Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Dec;16(12):6765–6774. doi: 10.1128/mcb.16.12.6765

Enhancement of lymphocyte responsiveness by a gain-of-function mutation of ZAP-70.

Q Zhao 1, A Weiss 1
PMCID: PMC231679  PMID: 8943331

Abstract

The protein tyrosine kinase ZAP-70 plays an essential role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 is associated with the receptor and is phosphorylated on many tyrosine residues, including tyrosine 292 (Y-292), in the region between the C-terminal SH2 domain and the kinase domain (interdomain B). Here we show that a mutation of Y-292 (292F) or deletion of interdomain B enhanced the ability of ZAP-70 to reconstitute B-cell receptor stimulation-dependent NF-AT induction in a B-cell line deficient in Syk. In contrast, in a T-cell line, expression of 292F led to basal NF-AT induction independent of T-cell receptor stimulation. These results demonstrate that the role of Y-292 is to negatively regulate the function of ZAP-70 in lymphocytes. This appears to be a dominant function of interdomain B because deletion of most of interdomain B also resulted in a mutant of ZAP-70 with enhanced ability to reconstitute Syk-deficient DT-40 B cells. Since our biochemical studies did not reveal an effect of the 292F mutation on either the kinase activity of ZAP-70 or on the ability of ZAP-70 to bind to the receptor, we propose a model in which Y-292 interacts with an inhibitory protein to negatively regulate ZAP-70 function.

