BLNK Binds Active H-Ras to Promote B Cell Receptor-mediated Capping and ERK Activation
2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês
10.1074/jbc.m809051200
ISSN1083-351X
AutoresYasuhiro Imamura, Akihisa Oda, Takashi Katahira, Kenji Bundo, Kelly A. Pike, Michael J. H. Ratcliffe, Daisuke Kitamura,
Tópico(s)CAR-T cell therapy research
ResumoCross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igα, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8α-Igα chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation. Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igα, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8α-Igα chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation. Signals from the B cell antigen receptor (BCR), 5The abbreviations used are: BCR, B cell receptor; ERK, extracellular signal-regulated protein kinase; RBD, Ras-binding domain; ITAM, immunoreceptor tyrosine-based activation motif; PLC, phospholipase C; PKC, protein kinase C; SH2, Src homology 2; GTPase, guanosine triphosphatase; GST, glutathione S-transferase, ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; MCS, multi-cloning site; FITC, fluorescein isothiocyanate; CLSM, Confocal laser scanning microscopy; TRITC, tetramethyl rhodamine isothiocyanate; WT, wild type; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase/ERK kinase; aa, amino acid(s). either bound with antigen or unbound, and the pre-B cell receptor (pre-BCR) play a critical role in B cell development, activation, and immune responses (1Rajewsky K. Nature. 1996; 381: 751-758Crossref PubMed Scopus (1382) Google Scholar). Such signals are primarily transduced from Igα/Igβ subunits, transmembrane proteins each containing the immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic regions (2Reth M. Wienands J. Annu. Rev. Immunol. 1997; 15: 453-479Crossref PubMed Scopus (370) Google Scholar). Tyrosine residues in the ITAM are phosphorylated by Src family kinases, such as Lyn, and serve as a docking site for Syk, a pivotal tyrosine kinase for this signaling. The ITAM-bound Syk becomes activated and phosphorylates and activates key signaling proteins, such as another tyrosine kinase Btk, phospholipase C (PLC) γ2, and the adaptor protein BLNK (3Kurosaki T. Nat. Rev. Immunol. 2002; 2: 354-363Crossref PubMed Scopus (175) Google Scholar). Upon phosphorylation by Btk, PLCγ2 produces inositol 1,4,5-triphosphate and diacylglycerol, which induce Ca2+ mobilization and activation of enzymes such as protein kinase C (PKC), respectively. BLNK (also known as SLP-65 or BASH) is an important adaptor protein selectively expressed in B-lineage cells (4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 5Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (234) Google Scholar, 6Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar, 7Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). BLNK is a cytoplasmic protein, but a part of it is constitutively bound to the plasma membrane through an N-terminal leucine zipper motif (8Kohler F. Storch B. Kulathu Y. Herzog S. Kuppig S. Reth M. Jumaa H. Nat. Immunol. 2005; 6: 204-210Crossref PubMed Scopus (46) Google Scholar) and transiently to a cytoplasmic domain of Igα through its C-terminal SH2 domain upon BCR-stimulation (9Engels N. Wollscheid B. Wienands J. Eur. J. Immunol. 2001; 31: 2126-2134Crossref PubMed Scopus (115) Google Scholar, 10Kabak S. Skaggs B.J. Gold M.R. Affolter M. West K.L. Foster M.S. Siemasko K. Chan A.C. Aebersold R. Clark M.R. Mol. Cell. Biol. 2002; 22: 2524-2535Crossref PubMed Scopus (112) Google Scholar). Previous reports indicated that a non-ITAM phosphotyrosine in Igα is necessary for the binding with the BLNK SH2 domain and/or for normal BLNK function in signaling and B cell activation (9Engels N. Wollscheid B. Wienands J. Eur. J. Immunol. 