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. 1992 Apr 15;89(8):3343–3346. doi: 10.1073/pnas.89.8.3343

Ras GTPase-activating protein: a substrate and a potential binding protein of the protein-tyrosine kinase p56lck.

K E Amrein 1, N Flint 1, B Panholzer 1, P Burn 1
PMCID: PMC48863  PMID: 1565625

Abstract

Ras GTPase-activating protein (GAP) is a cytoplasmic factor that regulates the GTPase activity of p21ras. Phosphorylation of GAP on tyrosine has recently been reported by several groups and may be an important step in linking signaling pathways involving p21ras and protein-tyrosine kinases. p56lck, a src-like protein-tyrosine kinase, seems to play a crucial role in T-cell development and T-cell activation. However, the molecular mechanisms of T-cell signaling involving p56lck and the substrates of p56lck have not yet been identified. To test whether GAP is a substrate of p56lck, in vitro kinase reactions were performed with purified, recombinant GAP and p56lck. We found that GAP became specifically phosphorylated on tyrosine within one tryptic peptide. Furthermore, coimmunoprecipitation studies provided evidence that the tyrosine-phosphorylated form of GAP is bound to p56lck. These results suggest that in T cells the function of GAP might be regulated through its phosphorylation on tyrosine and binding to the protein-tyrosine kinase p56lck.

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Selected References

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

  1. 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]
  2. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Brott B. K., Decker S., Shafer J., Gibbs J. B., Jove R. GTPase-activating protein interactions with the viral and cellular Src kinases. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):755–759. doi: 10.1073/pnas.88.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Choudhury G. G., Sylvia V. L., Pfeifer A., Wang L. M., Smith E. A., Sakaguchi A. Y. Human colony stimulating factor-1 receptor activates the C-raf-1 proto-oncogene kinase. Biochem Biophys Res Commun. 1990 Oct 15;172(1):154–159. doi: 10.1016/s0006-291x(05)80186-x. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., Esch F. S., Taylor S. S., Hunter T. Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro. J Biol Chem. 1984 Jun 25;259(12):7835–7841. [PubMed] [Google Scholar]
  7. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  8. Ellis C., Liu X. Q., Anderson D., Abraham N., Veillette A., Pawson T. Tyrosine phosphorylation of GAP and GAP-associated proteins in lymphoid and fibroblast cells expressing lck. Oncogene. 1991 Jun;6(6):895–901. [PubMed] [Google Scholar]
  9. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  10. Escobedo J. A., Navankasattusas S., Kavanaugh W. M., Milfay D., Fried V. A., Williams L. T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. doi: 10.1016/0092-8674(91)90409-r. [DOI] [PubMed] [Google Scholar]
  11. Halenbeck R., Crosier W. J., Clark R., McCormick F., Koths K. Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources. J Biol Chem. 1990 Dec 15;265(35):21922–21928. [PubMed] [Google Scholar]
  12. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  13. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Liu X. Q., Pawson T. The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains. Mol Cell Biol. 1991 May;11(5):2511–2516. doi: 10.1128/mcb.11.5.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Luo K. X., Sefton B. M. Cross-linking of T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site. Mol Cell Biol. 1990 Oct;10(10):5305–5313. doi: 10.1128/mcb.10.10.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Matsuda M., Mayer B. J., Fukui Y., Hanafusa H. Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science. 1990 Jun 22;248(4962):1537–1539. doi: 10.1126/science.1694307. [DOI] [PubMed] [Google Scholar]
  23. Mayer B. J., Hanafusa H. Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2638–2642. doi: 10.1073/pnas.87.7.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989 Jan 13;56(1):5–8. doi: 10.1016/0092-8674(89)90976-8. [DOI] [PubMed] [Google Scholar]
  25. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  26. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  27. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  28. Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
  29. Pawson T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene. 1988 Nov;3(5):491–495. [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Veillette A., Sleckman B. P., Ratnofsky S., Bolen J. B., Burakoff S. J. The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck. Eur J Immunol. 1990 Jun;20(6):1397–1400. doi: 10.1002/eji.1830200628. [DOI] [PubMed] [Google Scholar]
  37. Voronova A. F., Buss J. E., Patschinsky T., Hunter T., Sefton B. M. Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells. Mol Cell Biol. 1984 Dec;4(12):2705–2713. doi: 10.1128/mcb.4.12.2705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]

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