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• W2064216086 abstract "TcRζ/CD3 and TcRζ/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3α phosphorylation in the Vav-1–/– Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced GSK-3α phosphorylation was observed in Vav-1,2,3–/– T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells. TcRζ/CD3 and TcRζ/CD3-CD28 signaling requires the guanine nucleotide exchange factor (GEF) Vav-1 as well as the activation of phosphatidylinositol 3-kinase, protein kinase B (PKB/AKT), and its inactivation of glycogen synthase kinase-3 (GSK-3). Whether these two pathways are connected or operate independently of each other in T-cells has been unclear. Here, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3α phosphorylation in the Vav-1–/– Jurkat cell line J. Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced GSK-3α phosphorylation was observed in Vav-1,2,3–/– T-cells together with a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of GEF-inactive Vav-1 that inhibited interleukin-2 gene activation and a form of Src homology 2 domain-containing lymphocytic protein of 76-kDa (SLP-76) that is defective in binding to Vav-1. The pathway also was intact under conditions of c-Jun N-terminal kinase (JNK) inhibition and disruption of the actin cytoskeleton by cytochalasin D. Both events are down-stream targets of Vav-1. Overall, our findings indicate that the TcR and TcR-CD28 driven PKB-GSK-3 pathway can operate independently of Vav-1 in T-cells. T-cell activation is induced by ligation of the antigen-receptor (TcRζ/CD3) as well as co-receptors such as CD28. TcRζ/CD3 and CD4/CD8-lck initiate tyrosine phosphorylation, while TcRζ/CD3 and CD28 induce the production of D-3 lipids (1Rudd C.E. Schneider H. Nat. Rev. Immunol. 2003; 3: 544-556Crossref PubMed Scopus (307) Google Scholar, 2Abraham R.T. Weiss A. Nat. Rev. Immunol. 2004; 4: 301-308Crossref PubMed Scopus (396) Google Scholar). CD28 co-signals are needed for optimal cytokine production, proliferation, and effector function (3Bluestone J.A. Immunity. 1995; 2: 555-559Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 4June C.H. Bluestone J.A. Nadler L.M. Thompson C.B. Immunol. Today. 1994; 15: 321-331Abstract Full Text PDF PubMed Scopus (198) Google Scholar). CD28-deficient mice have reduced responses to antigen, highlighting the capacity of CD28 to lower the threshold of signaling (5Shahinian A. Pfeffer K. Lee K.P. Kundig T.M. Kishihara K. Wakeham A. Kawai K. Ohashi P.S. Thompson C.B. Mak T.W. Science. 1993; 261: 609-612Crossref PubMed Scopus (1171) Google Scholar). Primary responses exhibit more of a dependence on CD28 than do secondary responses, and the co-receptor can influence the differentiation of T helper 2 (Th2) versus T helper 1 (Th1) cell, increase cell survival, and prevent the induction of T-cell anergy (3Bluestone J.A. Immunity. 1995; 2: 555-559Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 6Dahl A.M. Klein C. Andres P.G. London C.A. Lodge M.P. Mulligan R.C. Abbas A.K. J. Exp. Med. 2000; 191: 2031-2038Crossref PubMed Scopus (52) Google Scholar, 7Schwartz R.H. Curr. Opin. Immunol. 1997; 9: 351-357Crossref PubMed Scopus (229) Google Scholar). The molecular basis of TcRζ/CD3 and CD28 signaling in T-cells has been the subject of much investigation. TcRζ/CD3 engagement with CD4/CD8-p56lck leads to the recruitment of ZAP-70 and the phosphorylation of multiple adaptors (8Rudd C.E. Cell. 1999; 96: 5-8Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 9Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar). TcR and CD28 ligation can increase the expression of lipid rafts or microdomains (10Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (842) Google Scholar, 11Martin M. Schneider H. Azouz A. Rudd C.E. J. Exp. Med. 2001; 194: 1675-1681Crossref PubMed Scopus (118) Google Scholar). Furthermore, CD28 can be distinguished from the TcR by virtue of the fact that it directly interacts with phosphatidylinositol 3-kinase (PI3K) 2The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; SH, Src homology; IL, interleukin; PKB, protein kinase B; GSK, glycogen synthase kinase-3; NFAT, nuclear factor of activated T-cell; DH, Dbl homology; JNK, c-Jun N-terminal kinase; WT, wild-type; FACS, fluorescence-activated cell sorter; RαM, rabbit anti-mouse; RαH, rabbit anti-hamster; GEF, guanine nucleotide exchange factor; FITC, fluorescein isothiocyanate. by means of classic p85 Src homology 2 (SH2) domain binding to a cytoplasmic YMNM motif (12Prasad K.V. Cai Y.C. Raab M. Duckworth B. Cantley L. Shoelson S.E. Rudd C.