fig 6, the tyrosine-phosphorylation level of 3BP2 was dramatically reduced when cotransfected with wild-type SHP-1 In contrast, neither the catalytically inactive SHP-1 (SHP-1 ⁄ C455S) nor mutant SHP-1 ⁄ Y566F exerted any detectable effect on the tyrosine phosphorylation of the cotransfected 3BP2 in Jurkat cells Because Tyr448 is the major phosphorylated site of 3BP2, SHP-1-mediated dephosphorylation of 3BP2 is expected to take place mainly on this tyrosine residue These results suggest that SHP-1 via Tyr566 recruits 3BP2 as its potential substrate for dephosphorylation during TCR signaling To further determine if the SHP-1-mediated dephosphorylation of 3BP2 affects its function in TCR, we cotransfected Jurkat cells with NFAT-luciferase reporter, 3BP2 and SHP-1 or SHP-1 mutants As shown in Fig 7, expression of 3BP2 resulted in both constitutive and anti-CD3 IgG-induced NFAT activation Expression of SHP-1, however, inhibited antiCD3-induced NFAT activation SHP-1 also nearly completely inhibited 3BP2-mediated NFAT activation in response to anti-CD3 stimulation in 3BP2-transfected cells Furthermore, SHP-1 also inhibited the constitutive NFAT activation in 3BP2-transfected cells but not the basal NFAT activity in the cells without 3BP2 3BP2-myc WB: anti-myc 62 Fig 3BP2 is dephosphorylated by SHP-1 in activated Jurkat T cells Jurkat T cells were transfected with 3BP2 and SHP-1 or its mutants as indicated Forty-eight hours after transfection, the cells (5 · 107 cell equivalents) were stimulated with anti-CD3 antibody (OKT3) at 37 °C for as described in Experimental procedures Whole-cell lysates were immunoprecipitated with anti-myc IgG and the precipitated proteins were subjected to western blot analysis with anti-phosphotyrosine (pTyr) and anti-myc IgG transfection In contrast, the catalytically inactive mutant SHP-1 ⁄ C455S or mutant SHP-1 ⁄ Y566F, which abolished its interaction with 3BP2, was incapable of suppressing the 3BP2-mediated NFAT activation in 3BP2 transfected cells Taken together, these results suggest that SHP-1 negatively regulates the function of 3BP2 in TCR signaling through dephosphorylation of 3BP2 on its Tyr448 residue Discussion It has been reported that SHP-1 plays a negative role in TCR signaling However, the precise mechanism by which SHP-1 regulates TCR signaling is largely unknown In this study, we reported the identification of a novel SHP-1-interacting adapter protein 3BP2 3BP2 is composed of an N-terminal PH domain, an SH3-binding proline-rich region, and a C-terminal SH2 domain In addition to SHP-1 reported here, the SH2 domain of 3BP2 has been shown to bind to several phosphorylated proteins including ZAP70, PLCc, LAT, Grb2 and Cbl26 3BP2 was initially identified as an Abl SH3 domain-binding protein of unknown function [25] Recently, 3BP2 has been shown to interact with the Syk and ZAP70 proteins of the Syk family of tyrosine kinases In addition, 3BP2 plays a positive adapter function on basal and TCR-mediated NFAT and AP-1 transcriptional activation in human Jurkat FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2201 Z Yu et al SH 3BP P- + 1Y 56 6F /S SH BP2 P- + 1C /S SH P1Y 56 6F 1C PSH 3B P SH + P1 SH P1 3B P2 ve cto r SHP-1 and 3BP2 in T cell receptor signaling Fig SHP-1 negatively regulates 3BP2-induced NFAT activation Jurkat T cells were cotransfected with the NFAT-luciferase reporter gene and empty vector, 3BP2–myc and SHP-1 or its mutants as indicated Twenty hours after transfection, the cells were incubated with either no addition, anti-CD3 IgG (OKT3) or PMA plus ionomycin at 37 °C for h as described in Experimental procedures Luciferase activity in cell extracts was assayed and the data were normalized by the maximal response obtained in the presence of PMA plus ionomycin