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Tec Kinases Mediate Sustained Calcium Influx via Site-specific Tyrosine Phosphorylation of the Phospholipase Cγ Src Homology 2-Src Homology 3 Linker

2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês

10.1074/jbc.m311985200

ISSN

1083-351X

Autores

Lisa A. Humphries, Carol Dangelmaier, Karen Sommer, Kevin R. Kipp, R. Kato, Natasha Griffith, Irene Bakman, Christoph W. Turk, James L. Daniel, David J. Rawlings,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr753 and Tyr759. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγ2 carboxyl-terminal sites, Tyr1197 and Tyr1217, was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker. Tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) is a crucial activation switch that initiates and maintains intracellular calcium mobilization in response to B cell antigen receptor (BCR) engagement. Although members from three distinct families of non-receptor tyrosine kinases can phosphorylate PLCγ in vitro, the specific kinase(s) controlling BCR-dependent PLCγ activation in vivo remains unknown. Bruton's tyrosine kinase (Btk)-deficient human B cells exhibit diminished inositol 1,4,5-trisphosphate production and calcium signaling despite a normal inducible level of total PLCγ2 tyrosine phosphorylation. This suggested that Btk might modify a critical subset of residues essential for PLCγ2 activity. To evaluate this hypothesis, we generated site-specific phosphotyrosine antibodies recognizing four putative regulatory residues within PLCγ2. Whereas all four sites were rapidly modified in response to BCR engagement in normal B cells, Btk-deficient B cells exhibited a marked reduction in phosphorylation of the Src homology 2 (SH2)-SH3 linker region sites, Tyr753 and Tyr759. Phosphorylation of both sites was restored by expression of Tec, but not Syk, family kinases. In contrast, phosphorylation of the PLCγ2 carboxyl-terminal sites, Tyr1197 and Tyr1217, was unaffected by the absence of functional Btk. Together, these data support a model whereby Btk/Tec kinases control sustained calcium signaling via site-specific phosphorylation of key residues within the PLCγ2 SH2-SH3 linker. Signals generated by the pre-B and mature B cell receptors are essential for B cell development, activation, and maintenance of mature B cell populations (1Lam K.P. Kuhn R. Rajewsky K. Cell. 1997; 90: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar). Engagement of the BCR 1The abbreviations used are: BCR, B cell antigen receptor; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; XLA, X-linked agammaglobulinemia; XID, X-linked immunodeficiency; SH2 and SH3, Src homology 2 and 3, respectively; WT, wild type; MOI, multiplicity of infection; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; PV, pervanadate; IP, immunoprecipitation; WCL, whole cell lysate. initiates the formation of a lipid-raft associated signaling complex, or "signalosome," containing tyrosine and serine/threonine kinases, adapter molecules, and lipid hydrolases including phospholipase Cγ isoforms. Together, these events promote a series of downstream signals including a sustained increase in intracellular calcium concentrations. PLCγ is essential for antigen receptor-mediated calcium mobilization (2Coggeshall K.M. McHugh J.C. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5660-5664Crossref PubMed Scopus (114) Google Scholar, 3Hempel W.M. DeFranco A.L. J. Immunol. 1991; 146: 3713-3720PubMed Google Scholar, 4Wilde J.I. Watson S.P. Cell. Signal. 2001; 13: 691-701Crossref PubMed Scopus (125) Google Scholar). Activated PLCγ hydrolyzes its substrate, phosphatidylinositol 4,5-bisphosphate, generating the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (5Hempel W.M. Schatzman R.C. DeFranco A.L. J. Immunol. 1992; 148: 3021-3027PubMed Google Scholar, 6Nishibe S. Wahl M.I. Hernandez-Sotomayor S.M. Tonks N.K. Rhee S.G. Carpenter G. Science. 