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2834-2838Crossref PubMed Scopus (269) Google Scholar, 13Pages F. Ragueneau M. Rottapel R. Truneh A. Nunes J. Imbert J. Olive D. Nature. 1994; 369: 327-329Crossref PubMed Scopus (347) Google Scholar, 14Hehner S.P. Hofmann T.G. Dienz O. Droge W. Schmitz M.L. J. Biol. Chem. 2000; 275: 18160-18171Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Additional proline residues of CD28 mediate supplemental binding to the Src homology 3 (SH3) domains of Grb-2 and p56lck (15Kim H.H. Tharayil M. Rudd C.E. J. Biol. Chem. 1998; 273: 296-301Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Holdorf A.D. Green J.M. Levin S.D. Denny M.F. Straus D.B. Link V. Changelian P.S. Allen P.M. Shaw A.S. J. Exp. Med. 1999; 190: 375-384Crossref PubMed Scopus (147) Google Scholar). Mutations in both sets of residues attenuate CD28-mediated signaling (16Holdorf A.D. Green J.M. Levin S.D. Denny M.F. Straus D.B. Link V. Changelian P.S. Allen P.M. Shaw A.S. J. Exp. Med. 1999; 190: 375-384Crossref PubMed Scopus (147) Google Scholar, 17Burr J.S. Savage N.D. Messah G.E. Kimzey S.L. Shaw A.S. Arch R.H. Green J.M. J. Immunol. 2001; 166: 5331-5335Crossref PubMed Scopus (130) Google Scholar, 18Okkenhaug K. Wu L. Garza K.M. La Rose J. Khoo W. Odermatt B. Mak T.W. Ohashi P.S. Rottapel R. Nat. Immunol. 2001; 2: 325-332Crossref PubMed Scopus (172) Google Scholar, 19Cai Y.C. Cefai D. Schneider H. Raab M. Nabavi N. Rudd C.E. Immunity. 1995; 3: 417-426Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 20Cefai D. Cai Y.C. Hu H. Rudd C. Int. Immunol. 1996; 8: 1609-1616Crossref PubMed Scopus (26) Google Scholar). In vivo re-constitution studies have provided mixed results in the role of the YMNM motif in interleukin-2 (IL-2) production and graft versus host responses (17Burr J.S. Savage N.D. Messah G.E. Kimzey S.L. Shaw A.S. Arch R.H. Green J.M. J. Immunol. 2001; 166: 5331-5335Crossref PubMed Scopus (130) Google Scholar, 18Okkenhaug K. Wu L. Garza K.M. La Rose J. Khoo W. Odermatt B. Mak T.W. Ohashi P.S. Rottapel R. Nat. Immunol. 2001; 2: 325-332Crossref PubMed Scopus (172) Google Scholar, 21Harada Y. Tokushima M. Matsumoto Y. Ogawa S. Otsuka M. Hayashi K. Weiss B.D. June C.H. Abe R. J. Immunol. 2001; 166: 3797-3803Crossref PubMed Scopus (78) Google Scholar). The production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate lipids by PI3K leads to the membrane recruitment and activation of protein kinase B (PKB/AKT) that in turn can phosphorylate and inactivate glycogen synthase kinase-3 (GSK-3) (22Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4681) Google Scholar, 23Lawlor M.A. Alessi D.R. J. Cell Sci. 2001; 114: 2903-2910Crossref PubMed Google Scholar). In resting T-cells, GSK-3 negatively regulates IL-2 transcription by phosphorylating nuclear factor of activated T-cells (NFATs), leading to its translocation from the nucleus (24Woodgett J.R. Curr. Opin Cell Biol. 2005; 17: 150-157Crossref PubMed Scopus (311) Google Scholar, 25Liang J. Slingerland J.M. Cell Cycle. 2003; 2: 339-345Crossref PubMed Scopus (698) Google Scholar). CD28 ligation can potentiate PKB and/or GSK-3 phosphorylation (26Appleman L.J. van Puijenbroek A.A. Shu K.M. Nadler L.M. Boussiotis V.A. J. Immunol. 2002; 168: 2729-2736Crossref PubMed Scopus (168) Google Scholar, 27Frauwirth K.A. Riley J.L. Harris M.H. Parry R.V. Rathwell J.C. Plas D.R. Elstrom R.L. June C.H. Thompson C.B. Immunity. 