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

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). 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In this we report that anti-CD3 and anti-CD3/CD28 can induce PKB and GSK-3 phosphorylation in the Vav-1–/– Jurkat cell line and in primary CD4-positive Vav-1–/– T-cells. Reduced but GSK-3 phosphorylation was also observed in Vav-1,2,3–/– T-cells, a complete loss of FOXO1 phosphorylation. Furthermore, PKB and GSK-3 phosphorylation was unperturbed in the presence of a GEF-inactive form of a that IL-2 gene of phosphorylation also observed in the presence of a form of that is defective in Vav-1 binding and in with cytochalasin to the findings indicate that TcR and TcR-CD28 can induce signaling independently of Vav-1 in T-cells. and Jurkat T-cell line was cell The Jurkat cell line and a from La and Vav-1–/– mice a from for Vav-1,2,3–/– mice provided by T-cells from from and Vav-1–/– mice in the of a of T-cells that than for as by used for the of the Vav-1,2,3–/– was from was from and CD28 with rabbit that the of primary was and CD28 from with rabbit that the of primary or for the and of and Jurkat with of the and of a and for in to the Jurkat in of the for in a and was a and the from of GSK-3 and PKB Jurkat or the Jurkat cell or 3 T-cells from of or Vav-1–/– mice in on a with the primary for and with for the by of in and by The used in from and secondary the of and PKB phosphorylation was as a of the of to protein expression by the or with the of cytochalasin for 1 in 1 and in and with 2 anti-CD3 for with or and was by on or for 1 was to the and with and on TcR was by T-cells for TcR in each as was used to anti-CD3 for 1 was to the and with by was by with and a the the PI3K and Vav-1 pathways in to TcR and TcR-CD28 we to that anti-CD3 and anti-CD3/CD28 induced PKB and GSK-3 phosphorylation and anti-CD3/CD28 used for of on Jurkat for was by of cell with a to of PKB or to and on GSK-3α and PKB phosphorylation by to the 2 versus was with versus also induced PKB phosphorylation to as or than observed with anti-CD3 3 versus with to PKB of protein expression In of the phosphorylation was with the of phosphorylation by versus The and was of phosphorylation results with the two GSK-3 GSK-3α was to the in T-cells. with PKB phosphorylation, anti-CD3 GSK-3α phosphorylation to the 2 versus phosphorylation was with versus also induced GSK-3α phosphorylation the of protein expression The that and anti-CD3/CD28 induce phosphorylation of PKB and GSK-3α in T-cells. Vav-1 expression was for PKB and GSK-3α phosphorylation, was in the Jurkat cell line Y. A. D. R.T. J. 2002; PubMed Scopus Google Scholar). and induced of PKB or GSK-3α phosphorylation in Vav-1–/– that with that observed in and versus of the of phosphorylation in the lower The of Vav-1 was by versus that anti-CD3 and anti-CD3/CD28 induction of PKB and GSK-3α phosphorylation in T-cells can independently of Vav-1 The of phosphorylation on Vav-1 expression with the in and signaling in the Y. A. D. R.T. J. 2002; PubMed Scopus Google Scholar). this pathway was intact in primary Vav-1–/– T-cells, we also PKB and GSK-3 phosphorylation in T-cells from of or Vav-1–/– for with or by of PKB versus and GSK-3α phosphorylation versus The of Vav-1 was by versus The in the of phosphorylation. a in PKB and GSK-3 phosphorylation in Vav-1–/– the was more than with T-cells a loss of GSK-3 phosphorylation in phosphorylation was that GSK-3 phosphorylation is on and that is a pathway that is this with the transcription factor FOXO1 phosphorylation was indicate that anti-CD3 and induction of PKB-GSK-3 phosphorylation can in the of Vav-1 expression and is reduced with the loss of the Vav-1 and the phosphorylation, we Vav-1 a domain with PKB and GSK-3α phosphorylation of the of the Jurkat with Vav-1 or the induced phosphorylation of PKB and GSK-3α in with or and and versus and also induced of phosphorylation in WT, and and and Vav-1 expression was with versus The expression of PKB and was by with the lower and lower GSK-3α and PKB phosphorylation by lower the of on the PKB-GSK-3 as by Cell. 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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, 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, 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, K.A. Riley J.L. Harris M.H. Parry R.V. J.C. Plas D.R. Elstrom R.L. June C.H. Thompson C.B. Immunity. 2002; 16: Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. Rudd C.E. Immunity. 2001; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, F. L. A. A. Olive D. O. J. Immunol. 2000; PubMed Scopus Google Scholar). findings that anti-CD3 and induced of PKB and GSK-3α phosphorylation in Vav-1–/– Jurkat and primary CD4-positive T-cells 1 and that Vav-1 is not for the activation of the PKB-GSK-3 pathway in T-cells. of phosphorylation observed in the presence of a Vav-1 domain that is well in its to IL-2 transcription that the domain has in TcR driven IL-2 transcription that is of the PKB-GSK-3 the of of the Vav-1 binding of a of and the PKB-GSK-3 in T-cells are to observed in activation of PKB in the of Vav-1 M. 2004; PubMed Scopus Google Scholar). 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