1990; 250: 1253-1256Crossref PubMed Scopus (503) Google Scholar). IP3 acts to open intracellular calcium stores promoting an initial, transient rise in intracellular calcium. Depletion of intracellular calcium stores triggers the opening of plasma membrane store-operated calcium channels (7Parekh A.B. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), resulting in an influx of extracellular calcium and a secondary, sustained calcium signal. The amplitude and duration of this sustained calcium signal is a key determinant of the specific transcription program initiated in response to antigen receptor engagement (8Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6187) Google Scholar, 9Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1563) Google Scholar, 10Healy J.I. Dolmetsch R.E. Timmerman L.A. Cyster J.G. Thomas M.L. Crabtree G.R. Lewis R.S. Goodnow C.C. Immunity. 1997; 6: 419-428Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). The most abundantly expressed PLCγ isoform in B lineage cells is PLCγ2. Chicken B lymphoma cells lacking PLCγ2 are unable to generate IP3 in response to BCR engagement, resulting in the abrogation of receptor-mediated calcium mobilization (11Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). Similarly, mice deficient in PLCγ2 have a defective response to BCR engagement and exhibit a block in B cell development (12Hashimoto A. Okada H. Jiang A. Kurosaki M. Greenberg S. Clark E.A. Kurosaki T. J. Exp. Med. 1998; 188: 1287-1295Crossref PubMed Scopus (183) Google Scholar, 13Hashimoto A. Takeda K. Inaba M. Sekimata M. Kaisho T. Ikehara S. Homma Y. Akira S. Kurosaki T. J. Immunol. 2000; 165: 1738-1742Crossref PubMed Scopus (139) Google Scholar, 14Wang D. Feng J. Wen R. Marine J.C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Despite its crucial role in BCR-dependent calcium mobilization, the molecular events regulating PLCγ2 activity remain incompletely defined. Activation of PLCγ isoforms correlates with an increase in protein tyrosine phosphorylation (15Wahl M.I. Daniel T.O. Carpenter G. Science. 1988; 241: 968-970Crossref PubMed Scopus (192) Google Scholar, 16Weiss A. Koretzky G. Schatzman J.N. Kadlecek T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5484-5488Crossref PubMed Scopus (279) Google Scholar), and inhibition of tyrosine kinases abolishes BCR-dependent IP3 production and calcium signaling (17Carter R.H. Park D.J. Rhee S.G. Fearon D.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2745-2749Crossref PubMed Scopus (165) Google Scholar). Consistent with this, members of the Src, Syk/Zap70, and Tec/Btk kinase families are each capable of phosphorylating PLCγ isoforms and peptide fragments in vitro (18Law C.L. Chandran K.A. Sidorenko S.P. Clark E.A. Mol. Cell. Biol. 1996; 16: 1305-1315Crossref PubMed Google Scholar, 19Ozdener F. Dangelmaier C. Ashby B. Kunapuli S.P. Daniel J.L. Mol. Pharmacol. 2002; 3: 672-679Crossref Scopus (48) Google Scholar, 20Rodriguez R. Matsuda M. Perisic O. Bravo J. Paul A. Jones N.P. Light Y. Swann K. Williams R.L. Katan M. J. Biol. Chem. 2001; 276: 47982-47992Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). The specific contribution of these kinases to BCR-mediated phosphorylation and activation of PLCγ in vivo, however, remains unknown (4Wilde J.I. Watson S.P. Cell. Signal. 2001; 13: 691-701Crossref PubMed Scopus (125) Google Scholar). Btk/Tec kinases specifically regulate the BCR-dependent sustained phase of calcium signaling (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 22Rawlings D.J. Clin. Immunol. 1999; 91: 243-253Crossref PubMed Scopus (87) Google Scholar, 23Scharenberg A.M. Kinet J.P. Cell. 1998; 94: 5-8Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Deficient function of Btk leads to the related immunodeficiencies, X-linked agammaglobulinemia (XLA) in humans and X-linked immuno-deficiency (XID) in mice (24Rawlings D.J. Witte O.N. Immunol. Rev. 1994; 138: 105-119Crossref PubMed Scopus (102) Google Scholar). Igm++/IgD+ B cells derived from XLA patients exhibit reduced IP3 levels and fail to generate a sustained calcium signal in response to BCR engagement. Reconstitution of XLA B cells with increasing doses of wild type Btk can restore and specifically enhance the sustained calcium signal (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). Despite the marked reduction in peak IP3 levels and calcium influx in XLA B cells, BCR-dependent PLCγ2 tyrosine phosphorylation is indistinguishable from that present in normal B cells (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 25Genevier H.C. Callard R.E. Clin. Exp. Immunol. 1997; 110: 386-391Crossref PubMed Scopus (18) Google Scholar). Similarly, Btk-deficient murine B-lymphocytes, mast cells, and platelets also exhibit diminished calcium mobilization and phosphoinositide hydrolysis despite normal receptor-mediated PLCγ2 tyrosine phosphorylation (26Forssell J. Nilsson A. Sideras P. Scand. J. Immunol. 2000; 52: 30-38Crossref PubMed Scopus (11) Google Scholar, 27Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Crossref PubMed Scopus (75) Google Scholar, 28Setoguchi R. Kinashi T. Sagara H. Hirosawa K. Takatsu K. Immunol. Lett. 1998; 64: 109-118Crossref PubMed Scopus (39) Google Scholar). One potential explanation for these contrasting observations is that Btk may regulate the sustained calcium signal via phosphorylation of a subset of key tyrosine residues essential for PLCγ2 activity. Recently, two groups identified up to four putative regulatory phosphorylation sites within PLCγ2 using in vitro kinase assays and reconstitution studies (20Rodriguez R. Matsuda M. Perisic O. Bravo J. Paul A. Jones N.P. Light Y. Swann K. Williams R.L. Katan M. J. Biol. Chem. 2001; 276: 47982-47992Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Watanabe D. Hashimoto S. Ishiai M. Matsushita M. Baba Y. Kishimoto T. Kurosaki T. Tsukada S. J. Biol. Chem. 2001; 276: 38595-38601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These sites included tyrosine residues within the SH2-SH3 linker region (Tyr753 and Tyr759) (20Rodriguez R. Matsuda M. Perisic O. Bravo J. Paul A. Jones N.P. Light Y. Swann K. Williams R.L. Katan M. J. Biol. Chem. 2001; 276: 47982-47992Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Watanabe D. Hashimoto S. Ishiai M. Matsushita M. Baba Y. Kishimoto T. Kurosaki T. Tsukada S. J. Biol. Chem. 2001; 276: 38595-38601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and at the carboxyl terminus (Tyr1197 and Tyr1217) (29Watanabe D. Hashimoto S. Ishiai M. Matsushita M. Baba Y. Kishimoto T. Kurosaki T. Tsukada S. J. Biol. Chem. 2001; 276: 38595-38601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Notably, the Tyr753 and Tyr759 PLCγ2 sites appeared analogous in location to defined PLCγ1 SH2-SH3 linker regulatory sites (30Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (448) Google Scholar). Reconstitution of PLCγ2-deficient cells with PLCγ2 containing mutations in the SH2-SH3 linker sites alone (20Rodriguez R. Matsuda M. Perisic O. Bravo J. Paul A. Jones N.P. Light Y. Swann K. Williams R.L. Katan M. J. Biol. Chem. 2001; 276: 47982-47992Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) or in all four sites concurrently (29Watanabe D. Hashimoto S. Ishiai M. Matsushita M. Baba Y. Kishimoto T. Kurosaki T. Tsukada S. J. Biol. Chem. 2001; 276: 38595-38601Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) led to the near ablation of BCR-dependent calcium signaling. Whereas these studies demonstrate a key role for these sites in regulating PLCγ2 functional activity, the kinases responsible for mediating site-specific modification in the context of receptor dependent signaling remain undefined. In the current