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. 1995 Aug;15(8):4337–4346. doi: 10.1128/mcb.15.8.4337

A functional T-cell receptor signaling pathway is required for p95vav activity.

J Wu 1, S Katzav 1, A Weiss 1
PMCID: PMC230673  PMID: 7623828

Abstract

Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade.

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Selected References

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  1. Boguski M. S., Bairoch A., Attwood T. K., Michaels G. S. Proto-vav and gene expression. Nature. 1992 Jul 9;358(6382):113–113. doi: 10.1038/358113a0. [DOI] [PubMed] [Google Scholar]
  2. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  3. Bram R. J., Crabtree G. R. Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. Nature. 1994 Sep 22;371(6495):355–358. doi: 10.1038/371355a0. [DOI] [PubMed] [Google Scholar]
  4. Bustelo X. R., Barbacid M. Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells. Science. 1992 May 22;256(5060):1196–1199. doi: 10.1126/science.256.5060.1196. [DOI] [PubMed] [Google Scholar]
  5. Bustelo X. R., Ledbetter J. A., Barbacid M. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Nature. 1992 Mar 5;356(6364):68–71. doi: 10.1038/356068a0. [DOI] [PubMed] [Google Scholar]
  6. Bustelo X. R., Suen K. L., Leftheris K., Meyers C. A., Barbacid M. Vav cooperates with Ras to transform rodent fibroblasts but is not a Ras GDP/GTP exchange factor. Oncogene. 1994 Aug;9(8):2405–2413. [PubMed] [Google Scholar]
  7. Bustelo X. R., Suen K. L., Michael W. M., Dreyfuss G., Barbacid M. Association of the vav proto-oncogene product with poly(rC)-specific RNA-binding proteins. Mol Cell Biol. 1995 Mar;15(3):1324–1332. doi: 10.1128/mcb.15.3.1324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan A. C., Iwashima M., Turck C. W., Weiss A. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell. 1992 Nov 13;71(4):649–662. doi: 10.1016/0092-8674(92)90598-7. [DOI] [PubMed] [Google Scholar]
  9. Clipstone N. A., Crabtree G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. doi: 10.1038/357695a0. [DOI] [PubMed] [Google Scholar]
  10. Coppola J., Bryant S., Koda T., Conway D., Barbacid M. Mechanism of activation of the vav protooncogene. Cell Growth Differ. 1991 Feb;2(2):95–105. [PubMed] [Google Scholar]
  11. Desai D. M., Newton M. E., Kadlecek T., Weiss A. Stimulation of the phosphatidylinositol pathway can induce T-cell activation. Nature. 1990 Nov 1;348(6296):66–69. doi: 10.1038/348066a0. [DOI] [PubMed] [Google Scholar]
  12. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  13. Durand D. B., Shaw J. P., Bush M. R., Replogle R. E., Belagaje R., Crabtree G. R. Characterization of antigen receptor response elements within the interleukin-2 enhancer. Mol Cell Biol. 1988 Apr;8(4):1715–1724. doi: 10.1128/mcb.8.4.1715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Emmel E. A., Verweij C. L., Durand D. B., Higgins K. M., Lacy E., Crabtree G. R. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science. 1989 Dec 22;246(4937):1617–1620. doi: 10.1126/science.2595372. [DOI] [PubMed] [Google Scholar]
  15. Evans G. A., Howard O. M., Erwin R., Farrar W. L. Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck. Biochem J. 1993 Sep 1;294(Pt 2):339–342. doi: 10.1042/bj2940339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fischer K. D., Zmuldzinas A., Gardner S., Barbacid M., Bernstein A., Guidos C. Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes. Nature. 1995 Mar 30;374(6521):474–477. doi: 10.1038/374474a0. [DOI] [PubMed] [Google Scholar]
  17. Fruman D. A., Klee C. B., Bierer B. E., Burakoff S. J. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3686–3690. doi: 10.1073/pnas.89.9.3686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galland F., Katzav S., Birnbaum D. The products of the mcf-2 and vav proto-oncogenes and of the yeast gene cdc-24 share sequence similarities. Oncogene. 1992 Mar;7(3):585–587. [PubMed] [Google Scholar]
  19. Goldsmith M. A., Dazin P. F., Weiss A. At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8613–8617. doi: 10.1073/pnas.85.22.8613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldsmith M. A., Desai D. M., Schultz T., Weiss A. Function of a heterologous muscarinic receptor in T cell antigen receptor signal transduction mutants. J Biol Chem. 1989 Oct 15;264(29):17190–17197. [PubMed] [Google Scholar]
  21. Goldsmith M. A., Weiss A. Isolation and characterization of a T-lymphocyte somatic mutant with altered signal transduction by the antigen receptor. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6879–6883. doi: 10.1073/pnas.84.19.6879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Graber M., Bockenstedt L. K., Weiss A. Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number. J Immunol. 1991 May 1;146(9):2935–2943. [PubMed] [Google Scholar]
