The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cγ1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation. Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cγ1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation. Stimulation of the T cell antigen receptor (TCR) 2The abbreviations used are: TCR, T cell antigen receptor; SLP-76, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa; Zap-70, ζ-associated protein of 70 kDa; IFN-γ, interferon-γ; PLC, phospholipase-C; PV, pervanadate; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; co-IP, co-immunoprecipitation; GEF, guanine nucleotide exchange factor; CH, calponin homology; HIV, human immunodeficiency virus; FBS, fetal bovine serum; HBSS, Hanks' buffered saline solution; SH, Src homology; PH, pleckstrin homology; TG, thapsigargin; CRAC, Ca2+ release-activated Ca2+ channel; NFAT, nuclear factor of activated T cells; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; Ly-GDI, lymphoid-specific guanine dissociation inhibitor. initiates a cascade of signaling events that lead to T cell activation. Calcium plays a central role in this process and has been studied intensively (1Lewis R.S. 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IP3 binds to and activates the inositol 1,4,5-trisphosphate receptor (IP3R), which results in Ca2+ release from the endoplasmic reticulum (ER) and the subsequent calcium influx from Ca2+ release-activated Ca2+ channel (CRAC) (1Lewis R.S. Annu. Rev. Immunol. 2001; 19: 497-521Crossref PubMed Scopus (707) Google Scholar, 7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (697) Google Scholar). The elevated cytoplasmic [Ca2+]i evokes a multitude of cellular responses, such as the NFAT-mediated gene expressions and the cell proliferation (8Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 9Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1572) Google Scholar). Vav1 is expressed specifically in hematopoietic cells as a 95-kDa protein, which plays pivotal roles as a guanine exchange factor (GEF) for small GTPases as well as a scaffold protein in the activation of hematopoietic cells (10Turner M. Billadeau D.D. Nat. Rev. Immunol. 2002; 2: 476-486Crossref PubMed Scopus (264) Google Scholar, 11Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar, 12Zugaza J.L. Lopez-Lago M.A. Caloca M.J. Dosil M. Movilla N. Bustelo X.R. J. Biol. Chem. 2002; 277: 45377-45392Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The importance of Vav1 is because of its multiple structural elements, including a calponin homology (CH) domain, an acidic motif, a Dbl homology domain, a pleckstrin homology (PH) domain, a cysteine-rich motif, and one single SH2 domain flanked by two SH3 domains responsible for signaling protein assembly (12Zugaza J.L. Lopez-Lago M.A. Caloca M.J. Dosil M. Movilla N. Bustelo X.R. J. Biol. Chem. 2002; 277: 45377-45392Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Katzav S. Blood. 2004; 103: 2443-2451Crossref PubMed Scopus (39) Google Scholar). Upon TCR engagement, Vav1 is phosphorylated on the key tyrosine residues in the acidic motif, leading to the exposure of active Dbl homology domain for GDP/GTP exchange activity (14Aghazadeh B. Lowry W.E. Huang X.Y. Rosen M.K. Cell. 2000; 102: 625-633Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Studies on vav1-/- T cells isolated from knock-out mice demonstrated that Vav1 is essential for normal T cell activation and proliferation (15Holsinger L.J. Graef I.A. Swat W. Chi T. Bautista D.M. Davidson L. Lewis R.S. Alt F.W. Crabtree G.R. Curr. Biol. 1998; 8: 563-572Abstract Full Text Full Text PDF PubMed Google Scholar, 16Costello P.S. Walters A.E. Mee P.J. Turner M. Reynolds L.F. Prisco A. Sarner N. Zamoyska R. Tybulewicz V.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3035-3040Crossref PubMed Scopus (217) Google Scholar, 17Tarakhovsky A. Turner M. Schaal S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz V.L. Nature. 