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. 1995 May;15(5):2393–2401. doi: 10.1128/mcb.15.5.2393

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

F G Gervais 1, A Veillette 1
PMCID: PMC230468  PMID: 7739523

Abstract

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

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Selected References

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  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. Abraham N., Veillette A. Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation. Mol Cell Biol. 1990 Oct;10(10):5197–5206. doi: 10.1128/mcb.10.10.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Albritton L. M., Tseng L., Scadden D., Cunningham J. M. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 1989 May 19;57(4):659–666. doi: 10.1016/0092-8674(89)90134-7. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  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. 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]
  8. 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]
  9. Carrera A. C., Alexandrov K., Roberts T. M. The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):442–446. doi: 10.1073/pnas.90.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cartier M., Chang M. W., Stanners C. P. Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells. Mol Cell Biol. 1987 May;7(5):1623–1628. doi: 10.1128/mcb.7.5.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chow L. M., Fournel M., Davidson D., Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature. 1993 Sep 9;365(6442):156–160. doi: 10.1038/365156a0. [DOI] [PubMed] [Google Scholar]
  12. Cooke M. P., Perlmutter R. M. Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. New Biol. 1989 Oct;1(1):66–74. [PubMed] [Google Scholar]
  13. Cooper J. A., Howell B. The when and how of Src regulation. Cell. 1993 Jun 18;73(6):1051–1054. doi: 10.1016/0092-8674(93)90634-3. [DOI] [PubMed] [Google Scholar]
  14. Davidson D., Chow L. M., Fournel M., Veillette A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn. J Exp Med. 1992 Jun 1;175(6):1483–1492. doi: 10.1084/jem.175.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Davidson D., Fournel M., Veillette A. Oncogenic activation of p59fyn tyrosine protein kinase by mutation of its carboxyl-terminal site of tyrosine phosphorylation, tyrosine 528. J Biol Chem. 1994 Apr 8;269(14):10956–10963. [PubMed] [Google Scholar]
  16. Gervais F. G., Chow L. M., Lee J. M., Branton P. E., Veillette A. The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site. Mol Cell Biol. 1993 Nov;13(11):7112–7121. doi: 10.1128/mcb.13.11.7112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hurley T. R., Hyman R., Sefton B. M. Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases. Mol Cell Biol. 1993 Mar;13(3):1651–1656. doi: 10.1128/mcb.13.3.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koretzky G. A., Kohmetscher M., Ross S. CD45-associated kinase activity requires lck but not T cell receptor expression in the Jurkat T cell line. J Biol Chem. 1993 Apr 25;268(12):8958–8964. [PubMed] [Google Scholar]
  19. 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]
  20. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Luo K. X., Sefton B. M. Analysis of the sites in p56lck whose phosphorylation is induced by tetradecanoyl phorbol acetate. Oncogene. 1990 Jun;5(6):803–808. [PubMed] [Google Scholar]
  22. 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]
  23. Marth J. D., Peet R., Krebs E. G., Perlmutter R. M. A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell. 1985 Dec;43(2 Pt 1):393–404. doi: 10.1016/0092-8674(85)90169-2. [DOI] [PubMed] [Google Scholar]
  24. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Peri K. G., Gervais F. G., Weil R., Davidson D., Gish G. D., Veillette A. Interactions of the SH2 domain of lymphocyte-specific tyrosine protein kinase p56lck with phosphotyrosine-containing proteins. Oncogene. 1993 Oct;8(10):2765–2772. [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. Pingel J. T., Thomas M. L. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell. 1989 Sep 22;58(6):1055–1065. doi: 10.1016/0092-8674(89)90504-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Resh M. D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994 Feb 11;76(3):411–413. doi: 10.1016/0092-8674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  32. Reske-Kunz A. B., Rüde E. Insulin-specific T cell hybridomas derived from (H-2b x H-2k)F1 mice preferably employ F1-unique restriction elements for antigen recognition. Eur J Immunol. 1985 Oct;15(10):1048–1054. doi: 10.1002/eji.1830151017. [DOI] [PubMed] [Google Scholar]
  33. Rudd C. E., Trevillyan J. M., Dasgupta J. D., Wong L. L., Schlossman S. F. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5190–5194. doi: 10.1073/pnas.85.14.5190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Schraven B., Kirchgessner H., Gaber B., Samstag Y., Meuer S. A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate. Eur J Immunol. 1991 Oct;21(10):2469–2477. doi: 10.1002/eji.1830211025. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Shaw A. S., Chalupny J., Whitney J. A., Hammond C., Amrein K. E., Kavathas P., Sefton B. M., Rose J. K. Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol Cell Biol. 1990 May;10(5):1853–1862. doi: 10.1128/mcb.10.5.1853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shenoy-Scaria A. M., Gauen L. K., Kwong J., Shaw A. S., Lublin D. M. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins. Mol Cell Biol. 1993 Oct;13(10):6385–6392. doi: 10.1128/mcb.13.10.6385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Soula M., Rothhut B., Camoin L., Guillaume J. L., Strosberg D., Vorherr T., Burn P., Meggio F., Fischer S., Fagard R. Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck. J Biol Chem. 1993 Dec 25;268(36):27420–27427. [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. 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]
  43. 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]
  44. Veillette A., Abraham N., Caron L., Davidson D. The lymphocyte-specific tyrosine protein kinase p56lck. Semin Immunol. 1991 May;3(3):143–152. [PubMed] [Google Scholar]
  45. 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]
  46. Veillette A., Caron L., Fournel M., Pawson T. Regulation of the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck by the non-catalytic SH2 and SH3 domains. Oncogene. 1992 May;7(5):971–980. [PubMed] [Google Scholar]
  47. Veillette A., Davidson D. Src-related protein tyrosine kinases and T-cell receptor signalling. Trends Genet. 1992 Feb;8(2):61–66. doi: 10.1016/0168-9525(92)90351-4. [DOI] [PubMed] [Google Scholar]
  48. Veillette A., Foss F. M., Sausville E. A., Bolen J. B., Rosen N. Expression of the lck tyrosine kinase gene in human colon carcinoma and other non-lymphoid human tumor cell lines. Oncogene Res. 1987 Sep-Oct;1(4):357–374. [PubMed] [Google Scholar]
  49. Veillette A., Horak I. D., Bolen J. B. Post-translational alterations of the tyrosine kinase p56lck in response to activators of protein kinase C. Oncogene Res. 1988 May;2(4):385–401. [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. Voronova A. F., Sefton B. M. Expression of a new tyrosine protein kinase is stimulated by retrovirus promoter insertion. Nature. 1986 Feb 20;319(6055):682–685. doi: 10.1038/319682a0. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Weil R., Veillette A. Intramolecular and extramolecular mechanisms repress the catalytic function of p56lck in resting T-lymphocytes. J Biol Chem. 1994 Sep 9;269(36):22830–22838. [PubMed] [Google Scholar]
  55. 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]
  56. 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]

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