2002; 16: 679-777Abstract Full Text Full Text PDF Scopus (762) Google Scholar). In this context, PKB has been implicated in the regulation of IL-2 production but not Th2 cytokines (28Kane L.P. Andres P.G. Howland K.C. Abbas A.K. Weiss A. Nat. Immunol. 2001; 2: 37-44Crossref PubMed Scopus (270) Google Scholar). PI3K-PKB also regulate Fas-mediated apoptosis (29Ohteki T. Parsons M. Zakarian A. Jones R.G. Nguyen L.T. Woodgett J.R. Ohashi P.S. J. Exp. Med. 2000; 192: 99-104Crossref PubMed Scopus (127) Google Scholar), glucose uptake, and glycolysis in T-cells (30Frauwirth K.A. Riley J.L. Harris M.H. Parry R.V. Rathmell J.C. Plas D.R. Elstrom R.L. June C.H. Thompson C.B. Immunity. 2002; 16: 769-777Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar). In addition to activating the PI3K pathway, TcRζ/CD3 and CD28 signaling is dependent on the guanine nucleotide exchange factor, Vav-1. It has a calponin homology domain, an acidic motif, a zinc finger-like region, two SH3 domains, and a SH2 domain (31Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (449) Google Scholar, 32Turner M. Billadeau D.D. Nat. Rev. Immunol. 2002; 2: 476-486Crossref PubMed Scopus (264) Google Scholar, 33Tybulewicz V.L. Curr. Opin. Immunol. 2005; 17: 267-274Crossref PubMed Scopus (279) Google Scholar, 34Collins T.L. Deckert M. Altman A. Immunol. Today. 1997; 18: 221-225Abstract Full Text PDF PubMed Scopus (96) Google Scholar). The SH2 domain of Vav-1 binds tyrosine residues within the adaptor SH2 domain-containing lymphocytic protein of 76 kDa (SLP-76) (35Musci M.A. Motto D.G. Ross S.E. Fang N. Koretzky G.A. J. Immunol. 1997; 159: 1639-1647PubMed Google Scholar, 36Raab M. Pfister S. Rudd C.E. Immunity. 2001; 15: 921-933Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). These residues each reside within a YESP motif, both of which are phosphorylated upon receptor ligation. The Dbl homology (DH) domain has GEF activity for the activation of the small GTPases Rac1 and Cdc42 (37Crespo P. Bustelo X.R. Aaronson D.S. Coso O.A. Lopez-Barahona M. Barbacid M. Gutkind J.S. Oncogene. 1996; 13: 455-460PubMed Google Scholar). Vav-1-deficient T-cells show defects in TcR capping and the induction of cytokine production (38Tarakhovsky A. Turner M. Shall S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz V.L. Nature. 1995; 374: 467-470Crossref PubMed Scopus (390) Google Scholar, 39Fischer K.D. Kong Y.Y. Nishina H. Tedford K. Marengere L.E. Kozieradzki I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. Biol. 1998; 8: 554-562Abstract Full Text Full Text PDF PubMed Google Scholar). Phosphorylation of Vav-1 by CD28 depends on residues 173–181 of the receptor (15Kim H.H. Tharayil M. Rudd C.E. J. Biol. Chem. 1998; 273: 296-301Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 40Klasen S. Pages F. Peyron J.F. Cantrell D.A. Olive D. Intl Immunol. 1998; 10: 481-489Crossref PubMed Scopus (49) Google Scholar). CD28 has been reported to boost TcR signaling via Vav-1-SLP-76 cooperation (41Michel F. Mangino G. Attal-Bonnefoy G. Tuosto L. Alcover A. Roumier A. Olive D. Acuto O. J. Immunol. 2000; 165: 3820-3829Crossref PubMed Scopus (79) Google Scholar). Furthermore, co-expression can drive NFAT translocation into the nucleus of COS cells in response to CD28 ligation (36Raab M. Pfister S. Rudd C.E. Immunity. 2001; 15: 921-933Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition, CD28 engagement can activate IκB kinase (IKK) complex and NF-κB activation in a Vav-1 dependent fashion (42Piccolella E. Spadaro F. Ramoni C. Marinari B. Costanzo A. Levrero M. Thomson L. Abraham R.T. Tuosto L. J. Immunol. 2003; 170: 2895-2903Crossref PubMed Scopus (45) Google Scholar). In keeping this, confocal microscopy has shown that endogenous VAV-1 and IKKα co-localize in response to CD28 stimulation (42Piccolella E. Spadaro F. Ramoni C. Marinari B. Costanzo A. Levrero M. Thomson L. Abraham R.T. Tuosto L. J. Immunol. 