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. 1994 Jul 1;301(Pt 1):41–47. doi: 10.1042/bj3010041

Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

M M Davidson 1, R J Haslam 1
PMCID: PMC1137140  PMID: 8037689

Abstract

In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding protein or Ca2+.

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Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Coorssen J. R., Davidson M. M., Haslam R. J. Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S. Cell Regul. 1990 Dec;1(13):1027–1041. doi: 10.1091/mbc.1.13.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coorssen J. R., Haslam R. J. GTP gamma S and phorbol ester act synergistically to stimulate both Ca(2+)-independent secretion and phospholipase D activity in permeabilized human platelets. Inhibition by BAPTA and analogues. FEBS Lett. 1993 Jan 25;316(2):170–174. doi: 10.1016/0014-5793(93)81209-i. [DOI] [PubMed] [Google Scholar]
  5. Daniel J. L., Molish I. R., Rigmaiden M., Stewart G. Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response. J Biol Chem. 1984 Aug 10;259(15):9826–9831. [PubMed] [Google Scholar]
  6. Erdödi F., Csortos C., Sparks L., Murányi A., Gergely P. Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets. Arch Biochem Biophys. 1992 Nov 1;298(2):682–687. doi: 10.1016/0003-9861(92)90466-a. [DOI] [PubMed] [Google Scholar]
  7. Fenner C., Traut R. R., Mason D. T., Wikman-Coffelt J. Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal Biochem. 1975 Feb;63(2):595–602. doi: 10.1016/0003-2697(75)90386-3. [DOI] [PubMed] [Google Scholar]
  8. Haslam R. J., Davidson M. M. Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets. Evidence of a role for a GTP-binding protein in platelet activation. FEBS Lett. 1984 Aug 20;174(1):90–95. doi: 10.1016/0014-5793(84)81084-4. [DOI] [PubMed] [Google Scholar]
  9. Haslam R. J., Davidson M. M. Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation. Biochem J. 1984 Sep 1;222(2):351–361. doi: 10.1042/bj2220351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haslam R. J., Lynham J. A., Fox J. E. Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets. Biochem J. 1979 Feb 15;178(2):397–406. doi: 10.1042/bj1780397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haslam R. J., Lynham J. A. Relationship between phosphorylation of blood platelet proteins and secretion of platelet granule constituents. I. Effects of different aggregating agents. Biochem Biophys Res Commun. 1977 Jul 25;77(2):714–722. doi: 10.1016/s0006-291x(77)80037-5. [DOI] [PubMed] [Google Scholar]
  12. Higashihara M., Takahata K., Kurokawa K., Ikebe M. The inhibitory effects of okadaic acid on platelet function. FEBS Lett. 1992 Jul 28;307(2):206–210. doi: 10.1016/0014-5793(92)80768-c. [DOI] [PubMed] [Google Scholar]
  13. Imaoka T., Lynham J. A., Haslam R. J. Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. J Biol Chem. 1983 Sep 25;258(18):11404–11414. [PubMed] [Google Scholar]
  14. Kaibuchi K., Takai Y., Sawamura M., Hoshijima M., Fujikura T., Nishizuka Y. Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J Biol Chem. 1983 Jun 10;258(11):6701–6704. [PubMed] [Google Scholar]
  15. Kamps M. P., Sefton B. M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 1989 Jan;176(1):22–27. doi: 10.1016/0003-2697(89)90266-2. [DOI] [PubMed] [Google Scholar]
  16. Kawamoto S., Bengur A. R., Sellers J. R., Adelstein R. S. In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C. J Biol Chem. 1989 Feb 5;264(4):2258–2265. [PubMed] [Google Scholar]
  17. Kitazawa T., Masuo M., Somlyo A. P. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9307–9310. doi: 10.1073/pnas.88.20.9307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lerea K. M. Thrombin-induced effects are selectively inhibited following treatment of intact human platelets with okadaic acid. Biochemistry. 1991 Jul 16;30(28):6819–6824. doi: 10.1021/bi00242a003. [DOI] [PubMed] [Google Scholar]
  19. Lyons R. M., Stanford N., Majerus P. W. Thrombin-induced protein phosphorylation in human platelets. J Clin Invest. 1975 Oct;56(4):924–936. doi: 10.1172/JCI108172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matsuzaki F., Matsumoto S., Yahara I., Yonezawa N., Nishida E., Sakai H. Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence. J Biol Chem. 1988 Aug 15;263(23):11564–11568. [PubMed] [Google Scholar]
  21. Morgan T. E., Lockerbie R. O., Minamide L. S., Browning M. D., Bamburg J. R. Isolation and characterization of a regulated form of actin depolymerizing factor. J Cell Biol. 1993 Aug;122(3):623–633. doi: 10.1083/jcb.122.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moriyama K., Nishida E., Yonezawa N., Sakai H., Matsumoto S., Iida K., Yahara I. Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA. J Biol Chem. 1990 Apr 5;265(10):5768–5773. [PubMed] [Google Scholar]
  23. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  24. Naka M., Nishikawa M., Adelstein R. S., Hidaka H. Phorbol ester-induced activation of human platelets is associated with protein kinase C phosphorylation of myosin light chains. Nature. 1983 Dec 1;306(5942):490–492. doi: 10.1038/306490a0. [DOI] [PubMed] [Google Scholar]
  25. Nishida E., Maekawa S., Sakai H. Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin. Biochemistry. 1984 Oct 23;23(22):5307–5313. doi: 10.1021/bi00317a032. [DOI] [PubMed] [Google Scholar]
  26. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  27. Ogawa K., Tashima M., Yumoto Y., Okuda T., Sawada H., Okuma M., Maruyama Y. Coding sequence of human placenta cofilin cDNA. Nucleic Acids Res. 1990 Dec 11;18(23):7169–7169. doi: 10.1093/nar/18.23.7169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohta Y., Nishida E., Sakai H., Miyamoto E. Dephosphorylation of cofilin accompanies heat shock-induced nuclear accumulation of cofilin. J Biol Chem. 1989 Sep 25;264(27):16143–16148. [PubMed] [Google Scholar]
  29. Ono S., Abe H., Nagaoka R., Obinata T. Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells. J Muscle Res Cell Motil. 1993 Apr;14(2):195–204. doi: 10.1007/BF00115454. [DOI] [PubMed] [Google Scholar]
  30. Painter R. G., Ginsberg M. H. Centripetal myosin redistribution in thrombin-stimulated platelets. Relationship to platelet Factor 4 secretion. Exp Cell Res. 1984 Nov;155(1):198–212. doi: 10.1016/0014-4827(84)90781-x. [DOI] [PubMed] [Google Scholar]
  31. Siffert W., Siffert G., Scheid P. Activation of Na+/H+ exchange in human platelets stimulated by thrombin and a phorbol ester. Biochem J. 1987 Jan 1;241(1):301–303. doi: 10.1042/bj2410301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tallant E. A., Wallace R. W. Characterization of a calmodulin-dependent protein phosphatase from human platelets. J Biol Chem. 1985 Jun 25;260(12):7744–7751. [PubMed] [Google Scholar]
  33. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tyers M., Rachubinski R. A., Stewart M. I., Varrichio A. M., Shorr R. G., Haslam R. J., Harley C. B. Molecular cloning and expression of the major protein kinase C substrate of platelets. Nature. 1988 Jun 2;333(6172):470–473. doi: 10.1038/333470a0. [DOI] [PubMed] [Google Scholar]
  35. Wagner P. D., Vu N. D. Regulation of norepinephrine secretion in permeabilized PC12 cells by Ca2(+)-stimulated phosphorylation. Effects of protein phosphatases and phosphatase inhibitors. J Biol Chem. 1990 Jun 25;265(18):10352–10357. [PubMed] [Google Scholar]
  36. Walker T. R., Watson S. P. Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C. Br J Pharmacol. 1992 Mar;105(3):627–631. doi: 10.1111/j.1476-5381.1992.tb09030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Walker T. R., Watson S. P. Synergy between Ca2+ and protein kinase C is the major factor in determining the level of secretion from human platelets. Biochem J. 1993 Jan 1;289(Pt 1):277–282. doi: 10.1042/bj2890277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamanishi J., Takai Y., Kaibuchi K., Sano K., Castagna M., Nishizuka Y. Synergistic functions of phorbol ester and calcium in serotonin release from human platelets. Biochem Biophys Res Commun. 1983 Apr 29;112(2):778–786. doi: 10.1016/0006-291x(83)91529-2. [DOI] [PubMed] [Google Scholar]
  39. Yonezawa N., Nishida E., Iida K., Yahara I., Sakai H. Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides. J Biol Chem. 1990 May 25;265(15):8382–8386. [PubMed] [Google Scholar]
  40. Yonezawa N., Nishida E., Maekawa S., Sakai H. Studies on the interaction between actin and cofilin purified by a new method. Biochem J. 1988 Apr 1;251(1):121–127. doi: 10.1042/bj2510121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yonezawa N., Nishida E., Sakai H. pH control of actin polymerization by cofilin. J Biol Chem. 1985 Nov 25;260(27):14410–14412. [PubMed] [Google Scholar]

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