  23. Gulbins E., Coggeshall K. M., Baier G., Katzav S., Burn P., Altman A. Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation. Science. 1993 May 7;260(5109):822–825. doi: 10.1126/science.8484124. [DOI] [PubMed] [Google Scholar]
  24. Gulbins E., Coggeshall K. M., Baier G., Telford D., Langlet C., Baier-Bitterlich G., Bonnefoy-Berard N., Burn P., Wittinghofer A., Altman A. Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides. Mol Cell Biol. 1994 Jul;14(7):4749–4758. doi: 10.1128/mcb.14.7.4749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gulbins E., Coggeshall K. M., Langlet C., Baier G., Bonnefoy-Berard N., Burn P., Wittinghofer A., Katzav S., Altman A. Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein. Mol Cell Biol. 1994 Feb;14(2):906–913. doi: 10.1128/mcb.14.2.906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gulbins E., Langlet C., Baier G., Bonnefoy-Berard N., Herbert E., Altman A., Coggeshall K. M. Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells. J Immunol. 1994 Mar 1;152(5):2123–2129. [PubMed] [Google Scholar]
  27. Gupta S., Weiss A., Kumar G., Wang S., Nel A. The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. Evidence for the existence of a second protein kinase C-dependent pathway in an Lck-negative Jurkat cell mutant. J Biol Chem. 1994 Jun 24;269(25):17349–17357. [PubMed] [Google Scholar]
  28. Hart M. J., Eva A., Zangrilli D., Aaronson S. A., Evans T., Cerione R. A., Zheng Y. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J Biol Chem. 1994 Jan 7;269(1):62–65. [PubMed] [Google Scholar]
  29. Hobert O., Jallal B., Schlessinger J., Ullrich A. Novel signaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction. J Biol Chem. 1994 Aug 12;269(32):20225–20228. [PubMed] [Google Scholar]
  30. Irving B. A., Weiss A. The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell. 1991 Mar 8;64(5):891–901. doi: 10.1016/0092-8674(91)90314-o. [DOI] [PubMed] [Google Scholar]
  31. Iwashima M., Irving B. A., van Oers N. S., Chan A. C., Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136–1139. doi: 10.1126/science.7509083. [DOI] [PubMed] [Google Scholar]
  32. Izquierdo M., Downward J., Graves J. D., Cantrell D. A. Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. Mol Cell Biol. 1992 Jul;12(7):3305–3312. doi: 10.1128/mcb.12.7.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. June C. H., Fletcher M. C., Ledbetter J. A., Schieven G. L., Siegel J. N., Phillips A. F., Samelson L. E. Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7722–7726. doi: 10.1073/pnas.87.19.7722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Katzav S., Cleveland J. L., Heslop H. E., Pulido D. Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential. Mol Cell Biol. 1991 Apr;11(4):1912–1920. doi: 10.1128/mcb.11.4.1912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Katzav S., Martin-Zanca D., Barbacid M. vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J. 1989 Aug;8(8):2283–2290. doi: 10.1002/j.1460-2075.1989.tb08354.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Katzav S., Sutherland M., Packham G., Yi T., Weiss A. The protein tyrosine kinase ZAP-70 can associate with the SH2 domain of proto-Vav. J Biol Chem. 1994 Dec 23;269(51):32579–32585. [PubMed] [Google Scholar]
  37. Kazanietz M. G., Bustelo X. R., Barbacid M., Kolch W., Mischak H., Wong G., Pettit G. R., Bruns J. D., Blumberg P. M. Zinc finger domains and phorbol ester pharmacophore. Analysis of binding to mutated form of protein kinase C zeta and the vav and c-raf proto-oncogene products. J Biol Chem. 1994 Apr 15;269(15):11590–11594. [PubMed] [Google Scholar]
  38. Khosravi-Far R., Chrzanowska-Wodnicka M., Solski P. A., Eva A., Burridge K., Der C. J. Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras. Mol Cell Biol. 1994 Oct;14(10):6848–6857. doi: 10.1128/mcb.14.10.6848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  40. Koretzky G. A., Picus J., Schultz T., Weiss A. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2037–2041. doi: 10.1073/pnas.88.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Koretzky G. A., Picus J., Thomas M. L., Weiss A. Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature. 1990 Jul 5;346(6279):66–68. doi: 10.1038/346066a0. [DOI] [PubMed] [Google Scholar]
  42. Margolis B., Hu P., Katzav S., Li W., Oliver J. M., Ullrich A., Weiss A., Schlessinger J. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. Nature. 1992 Mar 5;356(6364):71–74. doi: 10.1038/356071a0. [DOI] [PubMed] [Google Scholar]