1995; 374: 467-470Crossref PubMed Scopus (390) Google Scholar). In addition, the vav1-null cell line, J.Vav1, derived from Jurkat cells by somatic gene targeting approach, also exhibits pleiotropic defects in TCR-mediated signaling pathways (18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). T cell stimulation evokes a biphasic calcium flux as follows: calcium release from intracellular stores followed by calcium influx across the plasma membrane (7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (697) Google Scholar, 19Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1294) Google Scholar). IP3Rs dominantly control the initiation of IP3-induced calcium release, demonstrated by using antisense knockdown of IP3R to block calcium release from the ER (20Jayaraman T. Ondriasova E. Ondrias K. Harnick D.J. Marks A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6007-6011Crossref PubMed Scopus (132) Google Scholar). Jurkat T cells express three IP3R isoforms, IP3R-1, IP3R-2, and IP3R-3 (21Harnick D.J. Jayaraman T. Ma Y. Mulieri P. Go L.O. Marks A.R. J. Biol. Chem. 1995; 270: 2833-2840Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), which differ significantly in their sensitivity to IP3 (22Ramos-Franco J. Fill M. Mignery G.A. Biophys. J. 1998; 75: 834-839Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 23Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (342) Google Scholar). A tyrosine kinase, Fyn, was suggested to modulate IP3R channel activities (24Jayaraman T. Ondrias K. Ondriasova E. Marks A.R. Science. 1996; 272: 1492-1494Crossref PubMed Scopus (203) Google Scholar, 25Cui J. Matkovich S.J. deSouza N. Li S. Rosemblit N. Marks A.R. J. Biol. Chem. 2004; 279: 16311-16316Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Interactions between IP3R and other proteins, such as calmodulin (CaM), were reported to control the channel opening. Although some observations viewed CaM as an inhibitory protein of IP3R (26Patel S. Morris S.A. Adkins C.E. O'Beirne G. Taylor C.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11627-11632Crossref PubMed Scopus (100) Google Scholar, 27Cardy T.J. Taylor C.W. Biochem. J. 1998; 334: 447-455Crossref PubMed Scopus (66) Google Scholar, 28Kasri N.N. Parys J.B. Callewaert G. Missiaen L. De S.H. Biol. Res. 2004; 37: 577-582Crossref PubMed Scopus (8) Google Scholar), more recent study illustrated that the Ca2+-dependent association of CaM to IP3R is necessary for normal calcium release (29Kasri N.N. Torok K. Galione A. Garnham C. Callewaert G. Missiaen L. Parys J.B. De S.H. J. Biol. Chem. 2006; 281: 8332-8338Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Analysis of Vav1-modulated calcium mobilization has been emphasized on its recruitment and activation of PLC-γ1 (10Turner M. Billadeau D.D. Nat. Rev. Immunol. 2002; 2: 476-486Crossref PubMed Scopus (264) Google Scholar, 13Katzav S. Blood. 2004; 103: 2443-2451Crossref PubMed Scopus (39) Google Scholar, 30Braiman A. Barda-Saad M. Sommers C.L. Samelson L.E. EMBO J. 2006; 25: 774-784Crossref PubMed Scopus (96) Google Scholar, 31Reynolds L.F. Smyth L.A. Norton T. Freshney N. Downward J. Kioussis D. Tybulewicz V.L. J. Exp. Med. 2002; 195: 1103-1114Crossref PubMed Scopus (179) Google Scholar). Vav1, together with SLP-76 and other adapter proteins, stabilizes PLC-γ1-linker for activation of the T cell complex necessary for tyrosine phosphorylation and activation of PLC-γ1 (30Braiman A. Barda-Saad M. Sommers C.L. Samelson L.E. EMBO J. 2006; 25: 774-784Crossref PubMed Scopus (96) Google Scholar). On the other hand, Vav1 was reported to facilitate PLC-γ1 tyrosine phosphorylation via GEF-mediated phosphoinositide 3-kinase-dependent pathways (31Reynolds L.F. Smyth L.A. Norton T. Freshney N. Downward J. Kioussis D. Tybulewicz V.L. J. Exp. Med. 2002; 195: 1103-1114Crossref PubMed Scopus (179) Google Scholar). However, these models were challenged by the fact that oncVav1 (lacking 66 amino acids at the N terminus) or Vav1 bearing mutations in the CH domain failed to provoke calcium flux and NFAT(IL2) activity in T cells, although the domains necessary for GEF activity or complex formation remained intact (13Katzav S. Blood. 2004; 103: 2443-2451Crossref PubMed Scopus (39) Google Scholar, 18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar, 32Billadeau D.D. Mackie S.M. Schoon R.A. Leibson P.J. J. Immunol. 