2003; 170: 2895-2903Crossref PubMed Scopus (45) Google Scholar). Vav-1 has been reported to cooperate with protein kinase C theta to activate c-Jun kinase (JNK) (43Moller A. Dienz O. Hefner S.P. Groge W. Schmitz M.L. J. Biol. Chem. 2001; 276: 20022-20028Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), while Vav-1-deficient Jurkat cells (termed J.Vav1) show defects in Ca2+ mobilization as well as in the activation of JNK and transcription factors needed for interleukin-2 transcription (44Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). Given that TcR and TcR-CD28 generate signals that depend on Vav-1 and PI3K, an important question is whether these pathways are interconnected or operate independently of each other. Observations have been mixed in other systems. Vav-1 GEF activity has been reported to be dependent on inositol lipids due to pleckstrin homology domain binding and localization (45Han J. Luby-Phillips K. Das B. Shu X. Xia Y. Mosteller R.D. Murali-Krisha=na U. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar). By contrast, inhibition of PI3K does not inhibit Vav-3 phosphorylation in Vav-3-deficient cells (46Inabe K. Ishiai M. Scherenburg A.M. Freshney N. Downward J. Kurosaki T. J. Exp. Med. 2002; 195: 189-200Crossref PubMed Scopus (123) Google Scholar). Other studies have reported a partial or full role for Vav-1 in activating PI3K (47Manetz T.S. Gonzalez-Espinosa C. Arudchandran R. Xirasagar S. Tybulewicz V.Z.L. Rivera J. Mol. Cell. Biol. 2002; 21: 3763-3774Crossref Scopus (135) Google Scholar, 48Reynolds 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, 49Vigorito E. Clayton E. Turner M. Biochem. Soc. Trans. 2004; 32: 781-784Crossref PubMed Scopus (2) Google Scholar). BcR activation of protein kinase B phosphorylation occurs normally in Vav-1-deficient B-cells (49Vigorito E. Clayton E. Turner M. Biochem. Soc. Trans. 2004; 32: 781-784Crossref PubMed Scopus (2) Google Scholar). In this study, we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3 phosphorylation in the Vav-1–/– Jurkat cell line J.Vav.1 and in primary CD4-positive Vav-1–/– T-cells. Reduced but significant GSK-3 phosphorylation was also observed in Vav-1,2,3–/– T-cells, despite a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of a GEF-inactive form of Vav-1, a mutant that markedly inhibits IL-2 gene activation. Wild-type levels of phosphorylation were also observed in the presence of a form of SLP-76 that is defective in Vav-1 binding and in cells treated with cytochalasin D to disrupt the cytoskeleton. These findings indicate that TcR and TcR-CD28 can induce PKB/GSK-3 signaling independently of Vav-1 in T-cells. Cells and Reagents—The Jurkat T-cell line was maintained using standard cell culture techniques. The J.Vav1 Jurkat cell line (clone 15–11 (Vav-1+/+) and clone 14-9 (Vav-1–/–)) were a kind gift from Dr. Robert Abraham, Burnham Institute, La Jolla, CA. WT and Vav-1–/– BALB/c mice were a kind gift from Dr. Victor Tybulewicz, National Institute for Medical Research, London, UK. Vav-1,2,3–/– B10.BR mice were kindly provided by Dr. Martin Turner, Babraham Institute, Cambridge, UK. CD4+ T-cells were purified from splenocytes freshly isolated from WT and Vav-1–/– BALB/c mice using Mouse CD4 (L3T4) Dynabeads (Dynal Biotech, UK). This technique resulted in the purification of a population of T-cells that were greater than 95% positive for CD4 as determined by FACS staining. Splenocytes were used for the analysis of the Vav-1,2,3–/– mice. Cytochalasin D was purchased from Calbiochem (UK); SP600125 (JNK inhibitor) was purchased from Tocris (UK). Antibodies anti-human CD3 (OKT3) (American Type Culture Collection hybridoma) (2 μg/ml) and anti-human CD28 (9.3) (Bristol Meyers Squibb) (8 μg/ml) were cross-linked with rabbit anti-mouse (RαM) antibody (Southern Biotechnology), at concentrations that were half the sum of total primary stimulatory antibodies. For control RαM-only stimulations, 5 μg/ml was used. Anti-mouse CD3 (2C11) (2 μg/ml) and anti-mouse CD28 (PV-1) (8 μg/ml) from BioExpress were cross-linked with rabbit anti-hamster (RαH) antibody (Sigma, UK), at concentrations that were half the sum of total primary stimulatory antibodies or 5 μg/ml for the RαH-only control stimulations. Plasmids—pCMV3-myc-hVav (WT) and pCMV3-hVav-L213Q (GEF-inactive) (Martin Turner, Babraham Institute), 3×NFAT-AP1-Luc reporter construct (Steve Burakoff, Dana-Faber Institute of Cancer, Boston, MA), wild-type SLP-76 and Y113F (Paul Findell, Roche Biosciences, Palo Alto, CA) were cloned into SRα. Transfection—For transfections, 50 × 106 Jurkat cells were co-transfected with 50 μg of the indicated constructs and 25 μg of 3xNFAT-AP1-Luc reporter, using a BTX PrecisionPulse electroporator (250 V, 800 μF, and 200 Ω). Cells were incubated for 18 h in RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum at 37 °C, prior to stimulation. Luciferase Assay—For the luciferase assays, 0.5 × 106 Jurkat cells were incubated in 100 μl of RPMI 1640 medium containing 5% fetal calf serum plus the appropriate antibodies for 6 h at 37 °C in a 96-well plate. Cells were lysed, and luciferase activity was determined using a Luminat LB9507 luminometer (EG&G Berthold) and the luciferase assay system protocol from Promega. Analysis of GSK-3 and PKB Phosphorylation—1 × 106 Jurkat cells (standard or the J.Vav1 Jurkat cell line) or 3 × 106 CD4+ T-cells purified from spleens of WT or Vav-1–/– BALB/c mice were suspended in RPMI 1640 medium containing 2% fetal calf serum. Cells were incubated on a rotor with the appropriate primary stimulatory antibodies at 4 °C for 30 min and then incubated with cross-linking antibody (RαMorRαH) at 37 °C for the indicated times. Stimulations were terminated by addition of cold RPMI 1640 medium. Whole cells were pelleted, boiled in 3× SDS sample buffer, and proteins were separated by SDS-PAGE. The following antibodies were used in immunoblotting: anti-phospho-GSK-3α/β (serines 21/9) (Cell Signaling Technology) (1:1000), anti-GSK-3α/β (BIOSOURCE) (1:1000), anti-Vav-1 (Upstate Biotechnologies) (1:1000), anti-SLP-76 (hybridoma from American Type Culture Collection) (1:1000), anti-phospho-PKB (Thr-308) (Cell Signaling Technology) (1:1000), anti-PKB (Cell Signaling Technology) (1:1000), anti-phospho-FOXO1 (Cell Signaling Technology) (1:1000), and anti-mouse/rabbit horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) (1:5000). Proteins were visualized using the enhanced chemiluminescence (ECL) system (Amersham Biosciences). Quantification of GSK-3α- and PKB phosphorylation was done using ImageJ (NIH) densitometry software. Readings were expressed as a percent of the phosphorylated band of maximum intensity. Phosphorylation levels were normalized to total protein expression levels by plotting the ratio (percent phosphorylation)/(% total) × 100. Anti-CD3 Surface Clustering—Jurkat cells were either untreated or preincubated with the indicated concentration of cytochalasin D for 1 h. Cells were then resuspended in 1 ml cold FACS buffer (1% bovine serum albumin and 0.01% sodium azide in phosphate-buffered saline, pH 7.0) and incubated with 2 μg/ml anti-CD3 (OKT3) for 30 min at 4 °C. Cells were then washed twice with cold (0 h samples) or prewarmed (1-h samples) FACS buffer and FITC-conjugated anti-mouse IgG (Sigma) was added, followed by incubation immediately on ice or at 37 °C for 1 h. Cold FACS buffer was added to terminate the stimulations, and cells were fixed with 2% paraformaldehyde and then mounted on coverslips. TcR distribution was visualized by fluorescence microscopy. At least 200 T-cells were counted for TcR cap formation in each experiment. F-actin Content—Jurkat cells were treated as above except RαM was used to cross-link anti-CD3 (OKT3) for 1 h at 37°C. Cold medium was added to terminate the stimulations, and paraformaldehyde-fixed cells were permeabilized with 0.03% saponin/FACS buffer followed by 0.3% saponin/FACS buffer. F-actin content was quantified by staining with FITC-conjugated phalloidin (Sigma) and analyzed using a FACSCalibur (BD Biosciences). To investigate the