The results shown are means ± SE from three independent assays performed in two separate experiments T cells [26] However, the molecular mechanism by which 3BP2 regulates TCR signal transduction remains unclear We found that 3BP2 is a potential substrate of SHP-1 and SHP-1 is likely to negatively regulate 3BP2-mediated NFAT activation in TCR signaling In addition, we identified the major tyrosine phosphorylation site of 3BP2, Tyr448 Mutation of this tyrosine residue reduced 3BP2-mediated NFAT activation Thus, tyrosine phosphorylation is crucial for 3BP2 function in TCR signaling and dephosphorylation of the phosphorylated 3BP2 by SHP-1 negatively regulates 3BP2 activity Tyrosine phosphorylation of 3BP2 has been also demonstrated in mast cells in response to aggregation of high affinity IgE receptor [29,34], in NK cells upon stimulation with anti-FcR IgG [28] and recently in T cells upon TCR activation [35] In RBL2H3 mast cells, phosphorylation of Tyr448 of 3BP2 creates a binding site for the SH2 domain of Lyn, a Src family protein tyrosine kinase, and interaction of Lyn with 3BP2 positively regulates the kinase activity of Lyn [34] In NK cells, Tyr183 of 3BP2 is phosphorylated and binds Vav and PLCc during activation of NK cells through natural cytotoxicity receptors and this phosphorylation is necessary for the enhancement of natural cytotoxicity by 3BP2 [28] Qu et al [35] recently found that both Tyr183 and Tyr448 could be phosphorylated in response to TCR activation by anti2202 CD3 IgG together with PMA However, in our study, mutation of Tyr183 to phenylalanine did not have obvious effect on 3BP2 phosphorylation in response to anti-CD3 IgG-induced TCR activation in the absence of PMA This suggests that both cross-linking of TCR and direct activation of protein kinase C are required for the phosphorylation of Tyr183 of 3BP2 Thus, it is likely that 3BP2 is selectively activated in response to various upstream signalings Usually, the SH2 domain of SHP-1 associates with tyrosine-phosphorylated proteins during its interactions with other signal molecules Interestingly, the association of 3BP2 with SHP-1 is through the SH2 domain of 3BP2 and the tyrosine-phosphorylated phosphatase SHP-1 contains three tyrosine residues (Tyr538, Tyr543 and Tyr566) in its C-terminal tail It has been reported that at least two of these tyrosine residues could be phosphorylated in response to the stimulation of T cell-receptor [19], CSF receptor and c-Kit [36] However, the biochemical consequence and physiological significance of tyrosine-phosphorylation on SHP-1 remain elusive It has been suspected that tyrosine phosphorylation of SHP-1 may regulate its phosphatase activity as observed in other phosphatases [33] In this study, however, we found that phosphorylation of SHP-1 at tyrosine residues on its C-terminal tail confers to the phosphatase an ability to recruit adapter protein 3BP2 and thereby affects signaling Site-directed mutation experiments further revealed that 3BP2 interacts with SHP-1 through its phosphorylated Tyr566 residue The sequence surrounding Tyr566 (Tyr566 ⁄ Glu567 ⁄ Asn568) is strikingly similar to the optimal 3BP2 SH2 domain-binding motif (Tyr ⁄ Glu ⁄ Asn) [37] In this study, we demonstrated that SHP-1 interacts with 3BP2 through the tyrosine-phosphorylated C-terminal segment of the former and the SH2 domain of the latter This interaction allows 3BP2 to be dephosphorylated more efficiently by the catalytic domain of SHP-1 We thus defined a novel function for the C-terminal segment of SHP-1 It has been known that tyrosine phosphorylation of SHP-1 at its C-terminal segment also initiates interaction with adapter protein Grb2 and mSOS [23] However, this does not seem involve the catalytic activity of the enzyme and