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. 1993 Oct 1;295(Pt 1):41–48. doi: 10.1042/bj2950041

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

U Liebenhoff 1, D Brockmeier 1, P Presek 1
PMCID: PMC1134817  PMID: 7692843

Abstract

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.

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Selected References

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

  1. Ashby C. D., Walsh D. A. Purification and characterization of an inhibitor protein of cyclic AMP-dependent protein kinases. Methods Enzymol. 1974;38:350–358. doi: 10.1016/0076-6879(74)38051-2. [DOI] [PubMed] [Google Scholar]
  2. Bachelot C., Cano E., Grelac F., Saleun S., Druker B. J., Levy-Toledano S., Fischer S., Rendu F. Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors. Biochem J. 1992 Jun 15;284(Pt 3):923–928. doi: 10.1042/bj2840923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beemon K., Hunter T. Characterization of Rous sarcoma virus src gene products synthesized in vitro. J Virol. 1978 Nov;28(2):551–566. doi: 10.1128/jvi.28.2.551-566.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Brugge J. S., Erikson R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977 Sep 22;269(5626):346–348. doi: 10.1038/269346a0. [DOI] [PubMed] [Google Scholar]
  6. Cichowski K., McCormick F., Brugge J. S. p21rasGAP association with Fyn, Lyn, and Yes in thrombin-activated platelets. J Biol Chem. 1992 Mar 15;267(8):5025–5028. [PubMed] [Google Scholar]
  7. Clark E. A., Brugge J. S. Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets. Mol Cell Biol. 1993 Mar;13(3):1863–1871. doi: 10.1128/mcb.13.3.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  9. Collett M. S., Erikson E., Erikson R. L. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. doi: 10.1128/jvi.29.2.770-781.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dhar A., Shukla S. D. Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane. J Biol Chem. 1991 Oct 5;266(28):18797–18801. [PubMed] [Google Scholar]
  12. Eiseman E., Bolen J. B. src-related tyrosine protein kinases as signaling components in hematopoietic cells. Cancer Cells. 1990 Oct;2(10):303–310. [PubMed] [Google Scholar]
  13. Elmore M. A., Anand R., Horvath A. R., Kellie S. Tyrosine-specific phosphorylation of gpIIIa in platelet membranes. FEBS Lett. 1990 Sep 3;269(2):283–287. doi: 10.1016/0014-5793(90)81177-p. [DOI] [PubMed] [Google Scholar]
  14. Espino P. C., Harvey R., Schweickhardt R. L., White G. A., Smith A. E., Cheng S. H. The amino-terminal region of pp60c-src has a modulatory role and contains multiple sites of tyrosine phosphorylation. Oncogene. 1990 Mar;5(3):283–293. [PubMed] [Google Scholar]
  15. Feder D., Bishop J. M. Purification and enzymatic characterization of pp60c-src from human platelets. J Biol Chem. 1990 May 15;265(14):8205–8211. [PubMed] [Google Scholar]
  16. Ferrell J. E., Jr, Martin G. S. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol. 1988 Sep;8(9):3603–3610. doi: 10.1128/mcb.8.9.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Findik D., Presek P. Zn2+ enhances protein tyrosine kinase activity of human platelet membranes. FEBS Lett. 1988 Aug 1;235(1-2):51–56. doi: 10.1016/0014-5793(88)81232-8. [DOI] [PubMed] [Google Scholar]
  18. Findik D., Reuter C., Presek P. Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase. FEBS Lett. 1990 Mar 12;262(1):1–4. doi: 10.1016/0014-5793(90)80138-9. [DOI] [PubMed] [Google Scholar]
  19. Gentry L. E., Chaffin K. E., Shoyab M., Purchio A. F. Novel serine phosphorylation of pp60c-src in intact cells after tumor promoter treatment. Mol Cell Biol. 1986 Feb;6(2):735–738. doi: 10.1128/mcb.6.2.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Golden A., Brugge J. S. Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci U S A. 1989 Feb;86(3):901–905. doi: 10.1073/pnas.86.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Golden A., Nemeth S. P., Brugge J. S. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc Natl Acad Sci U S A. 1986 Feb;83(4):852–856. doi: 10.1073/pnas.83.4.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gould K. L., Hunter T. Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Mol Cell Biol. 1988 Aug;8(8):3345–3356. doi: 10.1128/mcb.8.8.3345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gould K. L., Woodgett J. R., Cooper J. A., Buss J. E., Shalloway D., Hunter T. Protein kinase C phosphorylates pp60src at a novel site. Cell. 1985 Oct;42(3):849–857. doi: 10.1016/0092-8674(85)90281-8. [DOI] [PubMed] [Google Scholar]
  24. Grondin P., Plantavid M., Sultan C., Breton M., Mauco G., Chap H. Interaction of pp60c-src, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets. J Biol Chem. 1991 Aug 25;266(24):15705–15709. [PubMed] [Google Scholar]
  25. Gutkind J. S., Lacal P. M., Robbins K. C. Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets. Mol Cell Biol. 1990 Jul;10(7):3806–3809. doi: 10.1128/mcb.10.7.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Horak I. D., Corcoran M. L., Thompson P. A., Wahl L. M., Bolen J. B. Expression of p60fyn in human platelets. Oncogene. 1990 Apr;5(4):597–602. [PubMed] [Google Scholar]
  27. Horvath A. R., Muszbek L., Kellie S. Translocation of pp60c-src to the cytoskeleton during platelet aggregation. EMBO J. 1992 Mar;11(3):855–861. doi: 10.1002/j.1460-2075.1992.tb05123.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huang M. M., Bolen J. B., Barnwell J. W., Shattil S. J., Brugge J. S. Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7844–7848. doi: 10.1073/pnas.88.17.7844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  30. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  31. Mitchell C. A., Jefferson A. B., Bejeck B. E., Brugge J. S., Deuel T. F., Majerus P. W. Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9396–9400. doi: 10.1073/pnas.87.23.9396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oda A., Druker B. J., Smith M., Salzman E. W. Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP. Am J Physiol. 1992 Mar;262(3 Pt 1):C701–C707. doi: 10.1152/ajpcell.1992.262.3.C701. [DOI] [PubMed] [Google Scholar]
  33. Okada M., Nakagawa H. A protein tyrosine kinase involved in regulation of pp60c-src function. J Biol Chem. 1989 Dec 15;264(35):20886–20893. [PubMed] [Google Scholar]
  34. Parise L. V., Criss A. B., Nannizzi L., Wardell M. R. Glycoprotein IIIa is phosphorylated in intact human platelets. Blood. 1990 Jun 15;75(12):2363–2368. [PubMed] [Google Scholar]
  35. Patschinsky T., Hunter T., Sefton B. M. Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus. J Virol. 1986 Jul;59(1):73–81. doi: 10.1128/jvi.59.1.73-81.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Presek P., Reuter C. Amiloride inhibits the protein tyrosine kinases associated with the cellular and the transforming src-gene products. Biochem Pharmacol. 1987 Sep 1;36(17):2821–2826. doi: 10.1016/0006-2952(87)90271-1. [DOI] [PubMed] [Google Scholar]
  37. Presek P., Reuter C., Findik D., Bette P. High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets. Biochim Biophys Acta. 1988 May 13;969(3):271–280. doi: 10.1016/0167-4889(88)90062-6. [DOI] [PubMed] [Google Scholar]
  38. Pumiglia K. M., Huang C. K., Feinstein M. B. Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets. Biochem Biophys Res Commun. 1990 Sep 14;171(2):738–745. doi: 10.1016/0006-291x(90)91208-a. [DOI] [PubMed] [Google Scholar]
  39. Pumiglia K. M., Lau L. F., Huang C. K., Burroughs S., Feinstein M. B. Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). Biochem J. 1992 Sep 1;286(Pt 2):441–449. doi: 10.1042/bj2860441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ralston R., Bishop J. M. The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7845–7849. doi: 10.1073/pnas.82.23.7845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ramamoorthy S., Balasubramanian A. S. Zn(2+)-dependent tyrosine phosphorylation of a 68-kDa protein and its differentiation from Mg(2+)-dependent tyrosine phosphorylation in sheep platelets. Arch Biochem Biophys. 1991 May 1;286(2):433–440. doi: 10.1016/0003-9861(91)90062-n. [DOI] [PubMed] [Google Scholar]
  42. Reuter C., Findik D., Presek P. Characterization of purified pp60c-src protein tyrosine kinase from human platelets. Eur J Biochem. 1990 Jun 20;190(2):343–350. doi: 10.1111/j.1432-1033.1990.tb15581.x. [DOI] [PubMed] [Google Scholar]
  43. Roth C. W., Richert N. D., Pastan I., Gottesman M. M. Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity. J Biol Chem. 1983 Sep 10;258(17):10768–10773. [PubMed] [Google Scholar]
  44. Schieven G., Martin G. S. Nonenzymatic phosphorylation of tyrosine and serine by ATP is catalyzed by manganese but not magnesium. J Biol Chem. 1988 Oct 25;263(30):15590–15593. [PubMed] [Google Scholar]
  45. Shattil S. J., Brugge J. S. Protein tyrosine phosphorylation and the adhesive functions of platelets. Curr Opin Cell Biol. 1991 Oct;3(5):869–879. doi: 10.1016/0955-0674(91)90062-4. [DOI] [PubMed] [Google Scholar]
  46. Smart J. E., Oppermann H., Czernilofsky A. P., Purchio A. F., Erikson R. L., Bishop J. M. Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src). Proc Natl Acad Sci U S A. 1981 Oct;78(10):6013–6017. doi: 10.1073/pnas.78.10.6013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Smilowitz H. M., Aramli L., Xu D., Epstein P. M. Phosphotyrosine phosphatase activity in human platelets. Life Sci. 1991;49(1):29–37. doi: 10.1016/0024-3205(91)90576-w. [DOI] [PubMed] [Google Scholar]

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