2001; 31: 2126-2134Crossref PubMed Scopus (115) Google Scholar, 10Kabak S. Skaggs B.J. Gold M.R. Affolter M. West K.L. Foster M.S. Siemasko K. Chan A.C. Aebersold R. Clark M.R. Mol. Cell. Biol. 2002; 22: 2524-2535Crossref PubMed Scopus (112) Google Scholar, 11Pike K.A. Ratcliffe M.J.H. J. Immunol. 2005; 174: 2012-2020Crossref PubMed Scopus (21) Google Scholar, 12Patterson H.C. Kraus M. Kim Y.M. Ploegh H. Rajewsky K. Immunity. 2006; 25: 55-65Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Upon phosphorylation on tyrosines, BLNK binds Btk and PLCγ2 through their SH2 domains and mediates PLCγ2 activation by Btk (13Kurosaki T. Tsukada S. Immunity. 2000; 12: 1-5Abstract Full Text Full Text PDF PubMed Google Scholar). BLNK also binds other signaling molecules such as Vav, Grb2, Syk, and HPK1 (4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 5Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (234) Google Scholar, 6Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar, 14Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar). BLNK has been shown to be necessary for BCR-mediated Ca2+ mobilization, for the activation of mitogen-activated protein kinases such as ERK, JNK, and p38 in a chicken B cell line DT40 (7Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), and for activation of transcription factors such as NF-AT and NF-κB in human or mouse B cells (4Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 15Tan J.E. Wong S.C. Gan S.K. Xu S. Lam K.P. J. Biol. Chem. 2001; 276: 20055-20063Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). BLNK plays a crucial role in pre-BCR-dependent progression of B cell development, BCR-mediated B cell survival, activation, proliferation, and T-independent immune responses (16Jumaa H. Wollscheid B. Mitterer M. Wienands J. Reth M. Nielsen P.J. Immunity. 1999; 11: 547-554Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 17Pappu R. Cheng A.M. Li B. Gong Q. Chiu C. Griffin N. White M. Sleckman B.P. Chan A.C. Science. 1999; 286: 1949-1954Crossref PubMed Scopus (250) Google Scholar, 18Hayashi K. Nittono R. Okamoto N. Tsuji S. Hara Y. Goitsuka R. Kitamura D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2755-2760Crossref PubMed Scopus (124) Google Scholar, 19Xu S. Tan J.E. Wong E.P. Manickam A. Ponniah S. Lam K.P. Int. Immunol. 2000; 12: 397-404Crossref PubMed Scopus (128) Google Scholar, 20Minegishi Y. Rohrer J. Coustan-Smith E. Lederman H.M. Pappu R. Campana D. Chan A.C. Conley M.E. Science. 1999; 286: 1954-1957Crossref PubMed Scopus (286) Google Scholar). A small guanosine triphosphatase (GTPase) Ras is also implicated in BCR signal transduction. The earliest report demonstrated that, after BCR cross-linking, Ras is co-localized with the aggregates of BCR (patches), and then with the cap, an assembly of patches at one pole of the cell (21Graziadei L. Riabowol K. Bar-Sagi D. Nature. 1990; 347: 396-400Crossref PubMed Scopus (72) Google Scholar). Then followed reports indicating rapid activation of Ras upon BCR cross-linking (22Lazarus A.H. Kawauchi K. Rapoport M.J. Delovitch T.L. J. Exp. Med. 1993; 178: 1765-1769Crossref PubMed Scopus (67) Google Scholar, 23Harwood A.E. Cambier J.C. J. Immunol. 