relationship between the PI3K and Vav-1 pathways in response to TcR and TcR-CD28 ligation, we initially wanted to confirm that anti-CD3 and anti-CD3/CD28 induced PKB and GSK-3 phosphorylation (Fig. 1). Anti-CD3, anti-CD28, and anti-CD3/CD28 antibodies were used for cross-linking of receptors on Jurkat cells for various times at 37 °C. This was followed by immunoblotting of cell lysates with a monoclonal antibody to phosphorylated Thr-308 of PKB (upper panels) or to phosphorylated Ser-21 and Ser-9 sites on GSK-3α and -β, respectively (middle panels). Anti-CD3 increased PKB phosphorylation by 5 min relative to the RαM control (Fig. 1, upper panel, lane 2 versus lane 1). Phosphorylation was enhanced with anti-CD28 co-ligation (lane 4 versus lane 2). Anti-CD28 alone also induced PKB phosphorylation to levels as high or higher than observed with anti-CD3 (lane 3 versus lane 2). Blotting with an antibody to non-phosphorylated PKB confirmed equal levels of protein expression (lower upper panel, lanes 1–16). In terms of the kinetics, phosphorylation was rapid with the majority of phosphorylation having occurred by 5min (lanes 2–4 versus lanes 5–16). The RαM control lane at 5, 15, 30, and 60 min was indicative of phosphorylation levels at time 0. Similar results were obtained with anti-GSK-3α/β blotting (Fig. 1, middle panels). Of the two GSK-3 isoforms, GSK-3α was preferentially phosphorylated relative to the GSK-3β isoform in T-cells. As with PKB phosphorylation, anti-CD3 increased GSK-3α phosphorylation relative to the RαM control (middle panel, lane 2 versus lane 1). This phosphorylation was increased with anti-CD28 co-ligation (lane 4 versus lane 2). Anti-CD28 also induced GSK-3α phosphorylation (lane 3). Blotting using an antibody against non-phosphorylated GSK-3α/β showed the level of protein expression (lanes 1–16). The observations confirm that anti-CD3, anti-CD28, and anti-CD3/CD28 induce rapid phosphorylation of PKB and GSK-3α in T-cells. To assess whether Vav-1 expression was required for PKB and GSK-3α phosphorylation, blotting was conducted in the Vav-1-deficient Jurkat cell line J.Vav.1 (Fig. 1) (44Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). Anti-CD3, -CD28, and -CD3/CD28 induced levels of PKB or GSK-3α phosphorylation in Vav-1–/– cells that were comparable with that observed in WT cells (upper and middle panels, lanes 17–32 versus lanes 1–16). Densitometric readings of the phosphorylated bands documented similar levels of increased phosphorylation (histograms in the lower panels). The absence of Vav-1 was confirmed by anti-Vav-1 blotting (lowest panel, lanes 17–32 versus 1–16). These data show that anti-CD3 and anti-CD3/CD28 induction of PKB and GSK-3α phosphorylation in T-cells can occur independently of Vav-1 expression. The independence of PKB/GSK-3 phosphorylation on Vav-1 expression contrasts with the previously reported defects in Ca2+ and JNK signaling in the same cells (44Cao Y. Janssen E.M. Duncan A.W. Altman A. Billadeau D. Abraham R.T. EMBO J. 2002; 21: 4809-4819Crossref PubMed Scopus (88) Google Scholar). To assess whether this pathway was intact in freshly isolated primary Vav-1–/– T-cells, we also assessed PKB and GSK-3 phosphorylation in CD4+ T-cells purified from spleens of WT or Vav-1–/– BALB/c mice. Cells were stimulated for 30 min with anti-CD3, anti-CD28, or anti-CD3/anti-CD28 followed by measurement of PKB (Fig. 2, upper panels, lanes 1–4 versus lanes 5–8) and GSK-3α phosphorylation (middle panels, lanes 1–4 versus lanes 5–8). The absence of Vav-1 was confirmed by blotting (lowest panel, lanes 5–8 versus lanes 1–4). The experiments showed no difference in the levels of phosphorylation. Occasionally, an experiment showed a slight reduction in PKB and GSK-3 phosphorylation in Vav-1–/– T-cells; however, the reduction was never more than 10% less compared with WT cells. By contrast, Vav-1,2,3-deficient T-cells showed a consistent partial loss of GSK-3 phosphorylation (lower panels). A 40–50% reduction in phosphorylation