thus the physiological meaning remains unclear Furthermore, like SHP-1, SHP-2 is also known to be phosphorylated at its C-terminal segment However, because these two enzymes share minimum sequence identity at their C-termini, in contrast to high sequence homologies in their SH2 and catalytic domains, we believe this may allow the enzymes interact with distinct proteins This may explain the often-opposite functions of the two enzymes FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) Z Yu et al Experimental procedures Reagents and antibodies Rabbit anti-SHP-1 polyclonal IgG was generated as described previously [38] Mouse anti-SHP-1 and anti-SHP-2 monoclonal IgG were obtained from Transduction Laboratories (Lexington, KY) An anti-(human CD3-a) (OKT3) monoclonal IgG was purified from the culture medium of OKT3 hybridomas by protein A–Sepharose affinity chromatography Rabbit anti-(mouse IgG) was obtained from BD Biosciences Pharmingen (San Diego, CA) Antiphosphotyrosine (4G10) and anti-myc (9E10) monoclonal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) Anti-hemagglutinin (anti-HA) monoclonal IgG (clone 12CA5) was prepared from the culture medium of hybridomas (ATCC, Manassas, VA) Anti-(mouse IgGhorseradish peroxidase) and anti-(rabbit IgG-horseradish peroxidase) were from Bio-Rad Laboratories (Hercules, CA) Nitrocellulose membrane Hybond-ECL was from Amersham Pharmacia Biotech (Little Chalfont, UK) Western Lightning Chemiluminescence Reagent kit was purchased from Perkin–Elmer Life Sciences Inc (Boston, MA) Protease inhibitor cocktail tablets were from Roche Diagnostics (Mannheim, Germany) Plasmids Plasmids expressing SHP-1 and its mutants were constructed as described previously [31,38] Myc-tagged 3BP2 plasmid and HA-tagged 3BP2 plasmid were constructed by amplifying the full-length 3BP2 encoding region using total RNA from Jurkat cells and inserting the amplified PCR product into the HindIII site of pcDNA3.1 ⁄ myc-His (–) C vector (Invitrogen, Carlsbad, CA) and pACTAG-2 vector (kindly provided by M Tremblay, McGill University) 3BP2 mutants were generated by PCR-based mutagenesis Fyn kinase in pRK5 vector was a kind gift from S Stamm (Max Planck Institute of Biochemistry, Germany) Lck and its activated mutant (Lck ⁄ Y505F) constructs were kindly provided by B Sefton and G Chiang (The Salk Institute for Biological Studies) NFAT-luciferase reporter was kindly provided by G Crabtree (Stanford University School of Medicine) Cell culture and transfection 293T cells and Jurkat T cells were maintained as described previously [31,39] 293T cells were transfected with different sets of plasmid DNAs using standard calcium phosphate precipitation methods In some experiments, the transfected cells were treated with 0.5 mm sodium pervanadate in regular medium for 30 Sodium pervanadate was prepared by mixing 100 mm sodium orthovanadate (Sigma, St Louis, MO) and 50 mm H2O2 (Sigma) and incubating the mixture SHP-1 and 3BP2 in T cell receptor signaling at room temperature for at least 30 Jurkat T cells (107 in 400 lL of medium) were transfected with 20–25 lg DNA by electroporation using a gene pulser (BTX Corp., San Diego, CA) at 260 V for 50 ms Empty vector was added to some samples to make an equal amount of DNA in each transfection Forty-eight hours after transfection, Jurkat T cells were washed and suspended in NaCl ⁄ Pi For stimulation, cells were incubated with lgỈmL)1 of OKT3 on ice for and then with 10 lgỈmL)1 of rabbit anti-(mouse IgG) for an additional The samples were then incubated at 37 °C for the indicated times Yeast two-hybrid screen The cDNA encoding the full-length of SHP-1 with Cys455 to Ser mutation (SHP-1-C455S) was PCR-amplified from the corresponding plasmid [40] and cloned in-frame downstream of