2000; 164: 3971-3981Crossref PubMed Scopus (46) Google Scholar). Therefore, the CH domain of Vav1 must play a GEF-independent role in TCR-mediated calcium response and NFAT activity. Sequence analysis predicted the CH domain to be involved in F-actin binding (33Castresana J. Saraste M. FEBS Lett. 1995; 374: 149-151Crossref PubMed Scopus (124) Google Scholar). Up to now, few proteins were reported to interact with the N terminus of Vav1, such as lymphoid-specific guanine dissociation inhibitor (Ly-GDI) (34Groysman M. Russek C.S. Katzav S. FEBS Lett. 2000; 467: 75-80Crossref PubMed Scopus (32) Google Scholar) and a polycomb family protein ENX-1 (35Hobert O. Jallal B. Ullrich A. Mol. Cell. Biol. 1996; 16: 3066-3073Crossref PubMed Scopus (127) Google Scholar). However, the function of the N-terminal domain of Vav1 (CH) and the mechanism by which Vav1 participates in calcium flux are poorly understood. In this study, we investigated the mechanism of Vav1 in regulating calcium signaling in T cells. We found that Vav1 associated with CaM. The binding region was mapped to the CH domain of Vav1, and the association was dependent on calcium. Tyrosine phosphorylation of Vav1 had no impact on the interaction. Importantly, we showed that Vav1, via its CH domain, predetermined calcium release from the intracellular store in cooperation with CaM. This study revealed a new binding partner of Vav1 CH domain and may help to understand the mechanisms of Vav1 in T cell calcium signaling. Reagents—The anti-IP3R-1 antibody was purchased from Calbiochem. The anti-CaM antibody was purchased from Upstate. The anti-PLC-γ1 Tyr(P)-783 antibody and anti-α-tubulin antibody were purchased from Sigma. The anti-CD3 mAb OKT3, anti-CD28, anti-Vav1, anti-Zap70, and goat anti-mouse IgG were described previously (18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). The Indo-1-AM, CaM-agarose beads, protein A-Sepharose 4 Fast Flow beads, Indo-1-AM, thapsigargin, ionomycin, and W-7 were purchased from Sigma. The 20 mm pervanadate solution was prepared by adding 2.3 μl of 30% H2O2 to 1 ml of 20 m m Na3VO4 and allowing the mixture to react for 5 min at room temperature. Plasmids—The pcDNA3.FLAG.Vav1, pcDNA3.FLAG.ΔCH. Vav1, and pEFΔPH.Vav1 were described elsewhere (32Billadeau D.D. Mackie S.M. Schoon R.A. Leibson P.J. J. Immunol. 2000; 164: 3971-3981Crossref PubMed Scopus (46) Google Scholar, 36Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar). The C-terminal SH3 domain (position 786-845) truncated Vav1 was obtained by PCR and cloned into pcDNA4/HisMax C (Invitrogen) by BamHI and XhoI. To generate the C-terminal SH3 plus IQ domain (position 705-845) truncated Vav1, pcDNA4/HisMax.C.Vav1 was digested with ApoI and filled into blunt end with Klenow, then digested with BamHI, and subsequently cloned into the pcDNA4/HisMax.C vector digested with BamHI and EcoRV. Vav1 or Vav1ΔCH fragment was cloned into HIV retrovirus expression vector by replacing the EGFP fragment using BamHI and XhoI. Nucleotide sequences of new constructs were confirmed by DNA sequencing. Cell Culture, Transfection, and Stimulation—Jurkat T leukemia cells and J.Vav1 cells were obtained as described previously (18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). Primary lymphocytes were kindly provided by the Institute of Blood Diseases (Tianjin, China). Jurkat T leukemia cells were grown in RPMI 1640 medium at 37 °C containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. For transient transfections, 2 × 107 Jurkat cells were electroporated with a BTX Electro-square Porator model ECM830 (BTX Inc., San Diego) at 310 mV, 10 ms with 30 μg of total DNA. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2 mm l-glutamine, and 1% (v/v) penicillin/streptomycin at 37 °C. 3 × 106 293T cells were transfected with a total of 25 μg of plasmid by the calcium phosphate precipitation method. The concentrations of the stimuli used in both luciferase reporter assays and biochemical assays were 1 μg/ml OKT3 (cross-linked with 1 μg/ml goat anti-mouse IgG), 5 μg/ml CD28, and 1 μm ionomycin. Luciferase Reporter Assay—The reporter constructs were described previously (18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). The dual-luciferase reporter assay system (Promega, Madison, WI) was used to determine the activity of firefly luciferase and Renilla luciferase according to the manufacturer's instructions. Luciferase activity in cell extracts was measured in a TD20/20 luminometer (Turner Designs Inc, Sunnyvale, CA) by injecting 100 μl of assay buffer and measuring light emission for 10 s after injection. Normalized luciferase activity was obtained by dividing the firefly luciferase activity by the Renilla luciferase activity. Retroviral Transduction—Recombinant retroviruses were generated as described previously (37Naldini L. Blomer U. Gage F.H. Trono D. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11382-11388Crossref PubMed Scopus (1278) Google Scholar). 293T cells were transfected with 40 μg of plasmids consisting of HIV trans, vesicular stomatitis virus G, and the HIV-Vav1/HIV-Vav1ΔCH using the calcium phosphate precipitation method. 48 h after the transfection, supernatants were collected and centrifuged to remove cell debris. The processed supernatants were then mixed with J.Vav1 cells and centrifuged at the speed of 2000 × g for 2 h at 25 °C. The mixtures were incubated at 37 °C for another 2 h. Supernatants were removed by centrifuge, and transduced cells were cultured in 37 °C incubator with RPMI 1640 supplemented with 10% FBS. Calcium Measurement—1 × 107 cells were resuspended in 1 ml of HBSS supplemented with 5 mm dextrose and buffered to pH 7.0 with HEPES. Indo-1-AM was added to the cell suspension to a final concentration of 5 mm, and the mixture was incubated for 30 min at 37 °C. An equal volume of HBSS (pH 7.4) containing 5 mm dextrose was then added, and the mixture was incubated for another 30 min at 37 °C. The Indo-1-loaded cells were washed with HBSS (pH 7.5) supplemented with 5 mm dextrose and 0.05% bovine serum albumin. Samples were prewarmed for 5 min at 25 °C. To determine changes in [Ca2+]i, a cuvette filled with 1 ml of Indo-1 loaded cells was mounted to a Cary Eclipse fluorescence spectrophotometer (Varian, Inc., Palo Alto, CA) and were excited at 352 nm (5 nm slit) while being imaged simultaneously at emission wavelengths of 398 and 490 nm (10 nm slit). Cells were stimulated with the indicated agents, and the concentrations of the stimuli were 1 μg/ml OKT3 (cross-linked with 1 μg/ml goat anti-mouse IgG), 100 μm pervanadate, 1 μm thapsigargin, or 1 μm ionomycin, respectively. Protein-Protein Interaction Assay—For pulldown assay, the whole-cell lysates were prepared in ice-cold RIPA buffer (25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaF, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin). After centrifuging at 10,000 rpm for 10 min, the cell lysates were incubated with CaM-agarose or control agarose for 2 h at 4 °C as described in the figure legends. After washing four times, the bound proteins were identified by SDS-PAGE followed by Western blotting. For co-immunoprecipitation assay, the cells were lysed and incubated with protein A-Sepharose beads plus antibodies against Vav1 or CaM, respectively, for 4 h at 4 °C. After washing three times, the bound proteins were analyzed by SDS-PAGE and Western blot. Vav1 Is Prerequisite to TCR-induced Intracellular Calcium Release—T cell stimulation evokes a biphasic calcium flux, the calcium release followed by calcium depletion-activated calcium entry (7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (697) Google Scholar, 19Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1294) Google Scholar). We firstly recorded cellular [Ca2+]i profiles of Jurkat cells, vav1-null cells (J.Vav1), and wild type Vav1 reconstituted J.Vav1 cells (J.WT), respectively. Upon TCR activation by OKT3, J.Vav1 cells exhibited a slightly attenuated early Ca2+ spike and failed to sustain the later Ca2+ plateau (Fig. 1A). The abnormal calcium flux of J. Vav1 cells may result from the combination of the following two processes: 1) defective Ca2+ release from intracellular store; and 2) impaired Ca2+ influx through CRAC. To dissect the two scenarios, we performed experiments in a Ca2+-free