study, receptor-mediated PLCγ2 activation was directly evaluated within the context of an intact cellular system through the use of antibodies specific for distinct PLCγ2 phosphotyrosyl regulatory residues. Our data demonstrate that at least four regulatory sites are phosphorylated in response to BCR engagement and that phosphorylation of the PLCγ2 SH2-SH3 linker region is entirely dependent upon Btk/Tec family kinases. This specific property provides a coherent explanation for the unique role of Btk/Tec kinases in immuno-receptor dependent sustained calcium signaling. Cells and Reagents—The human B cell lines, WT (LDN-1), XLA-1 (LDX-1) (7Parekh A.B. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and Ramos were cultured in RPMI 1640 with l-glutamine (CellGro) plus 5% fetal calf serum and 50 μm 2-mercaptoethanol. Total splenocytes were obtained by harvesting whole spleens from age-matched Balb/c and Balb/Xid mice. Splenocytes were prepared by mechanical disruption followed by depletion of erythrocytes by lysis with ammonium chloride solution and resuspension in serum-free RPMI plus supplement. Antibody reagents included F(ab′)2 goat anti-human IgM Fc5μ-specific and F(ab′)2 goat anti-murine IgM μ chain-specific (Jackson Laboratories); rabbit anti-murine Btk (31Wahl M.I. Fluckiger A.C. Kato R.M. Park H. Witte O.N. Rawlings D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11526-11533Crossref PubMed Scopus (109) Google Scholar); anti-phosphotyrosine monoclonal antibody 4G10, anti-Itk, anti-Tec, and anti-Zap-70 (Upstate Biotechnology, Inc., Lake Placid, NY); anti-PLCγ2 (Q20), anti-PLCγ1 (1249), and anti-Syk (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-phosphotyrosine monoclonal antibody PY20 (BD Biosciences); and anti-P-Y783-PLCγ1 (BIOSOURCE International). For analysis by flow cytometry, primary splenocytes were surface-stained by incubating cells with anti-B220 (CD45R), R-Phycoerythrin (1:200), and anti-IgM fluorescein isothiocyanate (1:100) (Pharmingen) at 4 °C for 20 min. WT and XLA-1 human B cells were surface-stained with human anti-IgM fluorescein isothiocyanate (1:100) (Southern Bio-technology). Data were collected on a FACScalibur flow cytometer (BD Biosciences) and analyzed with CELLQuest software (BD Biosciences). Cell Stimulation, Lysis, Immunoprecipitation, and Immunoblotting—Prior to stimulation, B cells were incubated in serum-free RPMI plus glutamine for 45 min at 37 °C. 1 × 107 cells were then stimulated with either pervanadate (150 μm) or human- or murine-specific IgM F(ab′)2 antibodies (10 μg/ml) for the indicated times. Pervanadate was prepared as previously described (32Wienands J. Larbolette O. Reth M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7865-7870Crossref PubMed Scopus (154) Google Scholar). Cells were lysed for 10 min on ice in a lysis buffer consisting of 200 mm boric acid, pH 8.0, 150 mm NaCl, 0.5% Triton X-100, 5 mm NaF, 5 mm EDTA, and 1 mm sodium orthovanadate plus protease inhibitors leupeptin and aprotinin (10 μg/ml each) and phenylmethylsulfonyl fluoride (1 mm). Cellular debris was spun down at 14,000 rpm at 4 °C for 15 min, and cleared lysates were used as whole cell lysate or for immunoprecipitation. PLCγ2 was immunoprecipitated from cells stimulated with pervanadate due to increased stability and efficiency of detection of specifically phosphorylated PLCγ2. In contrast, site-specific phosphorylation was more reliably detected via analysis of whole cell lysate from IgM-stimulated primary and transformed B cells. PLCγ2 was immunoprecipitated by using 2 μg of anti-PLCγ2 antibody (Q-20; Santa Cruz Biotechnology) followed by incubation with protein A-Sepharose for 2 h at 4 °C. All samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane. Pervanadate studies represent 5 × 105 cell equivalents/lane, whereas IgM studies represent 5 × 106 cell equivalents/lane. Western