the DNA binding domain of Lex A in pBTM-116-src vector [30] to form the bait construct (Lex A–SHP-1–C455S) [31] The human Jurkat cDNA library expressed as fusion proteins with the activation domain of GAL4 in the pACT2 vector was obtained from Clontech Laboratories (Palo Alto, CA) The bait DNA and library DNA were sequentially transformed into yeast strain L40a and 1.1 · 107 primary transformants were screened for growth on medium lacking leucine, tryptophan and histidine The positive colonies were further screened for the expression of b-galactosidase The plasmid DNA was recovered from His+ ⁄ LacZ+ colonies and identified by DNA sequencing Immunoprecipitation and immunoblot analysis Immunoprecipitation and western blot experiments were carried out as described previously [31] Briefly, cells were washed with cold NaCl ⁄ Pi once and lysed in a lysis buffer containing 50 mm Hepes (pH 7.4), 150 mm NaCl, 1% Triton X-100, mm b-mercaptoethanol, 0.5 mm vanadate and an EDTA-free mixture of protease inhibitors The samples were centrifuged at 20 000 g for 10 at °C An aliquot of this whole-cell lysate was removed and the remaining lysate was subjected to immunoprecipitation For immunoprecipitations, cell lysates were incubated with optimal concentrations of antibodies for h at °C, followed by incubation with 50 lL of 50% suspension of Protein A– Sepharose CL-4B beads for h The Sepharose CL-4B beads were washed at °C with lysis buffer four times The proteins were resolved on a SDS ⁄ PAGE gel and transferred to nitrocellulose membranes (Hybond-ECL) The membranes were blocked with 5% milk in Tris-buffered saline (TBS) (pH 7.6) overnight and then incubated with the first antibodies for h After washing four times with TBS containing 0.05% Tween-20 (TBS-T), the membranes were incubated with the second antibody conjugated to horseradish peroxidase for h and then washed four times with TBS-T The blots were developed using the western FEBS Journal 273 (2006) 2195–2205 ª 2006 National Research Council Canada (CNRC) 2203 SHP-1 and 3BP2 in T cell receptor signaling Z Yu et al Lightning Chemiluminescence Reagent kit (Roche) according to the manufacturer’s instruction Expression, purification of GST fusion proteins, and GST pull-down For the construction of a plasmid expressing GST)3BP2– SH2 domain fusion protein, the cDNA fragment encoding amino acid residues 452–561 of 3BP2 was amplified by PCR and inserted into pGEX-5X1 vector (Amersham Pharmacia Biotech) Construction of GST–SHP-1–2SH2 has been described previously [41] Fusion proteins were expressed in Escherichia coli strain DH5a by induction with 25 lm isopropyl-d-thiogalactopyranoside at 25 °C for 16 h and purified as described previously [42] For binding assays, glutathione–Sepharose beads with  lg of bound GST or GST fusion protein were incubated at °C for h with mL of cell lysates The beads were washed four times with the lysis buffer and the bound proteins were analyzed by SDS ⁄ PAGE and western blot NFAT reporter assay Jurkat T cells (2 · 107) were transiently transfected with lg of pNFAT-luciferase and 20 lg of indicated plasmids by electroporation Twenty hours after transfection, cells were aliquoted into a 12-well plate in mL of culture medium and triplicate samples were either left unstimulated, stimulated with OKT3 (2 lgỈmL)1) or with PMA (50 ngỈmL)1) plus lm ionomycin for h Cells were then harvested and washed 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expected that the tyrosine-phosphorylated 3BP2 is a potential substrate for activated SHP-1 during their interaction The... this study, we demonstrated that SHP-1 interacts with 3BP2 through the tyrosine-phosphorylated C-terminal segment of the former and the SH2 domain of the latter This interaction allows 3BP2 to