Full Text

The Full Text of this article is available as a PDF (533.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abraham N., Miceli M. C., Parnes J. R., Veillette A. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature. 1991 Mar 7;350(6313):62–66. doi: 10.1038/350062a0. [DOI] [PubMed] [Google Scholar]
  2. Arpaia E., Shahar M., Dadi H., Cohen A., Roifman C. M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell. 1994 Mar 11;76(5):947–958. doi: 10.1016/0092-8674(94)90368-9. [DOI] [PubMed] [Google Scholar]
  3. Bolen J. B. Protein tyrosine kinases in the initiation of antigen receptor signaling. Curr Opin Immunol. 1995 Jun;7(3):306–311. doi: 10.1016/0952-7915(95)80103-0. [DOI] [PubMed] [Google Scholar]
  4. Cambier J. C., Pleiman C. M., Clark M. R. Signal transduction by the B cell antigen receptor and its coreceptors. Annu Rev Immunol. 1994;12:457–486. doi: 10.1146/annurev.iy.12.040194.002325. [DOI] [PubMed] [Google Scholar]
  5. Chan A. C., Dalton M., Johnson R., Kong G. H., Wang T., Thoma R., Kurosaki T. Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J. 1995 Jun 1;14(11):2499–2508. doi: 10.1002/j.1460-2075.1995.tb07247.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan A. C., Irving B. A., Fraser J. D., Weiss A. The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9166–9170. doi: 10.1073/pnas.88.20.9166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Chan A. C., Kadlecek T. A., Elder M. E., Filipovich A. H., Kuo W. L., Iwashima M., Parslow T. G., Weiss A. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science. 1994 Jun 10;264(5165):1599–1601. doi: 10.1126/science.8202713. [DOI] [PubMed] [Google Scholar]
  9. Cheng A. M., Rowley B., Pao W., Hayday A., Bolen J. B., Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature. 1995 Nov 16;378(6554):303–306. doi: 10.1038/378303a0. [DOI] [PubMed] [Google Scholar]
  10. Choi M. S., Brines R. D., Holman M. J., Klaus G. G. Induction of NF-AT in normal B lymphocytes by anti-immunoglobulin or CD40 ligand in conjunction with IL-4. Immunity. 1994 Jun;1(3):179–187. doi: 10.1016/1074-7613(94)90096-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. DeFranco A. L. Transmembrane signaling by antigen receptors of B and T lymphocytes. Curr Opin Cell Biol. 1995 Apr;7(2):163–175. doi: 10.1016/0955-0674(95)80024-7. [DOI] [PubMed] [Google Scholar]
  13. Duplay P., Thome M., Hervé F., Acuto O. p56lck interacts via its src homology 2 domain with the ZAP-70 kinase. J Exp Med. 1994 Apr 1;179(4):1163–1172. doi: 10.1084/jem.179.4.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Elder M. E., Lin D., Clever J., Chan A. C., Hope T. J., Weiss A., Parslow T. G. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science. 1994 Jun 10;264(5165):1596–1599. doi: 10.1126/science.8202712. [DOI] [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. Harrison M. L., Isaacson C. C., Burg D. L., Geahlen R. L., Low P. S. Phosphorylation of human erythrocyte band 3 by endogenous p72syk. J Biol Chem. 1994 Jan 14;269(2):955–959. [PubMed] [Google Scholar]
  18. Hatada M. H., Lu X., Laird E. R., Green J., Morgenstern J. P., Lou M., Marr C. S., Phillips T. B., Ram M. K., Theriault K. Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature. 1995 Sep 7;377(6544):32–38. doi: 10.1038/377032a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Izquierdo M., Leevers S. J., Marshall C. J., Cantrell D. p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes. J Exp Med. 1993 Oct 1;178(4):1199–1208. doi: 10.1084/jem.178.4.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kong G. H., Bu J. Y., Kurosaki T., Shaw A. S., Chan A. C. Reconstitution of Syk function by the ZAP-70 protein tyrosine kinase. Immunity. 1995 May;2(5):485–492. doi: 10.1016/1074-7613(95)90029-2. [DOI] [PubMed] [Google Scholar]
  23. Kong G., Dalton M., Bubeck Wardenburg J., Straus D., Kurosaki T., Chan A. C. Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function. Mol Cell Biol. 1996 Sep;16(9):5026–5035. doi: 10.1128/mcb.16.9.5026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Luo K., Sefton B. M. Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells. Mol Cell Biol. 1992 Oct;12(10):4724–4732. doi: 10.1128/mcb.12.10.4724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Madrenas J., Wange R. L., Wang J. L., Isakov N., Samelson L. E., Germain R. N. Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science. 1995 Jan 27;267(5197):515–518. doi: 10.1126/science.7824949. [DOI] [PubMed] [Google Scholar]
  26. Negishi I., Motoyama N., Nakayama K., Nakayama K., Senju S., Hatakeyama S., Zhang Q., Chan A. C., Loh D. Y. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature. 1995 Aug 3;376(6539):435–438. doi: 10.1038/376435a0. [DOI] [PubMed] [Google Scholar]
  27. Neumeister E. N., Zhu Y., Richard S., Terhorst C., Chan A. C., Shaw A. S. Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins. Mol Cell Biol. 1995 Jun;15(6):3171–3178. doi: 10.1128/mcb.15.6.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Perlmutter R. M., Levin S. D., Appleby M. W., Anderson S. J., Alberola-Ila J. Regulation of lymphocyte function by protein phosphorylation. Annu Rev Immunol. 1993;11:451–499. doi: 10.1146/annurev.iy.11.040193.002315. [DOI] [PubMed] [Google Scholar]
