Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Sep;16(9):4996–5003. doi: 10.1128/mcb.16.9.4996

Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

U D'Oro 1, K Sakaguchi 1, E Appella 1, J D Ashwell 1
PMCID: PMC231501  PMID: 8756658

Abstract

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

Full Text

The Full Text of this article is available as a PDF (486.1 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. Amrein K. E., Sefton B. M. Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4247–4251. doi: 10.1073/pnas.85.12.4247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Appleby M. W., Gross J. A., Cooke M. P., Levin S. D., Qian X., Perlmutter R. M. Defective T cell receptor signaling in mice lacking the thymic isoform of p59fyn. Cell. 1992 Sep 4;70(5):751–763. doi: 10.1016/0092-8674(92)90309-z. [DOI] [PubMed] [Google Scholar]
  4. Bei R., Kantor J., Kashmiri S. V., Schlom J. Serological and biochemical characterization of recombinant baculovirus carcinoembryonic antigen. Mol Immunol. 1994 Jul;31(10):771–780. doi: 10.1016/0161-5890(94)90151-1. [DOI] [PubMed] [Google Scholar]
  5. Bennett B. D., Cowley S., Jiang S., London R., Deng B., Grabarek J., Groopman J. E., Goeddel D. V., Avraham H. Identification and characterization of a novel tyrosine kinase from megakaryocytes. J Biol Chem. 1994 Jan 14;269(2):1068–1074. [PubMed] [Google Scholar]
  6. Bergman M., Mustelin T., Oetken C., Partanen J., Flint N. A., Amrein K. E., Autero M., Burn P., Alitalo K. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J. 1992 Aug;11(8):2919–2924. doi: 10.1002/j.1460-2075.1992.tb05361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  8. Burns C. M., Sakaguchi K., Appella E., Ashwell J. D. CD45 regulation of tyrosine phosphorylation and enzyme activity of src family kinases. J Biol Chem. 1994 May 6;269(18):13594–13600. [PubMed] [Google Scholar]
  9. Cahir McFarland E. D., Hurley T. R., Pingel J. T., Sefton B. M., Shaw A., Thomas M. L. Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1402–1406. doi: 10.1073/pnas.90.4.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheng H. C., Nishio H., Hatase O., Ralph S., Wang J. H. A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases. J Biol Chem. 1992 May 5;267(13):9248–9256. [PubMed] [Google Scholar]
  11. Cooke M. P., Abraham K. M., Forbush K. A., Perlmutter R. M. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn). Cell. 1991 Apr 19;65(2):281–291. doi: 10.1016/0092-8674(91)90162-r. [DOI] [PubMed] [Google Scholar]
  12. Couture C., Baier G., Oetken C., Williams S., Telford D., Marie-Cardine A., Baier-Bitterlich G., Fischer S., Burn P., Altman A. Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation. Mol Cell Biol. 1994 Aug;14(8):5249–5258. doi: 10.1128/mcb.14.8.5249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Danielian S., Alcover A., Polissard L., Stefanescu M., Acuto O., Fischer S., Fagard R. Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck. Eur J Immunol. 1992 Nov;22(11):2915–2921. doi: 10.1002/eji.1830221124. [DOI] [PubMed] [Google Scholar]
  14. Deans J. P., Kanner S. B., Torres R. M., Ledbetter J. A. Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase. Eur J Immunol. 1992 Mar;22(3):661–668. doi: 10.1002/eji.1830220308. [DOI] [PubMed] [Google Scholar]
  15. Desai D. M., Sap J., Silvennoinen O., Schlessinger J., Weiss A. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. 1994 Sep 1;13(17):4002–4010. doi: 10.1002/j.1460-2075.1994.tb06716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Glaichenhaus N., Shastri N., Littman D. R., Turner J. M. Requirement for association of p56lck with CD4 in antigen-specific signal transduction in T cells. Cell. 1991 Feb 8;64(3):511–520. doi: 10.1016/0092-8674(91)90235-q. [DOI] [PubMed] [Google Scholar]
  18. Imamoto A., Soriano P. Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice. Cell. 1993 Jun 18;73(6):1117–1124. doi: 10.1016/0092-8674(93)90641-3. [DOI] [PubMed] [Google Scholar]
  19. Imbert V., Farahifar D., Auberger P., Mary D., Rossi B., Peyron J. F. Stimulation of the T-cell antigen receptor-CD3 complex signaling pathway by the tyrosine phosphatase inhibitor pervanadate is mediated by inhibition of CD45: evidence for two interconnected Lck/Fyn- or zap-70-dependent signaling pathways. J Inflamm. 1996;46(2):65–77. [PubMed] [Google Scholar]
  20. Johansen T. E., Schøller M. S., Tolstoy S., Schwartz T. W. Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors. FEBS Lett. 1990 Jul 16;267(2):289–294. doi: 10.1016/0014-5793(90)80947-h. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Karnitz L., Sutor S. L., Torigoe T., Reed J. C., Bell M. P., McKean D. J., Leibson P. J., Abraham R. T. Effects of p56lck deficiency on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line. Mol Cell Biol. 1992 Oct;12(10):4521–4530. doi: 10.1128/mcb.12.10.4521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klages S., Adam D., Class K., Fargnoli J., Bolen J. B., Penhallow R. C. Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2597–2601. doi: 10.1073/pnas.91.7.2597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Marth J. D., Cooper J. A., King C. S., Ziegler S. F., Tinker D. A., Overell R. W., Krebs E. G., Perlmutter R. M. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol. 1988 Feb;8(2):540–550. doi: 10.1128/mcb.8.2.540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Molina T. J., Kishihara K., Siderovski D. P., van Ewijk W., Narendran A., Timms E., Wakeham A., Paige C. J., Hartmann K. U., Veillette A. Profound block in thymocyte development in mice lacking p56lck. Nature. 1992 May 14;357(6374):161–164. doi: 10.1038/357161a0. [DOI] [PubMed] [Google Scholar]