  43. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Musacchio A., Gibson T., Rice P., Thompson J., Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993 Sep;18(9):343–348. doi: 10.1016/0968-0004(93)90071-t. [DOI] [PubMed] [Google Scholar]
  45. Nishibe S., Wahl M. I., Hernández-Sotomayor S. M., Tonks N. K., Rhee S. G., Carpenter G. Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation. Science. 1990 Nov 30;250(4985):1253–1256. doi: 10.1126/science.1700866. [DOI] [PubMed] [Google Scholar]
  46. O'Keefe S. J., Tamura J., Kincaid R. L., Tocci M. J., O'Neill E. A. FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature. 1992 Jun 25;357(6380):692–694. doi: 10.1038/357692a0. [DOI] [PubMed] [Google Scholar]
  47. Ohashi P. S., Mak T. W., Van den Elsen P., Yanagi Y., Yoshikai Y., Calman A. F., Terhorst C., Stobo J. D., Weiss A. Reconstitution of an active surface T3/T-cell antigen receptor by DNA transfer. Nature. 1985 Aug 15;316(6029):606–609. doi: 10.1038/316606a0. [DOI] [PubMed] [Google Scholar]
  48. Owaki H., Varma R., Gillis B., Bruder J. T., Rapp U. R., Davis L. S., Geppert T. D. Raf-1 is required for T cell IL2 production. EMBO J. 1993 Nov;12(11):4367–4373. doi: 10.1002/j.1460-2075.1993.tb06121.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Price L. S., Norman J. C., Ridley A. J., Koffer A. The small GTPases Rac and Rho as regulators of secretion in mast cells. Curr Biol. 1995 Jan 1;5(1):68–73. doi: 10.1016/s0960-9822(95)00018-2. [DOI] [PubMed] [Google Scholar]
  50. Ravichandran K. S., Lorenz U., Shoelson S. E., Burakoff S. J. Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol. 1995 Feb;15(2):593–600. doi: 10.1128/mcb.15.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rayter S. I., Woodrow M., Lucas S. C., Cantrell D. A., Downward J. p21ras mediates control of IL-2 gene promoter function in T cell activation. EMBO J. 1992 Dec;11(12):4549–4556. doi: 10.1002/j.1460-2075.1992.tb05556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Reibel L., Dorseuil O., Stancou R., Bertoglio J., Gacon G. A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation. Biochem Biophys Res Commun. 1991 Mar 15;175(2):451–458. doi: 10.1016/0006-291x(91)91585-z. [DOI] [PubMed] [Google Scholar]
  53. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  54. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  55. Romeo C., Amiot M., Seed B. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor zeta chain. Cell. 1992 Mar 6;68(5):889–897. doi: 10.1016/0092-8674(92)90032-8. [DOI] [PubMed] [Google Scholar]
  56. Schlessinger J. How receptor tyrosine kinases activate Ras. Trends Biochem Sci. 1993 Aug;18(8):273–275. doi: 10.1016/0968-0004(93)90031-h. [DOI] [PubMed] [Google Scholar]
  57. Sieh M., Batzer A., Schlessinger J., Weiss A. GRB2 and phospholipase C-gamma 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol Cell Biol. 1994 Jul;14(7):4435–4442. doi: 10.1128/mcb.14.7.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sieh M., Bolen J. B., Weiss A. CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck. EMBO J. 1993 Jan;12(1):315–321. doi: 10.1002/j.1460-2075.1993.tb05659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Straus D. B., Weiss A. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell. 1992 Aug 21;70(4):585–593. doi: 10.1016/0092-8674(92)90428-f. [DOI] [PubMed] [Google Scholar]
  60. Tarakhovsky A., Turner M., Schaal S., Mee P. J., Duddy L. P., Rajewsky K., Tybulewicz V. L. Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav. Nature. 1995 Mar 30;374(6521):467–470. doi: 10.1038/374467a0. [DOI] [PubMed] [Google Scholar]
  61. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  62. Weiss A., Stobo J. D. Requirement for the coexpression of T3 and the T cell antigen receptor on a malignant human T cell line. J Exp Med. 1984 Nov 1;160(5):1284–1299. doi: 10.1084/jem.160.5.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Woodrow M., Clipstone N. A., Cantrell D. p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells. J Exp Med. 1993 Nov 1;178(5):1517–1522. doi: 10.1084/jem.178.5.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhang R., Alt F. W., Davidson L., Orkin S. H., Swat W. Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature. 1995 Mar 30;374(6521):470–473. doi: 10.1038/374470a0. [DOI] [PubMed] [Google Scholar]
  65. Zmuidzinas A., Fischer K. D., Lira S. A., Forrester L., Bryant S., Bernstein A., Barbacid M. The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro. EMBO J. 1995 Jan 3;14(1):1–11. doi: 10.1002/j.1460-2075.1995.tb06969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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