solution prior to supplement of 2 mm Ca2+ (Fig. 1B, top bar). Therefore, the initial Ca2+ spike represents the Ca2+ release from the intracellular store, and the subsequent [Ca2+]i elevation after the supplementation of 2 mm Ca2+ reflects the Ca2+ influx via CRAC. As seen in Fig. 1B, OKT3-stimulated J.Vav1 cells presented a delayed and attenuated spike of [Ca2+]i in comparison with that of Jurkat and J.WT cells. Upon addition of extracellular Ca2+ (at 400 s post-stimulation), all three kinds of cells displayed a rapid increase in intracellular calcium concentration, indicating comparable Ca2+ influx capabilities in cells with or without Vav1. Thus, the abnormal [Ca2+]i profile of J.Vav1 cells is largely because of the defect of initial calcium release, suggesting that Vav1 is essential for the TCR-mediated Ca2+ release from intracellular store. The deficiency in TCR-stimulated Ca2+ release in J.Vav1 cells is thought to be the consequence of the impaired activation of PLC-γ1, represented by tyrosine phosphorylation on both Tyr-783 (38Irvin 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) and Tyr-775 residues (39Serrano C.J. Graham L. DeBell K. Rawat R. Veri M.C. Bonvini E. Rellahan B.L. Reischl I.G. J. Immunol. 2005; 174: 6233-6237Crossref PubMed Scopus (37) Google Scholar). Indeed, the defective tyrosine phosphorylation of PLC-γ1 Tyr-783 was observed in J.Vavl cells (30Braiman A. Barda-Saad M. Sommers C.L. Samelson L.E. EMBO J. 2006; 25: 774-784Crossref PubMed Scopus (96) Google Scholar). However, when we treated J.Vav1 cells with pervanadate (PV), a potent protein-tyrosine phosphatase inhibitor, to induce PLC-γ1 phosphorylation (40Secrist J.P. Burns L.A. Karnitz L. Koretzky G.A. Abraham R.T. J. Biol. Chem. 1993; 268: 5886-5893Abstract Full Text PDF PubMed Google Scholar), the calcium defects still remained in J.Vav1 cells (Fig. 2A). Meanwhile, Jurkat and J.WT presented similar intracellular calcium release in response to PV, demonstrating the indispensable role that Vav1 plays in calcium release. The activation of PLC-γ1 was monitored by the phosphorylation of Tyr-783 (Fig. 2B, upper panel), and J.Vav1 displayed equal trends to that of wild type cells at 2, 5, and 10 min post-induction by PV (Fig. 2B, lower panel). Considering that both N- and C-SH2 domains of PLC-γ1 were shown to be dispensable for PV-induced phosphorylation (41Stoica B. DeBell K.E. Graham L. Rellahan B.L. Alava M.A. Laborda J. Bonvini E. J. Immunol. 1998; 160: 1059-1066PubMed Google Scholar), the remarkable defect of calcium release seen in J.Vav1 cells was independent of PLC-γ1 activation. The possible explanation is that Vav1 may bear other machineries to regulate Ca2+ mobilization in addition and in parallel to facilitating PLC-γ1 phosphorylation. This result was supported by our previous observation in which defect calcium profiles were seen in J.Vav1 despite normal IP3 production comparable with that of Jurkat (18Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D.D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). Therefore, Vav1 is prerequisite to ensure a normal calcium release upon TCR-induced PLC-γ1 activation and IP3 production. vav1-null Cells Possess Intact Intracellular Calcium Store and IP3R-1 Expression—Because J.Vav1 cells are derived from the Jurkat E6 cell line by the gene targeting approach, the possibility should be noted that J.Vav1 cells have disrupted calcium store or gene expression, which can lead to the abnormal calcium signals observed above. Thus, we inspected the intracellular calcium stores and calcium channel expression of ER. ER calcium pools were measured indirectly by treating cells with a known inhibitor of sarcoplasmic reticulum Ca2+-ATPase pumps, thapsigargin (TG), which causes passive calcium depletion from the ER lumen into the cytoplasm. As presented in Fig. 3A, the magnitude of TG-releasable calcium in J.Vav1 cells resembled that of Jurkat and J.WT cells. Moreover, the Ca2+ ionophore, ionomycin, evoked the same calcium release patterns in cells with or without Vav1 (data not shown), indicating the equal calcium stores in J.Vav1, Jurkat, and J.WT cells. Considering