blot analysis was performed using standard procedures as previously described (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). ECL was used for antibody detection according to the manufacturer's instructions (Amersham Biosciences). Generation of Phosphospecific Antibodies and Enzyme-linked Immunosorbent Assay—PLCγ2 Tyr753, Tyr759, Tyr1197, and Tyr1217 phosphospecific antibodies were generated by inoculating rabbits with 100 μg of phosphopeptide conjugated to keyhole limpet hemocyanin as immunogen with Freund's complete adjuvant. Phosphopeptide sequences used were as follows: PLCγ2 Tyr753 and Tyr759 (see Fig. 2), Tyr1197 (PVLESEEELpYSSCRQLRRRQ, where pY represents phosphotyrosine), and Tyr1217 (CELNNQLFLpYDTHQNLRNAN). Initial injection was followed by four booster injections every 2–3 weeks in Freund's incomplete adjuvant. To obtain affinity-purified antibodies, sera were first absorbed to a nonphosphorylated peptide column. Column flow-through was then run over a phosphopeptide column to isolate phosphospecific antibodies for use in our assays (31Wahl M.I. Fluckiger A.C. Kato R.M. Park H. Witte O.N. Rawlings D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11526-11533Crossref PubMed Scopus (109) Google Scholar). To evaluate antibody reactivity by enzyme-linked immunosorbent assay, microtiter wells were coated with nonphosphorylated or phosphorylated peptide species. Site-directed Mutagenesis and Generation of Recombinant Vaccinia Virus—Mutagenesis of full-length human PLCγ2 cDNA was carried out in the pBlueScript (pBS) vector using the QuikChange™ site-directed mutagenesis kit (Stratagene). Primers were designed in which a single nucleotide change resulted in a single amino acid change (tyrosine to phenylalanine). The Y753F/Y759F double mutant was generated by sequential mutagenesis. Mutagenized constructs were digested and purified for cloning into the pSC66 vaccinia virus vector. Sequence analysis of pBS and pSC66 PLCγ2 WT and mutant constructs was performed to verify each mutation. Recombinant vaccinia virus expressing WT or mutant human PLCγ2 was generated as previously described (33Rawlings D.J. Scharenberg A.M. Park H. Wahl M.I. Lin S. Kato R.M. Fluckiger A.C. Witte O.N. Kinet J.P. Science. 1996; 271: 822-825Crossref PubMed Scopus (381) Google Scholar). Preparation of Recombinant Human PLCγ2—Full-length PLCγ2 was expressed in bacteria using the pCAL expression and purification system. Briefly, Escherichia coli BL21 were transformed with WT or mutant pCAL-PLCγ2 expression constructs. Transformed bacteria were grown at room temperature with constant agitation until reaching an optical density at 550 nm of 0.8–1.0. PLCγ2 expression was induced by the addition of a 0.1 mm concentration of "the inducer" (Molecular Research Laboratories, LLC). Bacteria were harvested, and recombinant PLCγ2 was purified using a calmodulin column as previously described (19Ozdener F. Dangelmaier C. Ashby B. Kunapuli S.P. Daniel J.L. Mol. Pharmacol. 2002; 3: 672-679Crossref Scopus (48) Google Scholar). PLCγ2 in Vitro IP3 Assay for Activity—Recombinant purified WT or mutant PLCγ2 was added to a reaction mixture containing 200 μm3H-labeled phosphatidylinositol 4,5-bisphosphate (25,000 dpm), 35 mm NaH2 PO4 (pH 6.8), 70 mm KCl, 2 mm MgCl2, 1 mm EDTA, 5 μg/ml bovine serum albumin, 5 mm dithiothreitol, and 0.6 mm CaCl2 in the presence of recombinant, purified Lck (purified recombinant human Gst-Lck provided by Alexander Y. Tsygankov, Temple University, Philadelphia, PA). ATP (25 μm final concentration) was added to selected reactions, and mixtures were incubated for 10 min at 25 °C. Reactions were stopped by transferring mixtures to an ice bath and adding 0.5 ml of chloroform/methanol/HCl (100:100:0.6) followed by 150 μl of 1 n HCl with 5 mm EDTA. Aqueous and organic phases were separated by centrifugation, and a portion of the upper aqueous