1993; 151: 4513-4522PubMed Google Scholar). Ras is constitutively bound to the inner surface of the plasma membrane through post-translational modifications initiated predominantly by farnesyltransferase. Upon receptor stimulation, Ras-bound GDP is rapidly replaced with GTP by Ras guanyl nucleotide exchange factors, and Ras becomes active. The GTP-bound Ras recruits several cytoplasmic enzymes to the plasma membrane, including Raf kinases such as Raf-1 and B-Raf. The membrane-recruited Raf is activated and stimulates downstream MEK-ERK signaling cascade leading to transcriptional activation of downstream genes. The active state of Ras is only transient because its own GTPase activity hydrolyzes GTP into GDP with the aid of GTPase-activating proteins. In BCR signal transduction, Ras is mainly activated by RasGRP3 with a minor contribution of RasGRP1, both being Ras guanyl nucleotide exchange factors activated upon binding to diacylglycerol (24Oh-hora M. Johmura S. Hashimoto A. Hikida M. Kurosaki T. J. Exp. Med. 2003; 198: 1841-1851Crossref PubMed Scopus (98) Google Scholar, 25Coughlin J.J. Stang S.L. Dower N.A. Stone J.C. J. Immunol. 2005; 175: 7179-7184Crossref PubMed Scopus (117) Google Scholar). Diacylglycerol-activated PKC also contributes to the activation of RasGRP3 by a site-specific phosphorylation (26Teixeira C. Stang S.L. Zheng Y. Beswick N.S. Stone J.C. Blood. 2003; 102: 1414-1420Crossref PubMed Scopus (107) Google Scholar, 27Aiba Y. Oh-hora M. Kiyonaka S. Kimura Y. Hijikata A. Mori Y. Kurosaki T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16612-16617Crossref PubMed Scopus (70) Google Scholar, 28Zheng Y. Liu H. Coughlin J. Zheng J. Li L. Stone J.C. Blood. 2005; 105: 3648-3654Crossref PubMed Scopus (94) Google Scholar). In B cells from RasGRP1/3 double null mutant mice, BCR ligation fails to induce activation of Ras and ERK as well as cell proliferation, indicating that BCR signaling through Ras is essential for B cell activation (25Coughlin J.J. Stang S.L. Dower N.A. Stone J.C. J. Immunol. 2005; 175: 7179-7184Crossref PubMed Scopus (117) Google Scholar). Although BLNK has been shown to be necessary for PLCγ2 activation in BCR signal transduction, BLNK-deficient (BLNK–) DT40 cells displayed normal Ras activation upon BCR cross-linking (7Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), 6Y. Imamura, A. Oda, T. Katahira, K. Bundo, and D. Kitamura, unpublished result. suggesting alternative pathways for RasGRP activation. Despite the normal Ras activation, BCR-induced ERK activation was markedly attenuated in the BLNK– DT40 cells (7Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). It was also shown that BLNK is required for efficient ERK activation in mouse splenic B cells when BCR stimulation is modest (29Xu S. Huo J. Chew W.K. Hikida M. Kurosaki T. Lam K.P. J. Immunol. 2006; 176: 4690-4698Crossref PubMed Scopus (10) Google Scholar). These results suggest that BLNK is required for ERK activation in a Ras-independent pathway or, alternatively, that BLNK may link the active Ras to a downstream signaling pathway leading to full ERK activation. Here we demonstrate that BLNK directly binds active Ras and that this binding is critical for BCR-Ras co-capping and prolonged ERK activation leading to Elk1 activation upon BCR cross-linking. Plasmid Constructions-pGST-cBLNK(1–62), pECFP-cBLNK, pECFP-cBLNK(Δ62), and pAT7-mBLNK have been described previously (14Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar, 30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Other vectors were constructed as follows. For pGST-cBLNK(1–158), the EcoRI-XhoI fragment from pHybLex/Zeo-cBLNK(1–158) (31Katahira T. Imamura Y. Kitamura D. Int. Immunol. 