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. 1995 Jun;15(6):3171–3178. doi: 10.1128/mcb.15.6.3171

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.

E N Neumeister 1, Y Zhu 1, S Richard 1, C Terhorst 1, A C Chan 1, A S Shaw 1
PMCID: PMC230549  PMID: 7760813

Abstract

ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.

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Selected References

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  1. 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]
  2. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burkhardt A. L., Stealey B., Rowley R. B., Mahajan S., Prendergast M., Fargnoli J., Bolen J. B. Temporal regulation of non-transmembrane protein tyrosine kinase enzyme activity following T cell antigen receptor engagement. J Biol Chem. 1994 Sep 23;269(38):23642–23647. [PubMed] [Google Scholar]
  4. Cambier J. C. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL) Immunol Today. 1995 Feb;16(2):110–110. doi: 10.1016/0167-5699(95)80105-7. [DOI] [PubMed] [Google Scholar]
  5. Cambier J. C. Signal transduction by T- and B-cell antigen receptors: converging structures and concepts. Curr Opin Immunol. 1992 Jun;4(3):257–264. doi: 10.1016/0952-7915(92)90074-o. [DOI] [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. Cobb B. S., Schaller M. D., Leu T. H., Parsons J. T. Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol Cell Biol. 1994 Jan;14(1):147–155. doi: 10.1128/mcb.14.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Exley M., Varticovski L., Peter M., Sancho J., Terhorst C. Association of phosphatidylinositol 3-kinase with a specific sequence of the T cell receptor zeta chain is dependent on T cell activation. J Biol Chem. 1994 May 27;269(21):15140–15146. [PubMed] [Google Scholar]
  14. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gauen L. K., Zhu Y., Letourneur F., Hu Q., Bolen J. B., Matis L. A., Klausner R. D., Shaw A. S. Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif. Mol Cell Biol. 1994 Jun;14(6):3729–3741. doi: 10.1128/mcb.14.6.3729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hemsley A., Arnheim N., Toney M. D., Cortopassi G., Galas D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. doi: 10.1093/nar/17.16.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Irving B. A., Chan A. C., Weiss A. Functional characterization of a signal transducing motif present in the T cell antigen receptor zeta chain. J Exp Med. 1993 Apr 1;177(4):1093–1103. doi: 10.1084/jem.177.4.1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  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. June C. H., Fletcher M. C., Ledbetter J. A., Samelson L. E. Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. J Immunol. 1990 Mar 1;144(5):1591–1599. [PubMed] [Google Scholar]
  21. Kanner S. B., Aruffo A., Chan P. Y. Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10484–10487. doi: 10.1073/pnas.91.22.10484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kazlauskas A. Receptor tyrosine kinases and their targets. Curr Opin Genet Dev. 1994 Feb;4(1):5–14. doi: 10.1016/0959-437x(94)90085-x. [DOI] [PubMed] [Google Scholar]
  23. Klausner R. D., Samelson L. E. T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell. 1991 Mar 8;64(5):875–878. doi: 10.1016/0092-8674(91)90310-u. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Letourneur F., Klausner R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell. 1992 Jun 26;69(7):1143–1157. doi: 10.1016/0092-8674(92)90636-q. [DOI] [PubMed] [Google Scholar]
  26. Letourneur F., Klausner R. D. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8905–8909. doi: 10.1073/pnas.88.20.8905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luo K., Hurley T. R., Sefton B. M. Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene. 1990 Jun;5(6):921–923. [PubMed] [Google Scholar]
  28. McGlade J., Brunkhorst B., Anderson D., Mbamalu G., Settleman J., Dedhar S., Rozakis-Adcock M., Chen L. B., Pawson T. The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion. EMBO J. 1993 Aug;12(8):3073–3081. doi: 10.1002/j.1460-2075.1993.tb05976.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mustelin T., Coggeshall K. M., Isakov N., Altman A. T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation. Science. 1990 Mar 30;247(4950):1584–1587. doi: 10.1126/science.2138816. [DOI] [PubMed] [Google Scholar]
  30. Nakamura N., Tanaka J., Sobue K. Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery. J Cell Sci. 1993 Dec;106(Pt 4):1057–1069. doi: 10.1242/jcs.106.4.1057. [DOI] [PubMed] [Google Scholar]
  31. Pawson T., Gish G. D. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. doi: 10.1016/0092-8674(92)90504-6. [DOI] [PubMed] [Google Scholar]
  32. Pleiman C. M., Abrams C., Gauen L. T., Bedzyk W., Jongstra J., Shaw A. S., Cambier J. C. Distinct p53/56lyn and p59fyn domains associate with nonphosphorylated and phosphorylated Ig-alpha. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4268–4272. doi: 10.1073/pnas.91.10.4268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ravichandran K. S., Lee K. K., Songyang Z., Cantley L. C., Burn P., Burakoff S. J. Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation. Science. 1993 Nov 5;262(5135):902–905. doi: 10.1126/science.8235613. [DOI] [PubMed] [Google Scholar]
  34. Richard S., Yu D., Blumer K. J., Hausladen D., Olszowy M. W., Connelly P. A., Shaw A. S. Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1. Mol Cell Biol. 1995 Jan;15(1):186–197. doi: 10.1128/mcb.15.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Romeo C., Seed B. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell. 1991 Mar 8;64(5):1037–1046. doi: 10.1016/0092-8674(91)90327-u. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schlessinger J., Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992 Sep;9(3):383–391. doi: 10.1016/0896-6273(92)90177-f. [DOI] [PubMed] [Google Scholar]
  41. Shaw A. S., Amrein K. E., Hammond C., Stern D. F., Sefton B. M., Rose J. K. The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain. Cell. 1989 Nov 17;59(4):627–636. doi: 10.1016/0092-8674(89)90008-1. [DOI] [PubMed] [Google Scholar]
  42. Shoelson S. E., Sivaraja M., Williams K. P., Hu P., Schlessinger J., Weiss M. A. Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation. EMBO J. 1993 Feb;12(2):795–802. doi: 10.1002/j.1460-2075.1993.tb05714.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shores E. W., Huang K., Tran T., Lee E., Grinberg A., Love P. E. Role of TCR zeta chain in T cell development and selection. Science. 1994 Nov 11;266(5187):1047–1050. doi: 10.1126/science.7526464. [DOI] [PubMed] [Google Scholar]
  44. Sloan-Lancaster J., Evavold B. D., Allen P. M. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells. Nature. 1993 May 13;363(6425):156–159. doi: 10.1038/363156a0. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Timson Gauen L. K., Kong A. N., Samelson L. E., Shaw A. S. p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain. Mol Cell Biol. 1992 Dec;12(12):5438–5446. doi: 10.1128/mcb.12.12.5438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van Etten R. A., Jackson P. K., Baltimore D., Sanders M. C., Matsudaira P. T., Janmey P. A. The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity. J Cell Biol. 1994 Feb;124(3):325–340. doi: 10.1083/jcb.124.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. Wegener A. M., Letourneur F., Hoeveler A., Brocker T., Luton F., Malissen B. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell. 1992 Jan 10;68(1):83–95. doi: 10.1016/0092-8674(92)90208-t. [DOI] [PubMed] [Google Scholar]
  51. Weiss A. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell. 1993 Apr 23;73(2):209–212. doi: 10.1016/0092-8674(93)90221-b. [DOI] [PubMed] [Google Scholar]
  52. Xing Z., Chen H. C., Nowlen J. K., Taylor S. J., Shalloway D., Guan J. L. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol Biol Cell. 1994 Apr;5(4):413–421. doi: 10.1091/mbc.5.4.413. [DOI] [PMC free article] [PubMed] [Google Scholar]

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