phase (200 μl) was removed for liquid scintillation counting. All experiments were performed in triplicate. PLCγ2 in Vitro Kinase Assay—NIH-3T3 cells (5 × 106) were infected with vaccinia virus expressing recombinant wild type or mutant PLCγ2 at an MOI of 10 for 12 h at 37 °C. Cells were harvested, and whole cell lysates were prepared as previously described under "Experimental Procedures." PLCγ2 was immunoprecipitated by using 2 μg of anti-PLCγ2 antibody (Q-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by incubation with protein A-Sepharose for 2 h. Sepharose beads were then incubated in a reaction mixture containing 200 mm Tris-HCl, pH 7.5, 0.4 mm EDTA, and 0.4 mm Na3VO4 in the presence or absence of 0.25 μg of active Lck kinase (Upstate Biotechnology) for 10 min at 30 °C. All other reagents including enzyme dilution buffer and magnesium/ATP mixture were prepared per the manufacturer's instructions. Reactions were stopped by adding SDS sample buffer and boiling samples for 10 min. Alternatively, recombinant PLCγ2 was incubated with 0.25 μg of active Lck in the presence or absence of 25 μm ATP in a kinase buffer (50 mm MOPS, pH 7.4, 5 mm MnCl2, 5mm MgCl2, 5 mm dithiothreitol) at 24 °C for 5 min. Reactions were stopped by the addition of SDS sample buffer and boiling. Calcium Mobilization Assays—A20 B cells (5 × 106) were loaded with 1 mm indo-1 acetoxymethylester (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C in loading medium (RPMI, 2% fetal calf serum, 10 mm HEPES). Indo-1 acetoxymethylester-loaded cells were washed and resuspended in a buffer of Hanks' balanced salt solution (HBSS (Sigma) with calcium) plus 10 mm HEPES, pH 7.0, prior to analysis. Cells were stimulated with either 10 μg/ml anti-human IgM (Jackson Laboratories), 10 μg/ml anti-murine IgG (Jackson Laboratories), or 150 μm pervanadate and monitored for 3 min followed by the addition ionomycin (1 μm) as a positive control. Intracellular calcium was measured using a bulk spectofluorimeter (Photon Technology International) and calculated as a ratio of 400/488-nm emission following 350-nm excitation. Data were analyzed with FELIX software (Photon Technology International). Total Tyrosine Phosphorylation of PLCγ2 Is Not Indicative of Its Signaling Function in XLA B Cells—To determine the role of Btk in PLCγ2 activation, we compared the calcium response of WT and XLA-1 human B cell lines (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar) stimulated with anti-IgM or the protein-tyrosine phosphatase inhibitor, pervanadate. Consistent with our previously published data, XLA-1 B cells exhibited reduced calcium mobilization in response to IgM engagement (Fig. 1A) Signaling via pervanadate requires the presence of a functional BCR transducer complex and promotes phosphorylation, recruitment, and activation of BCR-associated molecules (32Wienands J. Larbolette O. Reth M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7865-7870Crossref PubMed Scopus (154) Google Scholar, 34Wienands J. Schweikert J. Wollscheid B. Jumaa H. Nielsen P.J. Reth M. J. Exp. Med. 1998; 188: 791-795Crossref PubMed Scopus (235) Google Scholar). Pervanadate, however, represents a more potent mimic of BCR engagement due to irreversible oxidation of key cysteine residues on protein-tyrosine phosphatases, generating a more sustained phosphotyrosine signal (35Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar). Notably, despite the ability of pervanadate to produce a marked enhancement in the phosphotyrosine signal of total cellular proteins compared with anti-IgM (Fig. 1C), these events were not sufficient to rescue the calcium signaling defect in Btk-deficient cells (Fig. 1B). Tyrosine phosphorylation of PLCγ2 is commonly used as a measure of its activation status. We compared the total PLCγ2 phosphotyrosine signal in IgM or pervanadate-stimulated WT and Btk-deficient B cells. Consistent with previous results (21Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar), tyrosine phosphorylation of PLCγ2 in response to BCR engagement (as assessed by the panspecific anti-phosphotyrosine antibody 4G10) was indistinguishable between XLA-1 and WT B cells (Fig. 1D). Similarly, stimulation with pervanadate resulted in equivalent levels of PLCγ2 tyrosine phosphorylation in XLA-1 and WT B cells, and no significant differences were observed over an extended activation time course (Fig. 1E and data not shown). Similar results were obtained using an alternative, panspecific anti-phosphotyrosine antibody (PY20) and in studies using an independently derived XLA B cell line (data not shown). Thus, whereas the reduction in BCR-dependent IP3 production and calcium flux in Btk-deficient B cells clearly suggests a defect in the PLCγ2 pathway, these events do not correlate with the phosphotyrosine content of PLCγ2 as measured by conventional anti-phosphotyrosine antibodies. Generation of Site-specific Antibodies to Candidate Regulatory PLCγ2 Phosphorylation Sites—One explanation for these data is that Btk regulates PLCγ2 activity via phosphorylation of a subset of tyrosine residues and that a loss of these specific phosphorylation events is not readily detectable using antibodies that recognize global tyrosine phosphorylation. Initial attempts to identify PLCγ2 regulatory sites by mass spectrometry were problematic due to low stoichiometric concentrations and the generation of multiple phosphopeptide fragments. Other groups have reported similar difficulties using this technique (20Rodriguez R. Matsuda M. Perisic O. Bravo J. Paul A. Jones N.P. Light Y. Swann K. Williams R.L. Katan M. J. Biol. Chem. 2001; 276: 47982-47992Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We therefore chose to evaluate site-specific phosphorylation using phosphospecific antibodies generated against individual candidate sites. Potential phosphorylation sites were initially identified by comparing PLCγ2 with the better characterized, ubiquitously expressed PLCγ isoform, PLCγ1 (Fig. 2A). PLCγ1 contains three regulatory tyrosines: Tyr771 and Tyr783 within its SH2-SH3 linker region and Tyr1254 at the carboxyl terminus. These sites are inducibly phosphorylated in response to stimulation by growth factors (36Kim J.W. Sim S.S. Kim U.H. Nishibe S. Wahl M.I. Carpenter G. Rhee S.G. J. Biol. Chem. 1990; 265: 3940-3943Abstract Full Text PDF PubMed Google Scholar, 37Wahl M.I. Nishibe S. Kim J.W. Kim H. Rhee S.G. Carpenter G. J. Biol. Chem. 1990; 265: 3944-3948Abstract Full Text PDF PubMed Google Scholar) or CD3 cross-linking in T cells (38Park D.J. Rho H.W. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5453-5456Crossref PubMed Scopus (210) Google Scholar), and mutation of any one of these residues leads to dysregulated phospholipase activity (30Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (448) Google Scholar). Specifically, mutation of Tyr783 (to Phe) results in the complete abrogation of PLCγ1 function in fibroblasts and in reconstituted PLCγ1-deficient Jurkat T cells (30Kim H.K. Kim J.W. Zilberstein A. Margolis B. Kim J.G. Schlessinger J. Rhee S.G. Cell. 1991; 65: 435-441Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 39Irvin B.J. Williams B.L. Nilson A.E. Maynor H.O. Abraham R.T. Mol. Cell. Biol. 2000; 20: 9149-9161Crossref PubMed Scopus (113) Google Scholar). Although the PLCγ isoforms share a similar domain structure, they share only ∼60% overall amino acid sequence homology, including less than 30% homology within the SH2-SH3 linker. Comparison with PLCγ1 revealed three potential, similarly located, residues within the SH2-SH3 linker, including Tyr743, Tyr753, and Tyr759 (Fig. 2B); however, there was no distinct homology at the carboxyl terminus. Among the initia

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