IP3R-1 as a predominant player in IP3-induced calcium release in T lymphocytes (20Jayaraman T. Ondriasova E. Ondrias K. Harnick D.J. Marks A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6007-6011Crossref PubMed Scopus (132) Google Scholar), we also confirmed its expression level in J.Vav1 cells. As shown in the upper panel of Fig. 3B, the same IP3R-1 expression levels were observed in all the three cell lines, with equal protein loading verified by Zap-70 (lower panel) and α-tubulin (not shown). Moreover, proteomics analysis showed no detectable differences in proteins that related to calcium signaling between J.Vav1 cells and Jurkat cells. 3J. Yin and Y. Cao, unpublished data. Hence, the loss of Vav1 had no effect on ER calcium content nor on IP3R-1 expression, pinpointing the function of Vav1 in the course of TCR-induced calcium release rather than interfering with the intracellular Ca2+ store or protein expression. Vav1 Interacts with Calmodulin via CH Domain in a Ca2+-dependent and Activation-independent Manner—To explore the possible mechanisms that Vav1 modulates calcium release, we looked for Vav1 binding partners. CaM came to the scene as it was demonstrated to ubiquitously bind and regulate IP3R (29Kasri N.N. Torok K. Galione A. Garnham C. Callewaert G. Missiaen L. Parys J.B. De S.H. J. Biol. Chem. 2006; 281: 8332-8338Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 42Hill T.D. Campos-Gonzalez R. Kindmark H. Boynton A.L. J. Biol. Chem. 1988; 263: 16479-16484Abstract Full Text PDF PubMed Google Scholar, 43Missiaen L. Parys J.B. Weidema A.F. Sipma H. Vanlingen S. De S.P. Callewaert G. De S.H. J. Biol. Chem. 1999; 274: 13748-13751Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 44Yamada M. Miyawaki A. Saito K. Nakajima T. Yamamoto-Hino M. Ryo Y. Furuichi T. Mikoshiba K. Biochem. J. 1995; 308: 83-88Crossref PubMed Scopus (124) Google Scholar). CaM pulldown assay was performed by incubating CaM-conjugated agarose with cell lysates of Jurkat or J.Vav1, respectively, or using plain agarose beads as a negative control. The pulldown contents were resolved by immunoblot with anti-Vav1 antibody. As shown in Fig. 4A, Vav1 can be precipitated by CaM-conjugated agarose but not control agarose from Jurkat lysates. The binding was selective for Vav1, as incubation with J.Vav1 lysates was negative (Fig. 4A, 2nd from left). Total protein input was presented in the rightmost lane of Fig. 4A. The binding specificity was confirmed by competition assays in which increasing amounts of purified CaM were added to the pulldown mixture containing eukaryotically overexpressed Vav1 (Fig. 4B, left). In the competition experiment, decreased amounts of Vav1 were pulled down in the presence of 20 μg of CaM, and the Vav1 band turned undetectable when 50 μg of CaM was added to compete the binding. Meanwhile, Ca2+ dependence of Vav1-CaM association was also characterized. As shown in the right panel of Fig. 4B, Vav1 binds to CaM in the presence of external Ca2+, whereas chelating agent EGTA completely abolished the interaction, indicating that the association between Vav1 and CaM is Ca2+-dependent. To examine if Vav1 endogenously interacts with calmodulin, we performed co-immunoprecipitation (co-IP) experiments. As shown in Fig. 4C, co-IP performed with anti-Vav1 antibody showed the co-precipitation with CaM from lysates of both Jurkat cells and primary lymphocytes (Fig. 4C, lower panel, 1st and 2nd lanes). Likewise, co-IP using anti-CaM antibody displayed Vav1 from Jurkat and primary lymphocytes (Fig. 4C, upper panel, 5th and 6th lanes). J.Vav1 cell lysate was used in both cases as a vav1-null control (Fig. 4C, 3rd and 7th lanes), and preimmune IgG was applied as negative control (lane labeled IgG).The total protein input was shown as indicated (Fig. 4C, lysate). These data show that the endogenous CaM and Vav1 associate in vivo. Tyrosine phosphorylation and activation of Vav1 are early events in TCR engagement; therefore, we asked if Vav1-CaM interaction is modulated by the activation status of T cells. Jurkat cells were treated with solvent only, OKT3, or OKT3 plus anti-CD28 IgM as co-signal ligand, res