  29. Murphy S. M., Bergman M., Morgan D. O. Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae. Mol Cell Biol. 1993 Sep;13(9):5290–5300. doi: 10.1128/mcb.13.9.5290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mustelin T., Altman A. Dephosphorylation and activation of the T cell tyrosine kinase pp56lck by the leukocyte common antigen (CD45). Oncogene. 1990 Jun;5(6):809–813. [PubMed] [Google Scholar]
  31. Mustelin T., Coggeshall K. M., Altman A. Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6302–6306. doi: 10.1073/pnas.86.16.6302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mustelin T., Pessa-Morikawa T., Autero M., Gassmann M., Andersson L. C., Gahmberg C. G., Burn P. Regulation of the p59fyn protein tyrosine kinase by the CD45 phosphotyrosine phosphatase. Eur J Immunol. 1992 May;22(5):1173–1178. doi: 10.1002/eji.1830220510. [DOI] [PubMed] [Google Scholar]
  33. Nada S., Okada M., MacAuley A., Cooper J. A., Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991 May 2;351(6321):69–72. doi: 10.1038/351069a0. [DOI] [PubMed] [Google Scholar]
  34. Niklinska B. B., Hou D., June C., Weissman A. M., Ashwell J. D. CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling. Mol Cell Biol. 1994 Dec;14(12):8078–8084. doi: 10.1128/mcb.14.12.8078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Okada M., Nada S., Yamanashi Y., Yamamoto T., Nakagawa H. CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J Biol Chem. 1991 Dec 25;266(36):24249–24252. [PubMed] [Google Scholar]
  36. Ostergaard H. L., Shackelford D. A., Hurley T. R., Johnson P., Hyman R., Sefton B. M., Trowbridge I. S. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8959–8963. doi: 10.1073/pnas.86.22.8959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ostergaard H. L., Trowbridge I. S. Coclustering CD45 with CD4 or CD8 alters the phosphorylation and kinase activity of p56lck. J Exp Med. 1990 Jul 1;172(1):347–350. doi: 10.1084/jem.172.1.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roussel R. R., Brodeur S. R., Shalloway D., Laudano A. P. Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10696–10700. doi: 10.1073/pnas.88.23.10696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Samelson L. E., Phillips A. F., Luong E. T., Klausner R. D. Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4358–4362. doi: 10.1073/pnas.87.11.4358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Stein P. L., Lee H. M., Rich S., Soriano P. pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells. Cell. 1992 Sep 4;70(5):741–750. doi: 10.1016/0092-8674(92)90308-y. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Tsygankov A. Y., Bröker B. M., Fargnoli J., Ledbetter J. A., Bolen J. B. Activation of tyrosine kinase p60fyn following T cell antigen receptor cross-linking. J Biol Chem. 1992 Sep 15;267(26):18259–18262. [PubMed] [Google Scholar]
  44. Turner J. M., Brodsky M. H., Irving B. A., Levin S. D., Perlmutter R. M., Littman D. R. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 1990 Mar 9;60(5):755–765. doi: 10.1016/0092-8674(90)90090-2. [DOI] [PubMed] [Google Scholar]
  45. Veillette A., Bolen J. B., Bookman M. A. Alterations in tyrosine protein phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes. Mol Cell Biol. 1989 Oct;9(10):4441–4446. doi: 10.1128/mcb.9.10.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  47. Veillette A., Bookman M. A., Horak E. M., Samelson L. E., Bolen J. B. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989 Mar 16;338(6212):257–259. doi: 10.1038/338257a0. [DOI] [PubMed] [Google Scholar]
  48. Veillette A., Fournel M. The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation. Oncogene. 1990 Oct;5(10):1455–1462. [PubMed] [Google Scholar]
  49. Volarević S., Niklinska B. B., Burns C. M., June C. H., Weissman A. M., Ashwell J. D. Regulation of TCR signaling by CD45 lacking transmembrane and extracellular domains. Science. 1993 Apr 23;260(5107):541–544. doi: 10.1126/science.8475386. [DOI] [PubMed] [Google Scholar]
  50. Volarević S., Niklinska B. B., Burns C. M., Yamada H., June C. H., Dumont F. J., Ashwell J. D. The CD45 tyrosine phosphatase regulates phosphotyrosine homeostasis and its loss reveals a novel pattern of late T cell receptor-induced Ca2+ oscillations. J Exp Med. 1992 Sep 1;176(3):835–844. doi: 10.1084/jem.176.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Watts J. D., Sanghera J. S., Pelech S. L., Aebersold R. Phosphorylation of serine 59 of p56lck in activated T cells. J Biol Chem. 1993 Nov 5;268(31):23275–23282. [PubMed] [Google Scholar]
  52. Weaver C. T., Pingel J. T., Nelson J. O., Thomas M. L. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli. Mol Cell Biol. 1991 Sep;11(9):4415–4422. doi: 10.1128/mcb.11.9.4415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Winkler D. G., Park I., Kim T., Payne N. S., Walsh C. T., Strominger J. L., Shin J. Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5176–5180. doi: 10.1073/pnas.90.11.5176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zamoyska R., Derham P., Gorman S. D., von Hoegen P., Bolen J. B., Veillette A., Parnes J. R. Inability of CD8 alpha' polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo. Nature. 1989 Nov 16;342(6247):278–281. doi: 10.1038/342278a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES