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. 1996 Aug 15;318(Pt 1):207–212. doi: 10.1042/bj3180207

Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets.

A G Börsch-Haubold 1, R M Kramer 1, S P Watson 1
PMCID: PMC1217609  PMID: 8761473

Abstract

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol esterstimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 microM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST-MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.

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Selected References

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  1. Alessi D. R., Cohen P., Ashworth A., Cowley S., Leevers S. J., Marshall C. J. Assay and expression of mitogen-activated protein kinase, MAP kinase kinase, and Raf. Methods Enzymol. 1995;255:279–290. doi: 10.1016/s0076-6879(95)55031-3. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  3. Ambs P., Baccarini M., Fitzke E., Dieter P. Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2+ and phosphorylation. Biochem J. 1995 Oct 1;311(Pt 1):189–195. doi: 10.1042/bj3110189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Börsch-Haubold A. G., Kramer R. M., Watson S. P. Cytosolic phospholipase A2 is phosphorylated in collagen- and thrombin-stimulated human platelets independent of protein kinase C and mitogen-activated protein kinase. J Biol Chem. 1995 Oct 27;270(43):25885–25892. doi: 10.1074/jbc.270.43.25885. [DOI] [PubMed] [Google Scholar]
  5. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  6. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686–7689. doi: 10.1073/pnas.92.17.7686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Durstin M., Durstin S., Molski T. F., Becker E. L., Sha'afi R. I. Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3142–3146. doi: 10.1073/pnas.91.8.3142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fouda S. I., Molski T. F., Ashour M. S., Sha'afi R. I. Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2. Biochem J. 1995 Jun 15;308(Pt 3):815–822. doi: 10.1042/bj3080815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gonzalez F. A., Raden D. L., Davis R. J. Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem. 1991 Nov 25;266(33):22159–22163. [PubMed] [Google Scholar]
  10. Gould G. W., Cuenda A., Thomson F. J., Cohen P. The activation of distinct mitogen-activated protein kinase cascades is required for the stimulation of 2-deoxyglucose uptake by interleukin-1 and insulin-like growth factor-1 in KB cells. Biochem J. 1995 Nov 1;311(Pt 3):735–738. doi: 10.1042/bj3110735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halenda S. P., Zavoico G. B., Feinstein M. B. Phorbol esters and oleoyl acetoyl glycerol enhance release of arachidonic acid in platelets stimulated by Ca2+ ionophore A23187. J Biol Chem. 1985 Oct 15;260(23):12484–12491. [PubMed] [Google Scholar]
  12. Han J., Lee J. D., Bibbs L., Ulevitch R. J. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 1994 Aug 5;265(5173):808–811. doi: 10.1126/science.7914033. [DOI] [PubMed] [Google Scholar]
  13. Kocher M., Clemetson K. J. Staurosporine both activates and inhibits serine/threonine kinases in human platelets. Biochem J. 1991 Apr 15;275(Pt 2):301–306. doi: 10.1042/bj2750301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kramer R. M., Roberts E. F., Hyslop P. A., Utterback B. G., Hui K. Y., Jakubowski J. A. Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2. J Biol Chem. 1995 Jun 16;270(24):14816–14823. doi: 10.1074/jbc.270.24.14816. [DOI] [PubMed] [Google Scholar]
  15. Kramer R. M., Roberts E. F., Manetta J. V., Hyslop P. A., Jakubowski J. A. Thrombin-induced phosphorylation and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in human platelets. J Biol Chem. 1993 Dec 15;268(35):26796–26804. [PubMed] [Google Scholar]
  16. Kramer R. M., Roberts E. F., Strifler B. A., Johnstone E. M. Thrombin induces activation of p38 MAP kinase in human platelets. J Biol Chem. 1995 Nov 17;270(46):27395–27398. doi: 10.1074/jbc.270.46.27395. [DOI] [PubMed] [Google Scholar]
  17. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  18. Lazar D. F., Wiese R. J., Brady M. J., Mastick C. C., Waters S. B., Yamauchi K., Pessin J. E., Cuatrecasas P., Saltiel A. R. Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem. 1995 Sep 1;270(35):20801–20807. doi: 10.1074/jbc.270.35.20801. [DOI] [PubMed] [Google Scholar]
  19. Lin L. L., Lin A. Y., DeWitt D. L. Interleukin-1 alpha induces the accumulation of cytosolic phospholipase A2 and the release of prostaglandin E2 in human fibroblasts. J Biol Chem. 1992 Nov 25;267(33):23451–23454. [PubMed] [Google Scholar]
  20. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  21. Marshall C. J. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994 Feb;4(1):82–89. doi: 10.1016/0959-437x(94)90095-7. [DOI] [PubMed] [Google Scholar]
  22. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  23. Nemenoff R. A., Winitz S., Qian N. X., Van Putten V., Johnson G. L., Heasley L. E. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem. 1993 Jan 25;268(3):1960–1964. [PubMed] [Google Scholar]
  24. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  25. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  26. Pang L., Sawada T., Decker S. J., Saltiel A. R. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995 Jun 9;270(23):13585–13588. doi: 10.1074/jbc.270.23.13585. [DOI] [PubMed] [Google Scholar]
  27. Qiu Z. H., Leslie C. C. Protein kinase C-dependent and -independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2. J Biol Chem. 1994 Jul 29;269(30):19480–19487. [PubMed] [Google Scholar]
  28. Sa G., Murugesan G., Jaye M., Ivashchenko Y., Fox P. L. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem. 1995 Feb 3;270(5):2360–2366. doi: 10.1074/jbc.270.5.2360. [DOI] [PubMed] [Google Scholar]
  29. Siess W. Molecular mechanisms of platelet activation. Physiol Rev. 1989 Jan;69(1):58–178. doi: 10.1152/physrev.1989.69.1.58. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Waterman W. H., Sha'afi R. I. A mitogen-activated protein kinase independent pathway involved in the phosphorylation and activation of cytosolic phospholipase A2 in human neutrophils stimulated with tumor necrosis factor-alpha. Biochem Biophys Res Commun. 1995 Apr 6;209(1):271–278. doi: 10.1006/bbrc.1995.1499. [DOI] [PubMed] [Google Scholar]
  32. Xing M., Wilkins P. L., McConnell B. K., Mattera R. Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like HL60 cells. Linkage between impaired responses to agonists and absence of protein kinase C-dependent phosphorylation of cytosolic phospholipase A2. J Biol Chem. 1994 Jan 28;269(4):3117–3124. [PubMed] [Google Scholar]
  33. Xu X. X., Rock C. O., Qiu Z. H., Leslie C. C., Jackowski S. Regulation of cytosolic phospholipase A2 phosphorylation and eicosanoid production by colony-stimulating factor 1. J Biol Chem. 1994 Dec 16;269(50):31693–31700. [PubMed] [Google Scholar]

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