2006; 18: 545-553Crossref PubMed Scopus (2) Google Scholar) was inserted into the same sites of pGEX-5X-1 (Amersham Biosciences). For pApuroT7-cBLNK, the SnaBI-SalI fragment from pCAT7 was inserted into the same sites of pApuro2 vector to generate pApuroT7. The EcoRI-SalI cBLNK fragment from pCAT7-cBLNK was ligated into the EcoRI and SmaI sites of pApuroT7. For pApuroT7-cBLNK(Δ62) and pApuroT7-cBLNK(Δ158), cBLNK fragments were prepared by PCR using pCAT7-cBLNK as a template and primers cBLNK63 and cBLNK-A or primers cBLNK159 and cBLNK-A, respectively (Table 1) and cloned into the pApuroT7. For pApuroT7-cBLNK-S and pApuroT7-cBLNK(Δ133), a short form (cBLNK-S) (6Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar) and an incomplete cDNA encoding a chicken BLNK lacking first 133 aa (Δ133) 6Y. Imamura, A. Oda, T. Katahira, K. Bundo, and D. Kitamura, unpublished result. were cloned into pApuroT7. The following internal deletion mutants of cBLNK were created by the two-step PCR. Two neighboring fragments were amplified independently using pCAT7-cBLNK as a template and each pair of primers (Table 1) in the first step, and the two fragments (1 ng each) were annealed through their 3′-end homologies, elongated to the 3′ ends, and amplified by PCR with the primers cBLNK1–1 and cBLNK-A in the second step. The resultant fragments were cloned into pApuroT7. The following primers were used in the first PCR: for pApuroT7-cBLNK(Δ134–158), cBLNK1–1 and cBLNK163–128, cBLNK129–164, and cBLNK-A; for pApuroT7-cBLNK(Δ135–144), cBLNK1–1 and cBLNK150–128, cBLNK129–151, and cBLNK-A; and for pApuroT7-cBLNK(Δ153–157), cBLNK1–1 and cBLNK162–146, cBLNK148–164, and cBLNK-A.TABLE 1Primers used in this studyPrimer nameSequence (5′ to 3′)cBLNK63GGGAATTCACCTCCAAGTCTACCACGAAGGGcBLNK-AAGTAGGGAGGGCTGATTTTGCGGGcBLNK159GGGAATTCACCCAGTTCAGCCTTGCCCAGACcBLNK1-1GGGAATTCTATGGACAAGCTGAACAAACcBLNK163-128CAAGGCTGAACTGGGAATAGGGAATGAGGAGGGcBLNK129-164TCCTCATTCCCTATTCCCAGTTCAGCCTTGCCCcBLNK150-128GGGAGGAAGCTGATGGTGAGAAATAGGGAATGAGGAGGGcBLNK129-151TCCTCATTCCCTATTTCTCACCATCAGCTTCCTCCCATCcBLNK162-146GGCTGAACTGGGTGTGTTGATGGGAGGAAGCTGATGcBLNK148-164CTTCCTCCCATCAACACACCCAGTTCAGCCTTGCCC Open table in a new tab The following vectors were made by general insert vector ligation. pECFP-cBLNK(Δ158) and pECFP-cBLNK(Δ134–158). EcoRI-SalI fragments of pApuroT7-cBLNK(Δ158) or pApuroT7-cBLNK(Δ134–158) were inserted into the same sites of pECFP-C1 (Clontech). For pEYFP-H-Ras and pEYFP-H-Ras(61L), an EcoRI-SalI fragment of pCMV-H-Ras or a KpnI-SmaI fragment of pCMV-H-Ras(61L) were inserted into the same sites of pEYFP-C1 (Clontech), respectively. For pEYFP-H-Ras(17N), an EcoRI-BamHI fragment of pCMV-H-Ras(17N) was first cloned into pBluescript II SK(–), and the KpnI-BamHI fragment thereof was inserted into the same sites of pEYFP-C1. CD8α:IgαF3RBD and CD8α:IgαF3 were constructed as follows: Cla12L-mCD8α:chIgαF3 (11Pike K.A. Ratcliffe M.J.H. J. Immunol. 2005; 174: 2012-2020Crossref PubMed Scopus (21) Google Scholar) was digested with NgoMI and HindIII, and the smaller fragment was replaced with a synthesized fragment (MCS: 5′-GCCGGCCTGGAGAAACCTCGAGCTAGCGGATCCAAGCTT-3′; the NgoMI, XhoI, BamHI, and HindIII sites are underlined). The resultant plasmid, Cla12L-mCD8α:chIgαF3-MCS, was digested with XhoI and BamHI, and the smaller fragment was replaced with a BLNK-RBD fragment made by PCR with primers (5′-CCGCTCGAGCATTCCCTATTTCTAGAGGTG-3′; the XhoI site is underlined; and 5′-CGGGATCCGTCGACTACTGATGGTGACTGGTGCGA-3′; the BamHI and SalI sites are underlined) and chicken BLNK cDNA as a template. By these procedures, a chicken BLNK sequence including the RBD, FPISRGEYADNRTSHHQ(stop), was fused to the C-terminal end of the chicken IgαF3 with an insertion of two linker amino acids (Arg and Ala). A SacI-SalI fragment from the resultant plasmid (mCD8α:chIgαF3-RBD), EcoRI/SalI-digested