  30. Plas D. R., Johnson R., Pingel J. T., Matthews R. J., Dalton M., Roy G., Chan A. C., Thomas M. L. Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science. 1996 May 24;272(5265):1173–1176. doi: 10.1126/science.272.5265.1173. [DOI] [PubMed] [Google Scholar]
  31. Qian D., Griswold-Prenner I., Rosner M. R., Fitch F. W. Multiple components of the T cell antigen receptor complex become tyrosine-phosphorylated upon activation. J Biol Chem. 1993 Feb 25;268(6):4488–4493. [PubMed] [Google Scholar]
  32. Qian D., Mollenauer M. N., Weiss A. Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling. J Exp Med. 1996 Feb 1;183(2):611–620. doi: 10.1084/jem.183.2.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reth M. Antigen receptor tail clue. Nature. 1989 Mar 30;338(6214):383–384. doi: 10.1038/338383b0. [DOI] [PubMed] [Google Scholar]
  34. Richards J. D., Gold M. R., Hourihane S. L., DeFranco A. L., Matsuuchi L. Reconstitution of B cell antigen receptor-induced signaling events in a nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. J Biol Chem. 1996 Mar 15;271(11):6458–6466. doi: 10.1074/jbc.271.11.6458. [DOI] [PubMed] [Google Scholar]
  35. Samelson L. E., Klausner R. D. Tyrosine kinases and tyrosine-based activation motifs. Current research on activation via the T cell antigen receptor. J Biol Chem. 1992 Dec 15;267(35):24913–24916. [PubMed] [Google Scholar]
  36. Sloan-Lancaster J., Shaw A. S., Rothbard J. B., Allen P. M. Partial T cell signaling: altered phospho-zeta and lack of zap70 recruitment in APL-induced T cell anergy. Cell. 1994 Dec 2;79(5):913–922. doi: 10.1016/0092-8674(94)90080-9. [DOI] [PubMed] [Google Scholar]
  37. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  38. Straus D. B., Chan A. C., Patai B., Weiss A. SH2 domain function is essential for the role of the Lck tyrosine kinase in T cell receptor signal transduction. J Biol Chem. 1996 Apr 26;271(17):9976–9981. doi: 10.1074/jbc.271.17.9976. [DOI] [PubMed] [Google Scholar]
  39. Takata M., Sabe H., Hata A., Inazu T., Homma Y., Nukada T., Yamamura H., Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 1994 Mar 15;13(6):1341–1349. doi: 10.1002/j.1460-2075.1994.tb06387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Turner M., Mee P. J., Costello P. S., Williams O., Price A. A., Duddy L. P., Furlong M. T., Geahlen R. L., Tybulewicz V. L. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature. 1995 Nov 16;378(6554):298–302. doi: 10.1038/378298a0. [DOI] [PubMed] [Google Scholar]
  42. Venkataraman L., Francis D. A., Wang Z., Liu J., Rothstein T. L., Sen R. Cyclosporin-A sensitive induction of NF-AT in murine B cells. Immunity. 1994 Jun;1(3):189–196. doi: 10.1016/1074-7613(94)90097-3. [DOI] [PubMed] [Google Scholar]
  43. Wange R. L., Guitián R., Isakov N., Watts J. D., Aebersold R., Samelson L. E. Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem. 1995 Aug 11;270(32):18730–18733. doi: 10.1074/jbc.270.32.18730. [DOI] [PubMed] [Google Scholar]
  44. Wange R. L., Isakov N., Burke T. R., Jr, Otaka A., Roller P. P., Watts J. D., Aebersold R., Samelson L. E. F2(Pmp)2-TAM zeta 3, a novel competitive inhibitor of the binding of ZAP-70 to the T cell antigen receptor, blocks early T cell signaling. J Biol Chem. 1995 Jan 13;270(2):944–948. doi: 10.1074/jbc.270.2.944. [DOI] [PubMed] [Google Scholar]
  45. Wange R. L., Malek S. N., Desiderio S., Samelson L. E. Tandem SH2 domains of ZAP-70 bind to T cell antigen receptor zeta and CD3 epsilon from activated Jurkat T cells. J Biol Chem. 1993 Sep 15;268(26):19797–19801. [PubMed] [Google Scholar]
  46. Watts J. D., Affolter M., Krebs D. L., Wange R. L., Samelson L. E., Aebersold R. Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J Biol Chem. 1994 Nov 25;269(47):29520–29529. [PubMed] [Google Scholar]
  47. 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]
  48. Woodrow M. A., Rayter S., Downward J., Cantrell D. A. p21ras function is important for T cell antigen receptor and protein kinase C regulation of nuclear factor of activated T cells. J Immunol. 1993 May 1;150(9):3853–3861. [PubMed] [Google Scholar]
  49. 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]
  50. Wu J., Katzav S., Weiss A. A functional T-cell receptor signaling pathway is required for p95vav activity. Mol Cell Biol. 1995 Aug;15(8):4337–4346. doi: 10.1128/mcb.15.8.4337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Zhao Q., Schoborg R. V., Pintel D. J. Alternative splicing of pre-mRNAs encoding the nonstructural proteins of minute virus of mice is facilitated by sequences within the downstream intron. J Virol. 1994 May;68(5):2849–2859. doi: 10.1128/jvi.68.5.2849-2859.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Oers N. S., Killeen N., Weiss A. ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR zeta in murine thymocytes and lymph node T cells. Immunity. 1994 Nov;1(8):675–685. doi: 10.1016/1074-7613(94)90038-8. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES