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. 2000 Oct 15;351(Pt 2):429–437.

Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

J A Rosado 1, D Graves 1, S O Sage 1
PMCID: PMC1221379  PMID: 11023829

Abstract

We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets.

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Selected References

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  1. Alvarez J., Montero M., García-Sancho J. Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx. Biochem J. 1991 Feb 15;274(Pt 1):193–197. doi: 10.1042/bj2740193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babnigg G., Bowersox S. R., Villereal M. L. The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels. J Biol Chem. 1997 Nov 21;272(47):29434–29437. doi: 10.1074/jbc.272.47.29434. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Capacitative calcium entry. Biochem J. 1995 Nov 15;312(Pt 1):1–11. doi: 10.1042/bj3120001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bertagnolli M. E., Hudson L. A., Stetsenko G. Y. Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets. Biochem Biophys Res Commun. 1999 Jul 14;260(3):790–798. doi: 10.1006/bbrc.1999.0985. [DOI] [PubMed] [Google Scholar]
  5. Bird G. S., Putney J. W., Jr Inhibition of thapsigargin-induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement of a small G-protein in capacitative calcium entry. J Biol Chem. 1993 Oct 15;268(29):21486–21488. [PubMed] [Google Scholar]
  6. Camello C., Pariente J. A., Salido G. M., Camello P. J. Sequential activation of different Ca2+ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells. J Physiol. 1999 Apr 15;516(Pt 2):399–408. doi: 10.1111/j.1469-7793.1999.0399v.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cavallini L., Coassin M., Alexandre A. Two classes of agonist-sensitive Ca2+ stores in platelets, as identified by their differential sensitivity to 2,5-di-(tert-butyl)-1,4-benzohydroquinone and thapsigargin. Biochem J. 1995 Sep 1;310(Pt 2):449–452. doi: 10.1042/bj3100449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chintala S. K., Kyritsis A. P., Mohan P. M., Mohanam S., Sawaya R., Gokslan Z., Yung W. K., Steck P., Uhm J. H., Aggarwal B. B. Altered actin cytoskeleton and inhibition of matrix metalloproteinase expression by vanadate and phenylarsine oxide, inhibitors of phosphotyrosine phosphatases: modulation of migration and invasion of human malignant glioma cells. Mol Carcinog. 1999 Dec;26(4):274–285. [PubMed] [Google Scholar]
  9. 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]
  10. Clark E. A., Shattil S. J., Brugge J. S. Regulation of protein tyrosine kinases in platelets. Trends Biochem Sci. 1994 Nov;19(11):464–469. doi: 10.1016/0968-0004(94)90131-7. [DOI] [PubMed] [Google Scholar]
  11. Doni M. G., Cavallini L., Alexandre A. Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides. Biochem J. 1994 Oct 15;303(Pt 2):599–605. doi: 10.1042/bj3030599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fasolato C., Hoth M., Penner R. A GTP-dependent step in the activation mechanism of capacitative calcium influx. J Biol Chem. 1993 Oct 5;268(28):20737–20740. [PubMed] [Google Scholar]
  13. Fleming I., Fisslthaler B., Busse R. Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. J Biol Chem. 1996 May 3;271(18):11009–11015. doi: 10.1074/jbc.271.18.11009. [DOI] [PubMed] [Google Scholar]
  14. Fox J. E., Lipfert L., Clark E. A., Reynolds C. C., Austin C. D., Brugge J. S. On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton. J Biol Chem. 1993 Dec 5;268(34):25973–25984. [PubMed] [Google Scholar]
  15. Fukui Y., O'Brien M. C., Hanafusa H. Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix. Mol Cell Biol. 1991 Mar;11(3):1207–1213. doi: 10.1128/mcb.11.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. García L. J., Rosado J. A., González A., Jensen R. T. Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho. Biochem J. 1997 Oct 15;327(Pt 2):461–472. doi: 10.1042/bj3270461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Grimaldi M., Favit A., Alkon D. L. cAMP-induced cytoskeleton rearrangement increases calcium transients through the enhancement of capacitative calcium entry. J Biol Chem. 1999 Nov 19;274(47):33557–33564. doi: 10.1074/jbc.274.47.33557. [DOI] [PubMed] [Google Scholar]
  19. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54. doi: 10.1146/annurev.cb.10.110194.000335. [DOI] [PubMed] [Google Scholar]
  20. Hartwig J. H., Kung S., Kovacsovics T., Janmey P. A., Cantley L. C., Stossel T. P., Toker A. D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate. J Biol Chem. 1996 Dec 20;271(51):32986–32993. doi: 10.1074/jbc.271.51.32986. [DOI] [PubMed] [Google Scholar]
  21. Holda J. R., Blatter L. A. Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments. FEBS Lett. 1997 Feb 17;403(2):191–196. doi: 10.1016/s0014-5793(97)00051-3. [DOI] [PubMed] [Google Scholar]
  22. Hoth M., Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992 Jan 23;355(6358):353–356. doi: 10.1038/355353a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Molony L., Ng-Sikorski J., Hellberg C., Andersson T. Inhibitors of farnesyl and geranylgeranyl methyltransferases prevent beta 2 integrin-induced actin polymerization without affecting beta 2 integrin-induced Ca2+ signaling in neutrophils. Biochem Biophys Res Commun. 1996 Jun 25;223(3):612–617. doi: 10.1006/bbrc.1996.0943. [DOI] [PubMed] [Google Scholar]
  26. Pandol S. J., Schoeffield-Payne M. S. Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell. J Biol Chem. 1990 Aug 5;265(22):12846–12853. [PubMed] [Google Scholar]
  27. Patterson R. L., van Rossum D. B., Gill D. L. Store-operated Ca2+ entry: evidence for a secretion-like coupling model. Cell. 1999 Aug 20;98(4):487–499. doi: 10.1016/s0092-8674(00)81977-7. [DOI] [PubMed] [Google Scholar]
  28. Patya M., Lander H. M., Novogrodsky A. Hemin-induced cap formation in lymphocytes: inhibition by protein tyrosine kinase inhibitors. Exp Cell Res. 1993 Mar;205(1):159–164. doi: 10.1006/excr.1993.1070. [DOI] [PubMed] [Google Scholar]
  29. Randriamampita C., Tsien R. Y. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature. 1993 Aug 26;364(6440):809–814. doi: 10.1038/364809a0. [DOI] [PubMed] [Google Scholar]
  30. Rosado J. A., Jenner S., Sage S. O. A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling. J Biol Chem. 2000 Mar 17;275(11):7527–7533. doi: 10.1074/jbc.275.11.7527. [DOI] [PubMed] [Google Scholar]
  31. Rosado J. A., Sage S. O. Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton. Biochem J. 2000 Apr 1;347(Pt 1):183–192. [PMC free article] [PubMed] [Google Scholar]
  32. Rosado J. A., Sage S. O. Phosphoinositides are required for store-mediated calcium entry in human platelets. J Biol Chem. 2000 Mar 31;275(13):9110–9113. doi: 10.1074/jbc.275.13.9110. [DOI] [PubMed] [Google Scholar]
  33. Rozengurt E. Convergent signalling in the action of integrins, neuropeptides, growth factors and oncogenes. Cancer Surv. 1995;24:81–96. [PubMed] [Google Scholar]
  34. Sage S. O., Reast R., Rink T. J. ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store. Biochem J. 1990 Feb 1;265(3):675–680. doi: 10.1042/bj2650675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sargeant P., Farndale R. W., Sage S. O. ADP- and thapsigargin-evoked Ca2+ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets. J Biol Chem. 1993 Aug 25;268(24):18151–18156. [PubMed] [Google Scholar]
  36. Sargeant P., Farndale R. W., Sage S. O. The tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and genistein reduce thrombin-evoked tyrosine phosphorylation and Ca2+ entry in human platelets. FEBS Lett. 1993 Jan 11;315(3):242–246. doi: 10.1016/0014-5793(93)81172-v. [DOI] [PubMed] [Google Scholar]
  37. Schlessinger J. New roles for Src kinases in control of cell survival and angiogenesis. Cell. 2000 Feb 4;100(3):293–296. doi: 10.1016/s0092-8674(00)80664-9. [DOI] [PubMed] [Google Scholar]
  38. Stenberg P. E., Pestina T. I., Barrie R. J., Jackson C. W. The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets. Blood. 1997 Apr 1;89(7):2384–2393. [PubMed] [Google Scholar]
  39. Symons M. Rho family GTPases: the cytoskeleton and beyond. Trends Biochem Sci. 1996 May;21(5):178–181. [PubMed] [Google Scholar]
  40. Vostal J. G., Jackson W. L., Shulman N. R. Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. J Biol Chem. 1991 Sep 5;266(25):16911–16916. [PubMed] [Google Scholar]
  41. Waltenberger J., Uecker A., Kroll J., Frank H., Mayr U., Bjorge J. D., Fujita D., Gazit A., Hombach V., Levitzki A. A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling. Circ Res. 1999 Jul 9;85(1):12–22. doi: 10.1161/01.res.85.1.12. [DOI] [PubMed] [Google Scholar]
  42. Yule D. I., Kim E. T., Williams J. A. Tyrosine kinase inhibitors attenuate "capacitative" Ca2+ influx in rat pancreatic acinar cells. Biochem Biophys Res Commun. 1994 Aug 15;202(3):1697–1704. doi: 10.1006/bbrc.1994.2130. [DOI] [PubMed] [Google Scholar]
  43. Zhang F. L., Casey P. J. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996;65:241–269. doi: 10.1146/annurev.bi.65.070196.001325. [DOI] [PubMed] [Google Scholar]

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