pApuro2, and annealed oligonucleotides (5′-AATTCAGATCTACTAGTGAGCT-3′ and 5′-CACTAGTAGATCTG-3′; the EcoRI and SacI protruding ends are underlined) were ligated together to generate pApuro2-CD8α:IgαF3RBD. A ClaI-ClaI fragment from Cla12L-mCD8α:chIgαF3 was inserted into an EcoRI site of pApuro2 through end blunting to generate pApuro2-CD8α:IgαF3. An Aor51HI-SalI fragment from the mCD8α:chIgαF3-RBD or an Aor51HI-XhoI fragment from the mCD8α:chIgαF3-MCS was cloned into the pCAT7-neo vector (32Morimura T. Goitsuka R. Zhang Y. Saito I. Reth M. Kitamura D. J. Biol. Chem. 2000; 275: 36523-36531Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) between the Klenow-blunted EcoRI and SalI sites, to generate T7-IgαF3RBD and T7-IgαF3, respectively. Cell Culture and Stimulation-DT40 cells and BLNK– DT40 cells were cultured and stimulated with anti-chicken IgM antibody (M4) as described previously (7Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Cos-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. BLNK– DT40 cells were electroporated with pApuroT7- or pApuro2-based vectors and selected with puromycin as described previously (32Morimura T. Goitsuka R. Zhang Y. Saito I. Reth M. Kitamura D. J. Biol. Chem. 2000; 275: 36523-36531Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). BLNK– DT40 cells expressing mCD8α fusion receptors were stimulated with preincubation for 20 min on ice with rat anti-mouse CD8α monoclonal antibody (53–6.72), followed by incubation at 40 °C with goat anti-rat IgG F(ab′)2 (Jackson ImmunoResearch) for the indicated time periods. Luciferase Assay-Twenty μg of a vector expressing either the wild type or one of the mutant cBLNKs, as well as a mixture of the reporter vectors, 1 μg of pFA2-Elk1, 10 μg of pFR-Luc, and 1 μg of pRSV-β-gal were transfected into 5 × 106 BLNK– DT40 cells by electroporation. DT40 cells, BLNK– DT40 cells, and the latter stably transfected with BLNK-derived constructs or CD8α-IgαF3 constructs were transfected with the reporter vectors similarly. After 24 h, surface BCR or CD8α was stimulated as described above for 6 h, and the luciferase and β-galactosidase activities were measured as described previously (30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 33Katsuta H. Tsuji S. Niho Y. Kurosaki T. Kitamura D. J. Immunol. 1998; 160: 1547-1551PubMed Google Scholar). ProteinAnalysis-Expressionvectors carrying either the wild type or one of the various mutant cBLNKs or one of the Igα constructs, all tagged with the T7 epitope, were mixed with either pCMV-H-Ras(61L) or pCMV-H-Ras(17N), tagged with the FLAG epitope, and with TransIT-LT1 reagents (Mirus). These mixtures were co-transfected into 2 × 106 Cos-7 cells. Two days after transfection, the cells were harvested, and the cell extracts were immunoprecipitated with anti-T7 or anti-FLAG antibodies and analyzed by Western blotting as described previously (14Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar, 30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). GST fusion proteins of cBLNK were produced in Escherichia coli and used in GST pull-down assays and Western blotting as described previously (14Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar, 30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). To examine the association of endogenous BLNK and Ras proteins, the lysates of B cells purified from mouse spleens using MACS B cell isolation kit (Miltenyi Biotec) were subjected to immunoprecipitation using rabbit anti-BLNK antibody (18Hayashi K. Nittono R. Okamoto N. Tsuji S. Hara Y. Goitsuka R. Kitamura D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2755-2760Crossref PubMed Scopus (124) Google Scholar) or control rabbit serum, and the precipitates were analyzed by Western blotting. Antibodies-The following antibodies were purchased: mouse monoclonal anti-T7 tag (Novagen), goat anti-GST (GE Healthcare), mouse monoclonal anti-FLAG (M5; Sigma-Aldrich), anti-pan Ras (Ab-3; Oncogene), anti-phospho-specific ERK (Cell Signaling), mouse monoclonal anti-Bcl-2 (Transduction Laboratories), anti-mouse CD8α (53–6.72), goat anti-rat IgG or anti-mouse IgM F(ab′)2 fragments (Jackson ImmunoResearch). Flow Cytometry-DT40-derived cells were stained with FITC-conjugated goat anti-chicken IgM antibody (Bethyl Laboratories) or FITC anti-mouse CD8α (53–6.72) and analyzed with FACSCalibur™ (Becton Dickinson) as described previously (34Hayashi K. Yamamoto M. Nojima T. Goitsuka R. Kitamura D. Immunity. 2003; 18: 825-836Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For the analysis of BCR-induced apoptosis, DT40 and DT40-derived cells (2 × 105/well) were stimulated in culture medium with or without anti-IgM (M4; 1.25 μg/ml) for 48 h in 12-well plates and then washed with the incubation buffer (10 mm Hepes/NaOH, pH 7.4, 140 mm NaCl, 5 mm CaCl2). Then the cells were stained with annexin V-biotin labeling solution (Roche Applied Science) and propidium iodide (1 μg/ml) for 15 min on ice. Then the cells were washed, stained with FITC-conjugated streptavidin (eBioscience) for 20 min on ice, and analyzed by flow cytometry. Confocal Laser Scanning Microscopy (CLSM)-CLSM was performed for cells transfected with the CFP- and YFP-tagged constructs as described previously (30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). DT40 cells and their transfectants were stimulated as described above, washed in cold phosphate-buffered saline, fixed with paraformaldehyde, and stained with anti-chicken IgM-FITC (Bethyl Laboratories) or with anti-rat IgG-FITC (Southern Biotechnology Associates) for staining of rat anti-mouse CD8-labeled cells. Then the cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline, incubated with the blocking reagent (0.1% goat serum), and stained with anti-Ras (Ab-3), followed by and goat anti-mouse IgG-TRITC (Jackson ImmunoResearch). The stained cells were placed onto glass slides, covered with thin glass, and inspected with CLSM. Excitation wavelengths for FITC and TRITC were 488 and 568 nm, respectively. Emission signals were detected between 495 and 540 nm for FITC and between 585 and 610 nm for TRITC. Each result shown here is a representative of the data from the same experiments repeated at least and mostly more than twice. Identification of H-Ras as a Binding Partner of BLNK-To elucidate the mechanism of BLNK action in the BCR signal transduction, we sought to identify proteins interacting with BLNK by the yeast two-hybrid system. We screened the cDNA library from the chicken B cell line DT40 with a "bait" consisting of a 158-aa region of chicken BLNK (BLNK(1–158) and identified several independent clones as reported previously (31Katahira T. Imamura Y. Kitamura D. Int. Immunol. 2006; 18: 545-553Crossref PubMed Scopus (2) Google Scholar). From these clones, we identified one that interacts with BLNK(1–158), but not with a 62-aa region of BLNK (BLNK(1–62)), in the two-hybrid system, and the insert of this clone turned out to be a cDNA encoding an H-Ras protein with a truncation of the first three amino acids. To verify the binding of BLNK and H-Ras in vitro, we performed a GST pull-down assay. Human H-Ras proteins, either constitutively active (61L) or inactive (17N) mutant forms, were transiently expressed in Cos-7 cells, and the cell lysates were subjected to binding with GST-fused BLNK(1–158) or BLNK(1–62) proteins. As shown in Fig. 1A, only the active form bound to the GST-BLNK(1–158) protein, and neither forms bound to GST-BLNK(1–62) or GST alone. We next expressed the H-Ras proteins and T7-tagged mouse BLNK protein transiently in Cos-7 cells, and the BLNK-bound complex was immunoprecipitated with anti-T7 tag antibody (α-T7). As shown in Fig. 1B, only the active form of H-Ras was co-precipitated with BLNK. With the same system, we showed that the active H-Ras also bound to a full-length chicken BLNK (long form) as well as the shorter form of chicken BLNK that lacks the 40–58 aa region (6Goitsuka R. Fujimura Y. Mamada H. Umeda A. Morimura T. Uetsuka K. Doi K. Tsuji S. Kitamura D. J. Immunol. 1998; 161: 5804-5808PubMed Google Scholar) and the BLNK with an N-terminal 133-aa deletion (Fig. 1C). These results indicate that the 134–158 aa region of chicken BLNK is responsible for the binding with the active H-Ras. We further examined the binding of endogenous Ras and BLNK proteins in mouse splenic B cells. As shown in Fig. 1D, immunoprecipitation with anti-BLNK antibody (α-BLNK) co-precipitated Ras protein from the cells stimulated with anti-IgM antibody but not from unstimulated cells. A control rabbit serum precipitated neither BLNK nor Ras. Thus, endogenous BLNK binds endogenous Ras in normal B cells after BCR stimulation. Co-localization of BLNK and the Constitutively Active H-Ras in Living Cells-To examine the binding of BLNK and Ras in living cells, we transfected expression vectors encoding CFP-tagged BLNK and YFP-tagged H-Ras proteins into Cos-7 cells and examined their intracellular localization with CLSM (Fig. 2). The CFP-tagged full length as well as deletion mutants of chicken BLNK lacking 1–62 (Δ62), 1–158 (Δ158), or 134–158 (Δ134–158) aa regions, respectively, were diffusely distributed throughout the cells with occasional exclusion from nuclei (Fig. 2A and Ref. 30Imamura Y. Katahira T. Kitamura D. J. Biol. Chem. 2004; 279: 26425-26432Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In contrast, YFP-tagged wild type or the constitutively active (61L) or inactive (17N) forms of H-Ras were localized at plasma membrane, with accumulation in ruffles and lamellipodia, as well as at Golgi apparatus, as described previously (35Choy E. Chiu V.K. Silletti J. Feoktistov M. Morimoto T. Michaelson D. Ivanov I.E. Philips M.R. Cell. 1999; 98: 69-80Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 36Jiang X. Sorkin A. Mol. Biol. Cell. 2002; 13: 1522-1535Crossref PubMed Scopus (170) Google Scholar). When BLNK and wild type H-Ras were co-expressed, BLNK was distributed in the cytoplasm but with a noticeable accumulation at lamellipodia and Golgi area where H-Ras was co-localized, suggesting that the active H-Ras at these sites is associated with BLNK (Fig. 2B, top row). When BLNK was co-expressed with H-Ras(61L), H-Ras(61L) was mostly co-localized with BLNK in a diffuse cytoplasmic pattern (Fig. 2B, middle row), suggesting that the active Ras was trapped inside of the cell through binding with BLNK. By contrast, inactive H-Ras was mostly distributed at the periphery of the cells where BLNK was largely excluded (Fig. 2B, bottom row). The cellular localization pattern of co-expressed BLNK(Δ62) and H-Ras proteins was almost the same as that of wild type BLNK and H-Ras; BLNK(Δ62) was co-localized with wild type H-Ras at the peripheral lamellipodia, with the H-Ras(61L) diffusely throughout the cells and not co-localized with H-Ras(17N) (Fig. 2C; data not sh
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