was generally observed. This indicates that GSK-3 phosphorylation is partially dependent on Vav family members and that there is a second pathway that is Vav independent. Interestingly, this partial effect contrasted with the transcription factor FOXO1 where phosphorylation was completely lost (lower panel). These observations indicate that anti-CD3 and anti-CD3-CD28 induction of PKB-GSK-3 phosphorylation can occur normally in the absence of Vav-1 expression and is partially reduced with the loss of Vav-1,2,3. To examine further the relationship between Vav-1 and the PKB-GSK-3α phosphorylation, we next assessed whether Vav-1 lacking a functional DH domain (L213Q) could interfere with PKB and GSK-3α phosphorylation (Fig. 3). Mutation of lysine 213 inactivates the GEF activity of the DH domain. Jurkat cells were transfected with empty vector, wild-type Vav-1 (WT), or the L213Q mutant. Anti-CD3 induced phosphorylation of PKB and GSK-3α in cells transfected with WT or L213Q (Fig. 3A, upper and middle panels, lanes 3, 8, and 12 versus lanes 1, 2, 6, 7, and 11). Anti-CD3/CD28 also induced similar levels of phosphorylation in vector, WT, and L213Q transfected cells (upper and middle panels, lanes 5, 10, and 14). Vav-1 expression was evident with anti-Vav-1 blotting (lowest panel, lanes 6–14 versus lanes 1–5). The expression level of total PKB and GSK-3α/β was confirmed by blotting with the respective antibodies (upper lower panel and middle lower panels, lanes 1–14). GSK-3α and PKB phosphorylation levels were quantified by densitometry (see lower histograms). Despite the absence of an effect on the PKB-GSK-3 pathway, as described by others (31Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (449) Google Scholar, 38Tarakhovsky A. Turner M. Shall S. Mee P.J. Duddy L.P. Rajewsky K. Tybulewicz V.L. Nature. 1995; 374: 467-470Crossref PubMed Scopus (390) Google Scholar), expression of L213Q completely inhibited anti-CD3, anti-CD28, and anti-CD3/anti-CD28-induced IL-2 transcription (Fig. 3B). This confirmed the inhibitory effect of the mutant on T-cell function. These observations indicate that the TcR and TcR-CD28-PKB-GSK-3 pathway is intact in the presence of a Vav-1 DH domain mutant that can potently inhibit IL-2 transcription in T-cells. This indicates that the inhibitory effect of the DH domain mutant on IL-2 transcription occurs independently of the PKB-GSK-3 pathway. Vav-1 has also been shown to interact with the adaptor protein SLP-76 (50Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 51Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 52Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), an interaction mediated by the Vav-1 SH2 domain binding to two specific YESP sites on SLP-76 (52Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Previous studies have shown that Vav-1 and SLP-76 can cooperate in the induction of IL-2 transcription, an effect that is lost with substitution of the YESP sites (Y113F) (50Wu J. Motto D.G. Koretzky G.A. Weiss A. Immunity. 1996; 4: 593-602Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 51Onodera H. Motto D.G. Koretzky G.A. Rothstein D.M. J. Biol. Chem. 1996; 271: 22225-22230Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 52Raab M. da Silva A.J. Findell P.R. Rudd C.E. Immunity. 1997; 6: 155-164Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). We therefore next assessed whether PKB and GSK-3 phosphorylation could operate independently of the Vav-1 binding to SLP-76. Neither WT SLP-76 nor DN Y113F SLP-76 had an effect on anti-CD3, anti-CD28, or anti-CD3/anti-CD28 induced phosphorylation of PKB and GSKα (Fig. 4A, upper and middle panels, lanes 2–4 versus lanes 6–8 versus lanes 10–12). SLP" @default.
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• W2064216086 date "2006-10-01" @default.
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• W2064216086 title "TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1" @default.
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