title: Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf-1 or B-Raf.
abstract: Extracellular signal-regulated kinase (ERK) activation pathways have been well characterized in a number of cell types but very few data are available for platelets. The thrombin-induced signaling pathway leading to ERK2 activation in platelets is largely uncharacterized. In this study, we investigated the kinases involved in thrombin-induced ERK2 activation in conditions of maximal ERK2 activation. We found that thrombin-induced mitogen-activated protein kinase/ERK kinase (MEK)1/2 activation was necessary for ERK2 phosphorylation. We obtained strong evidence that conventional protein kinase Cs (PKCs) and calcium are involved in thrombin-induced ERK2 activation. First, ERK2 and MEK1/2 phosphorylation was totally inhibited by low concentrations (1 microM) of RO318425, a specific inhibitor of conventional PKCs. Second, Ca(2+), from either intracellular pools or the extracellular medium, was necessary for ERK2 activation and conventional PKC activation, excluding the involvement of a new class of calcium-insensitive PKCs. Third, LY294002 and wortmannin had no significant effect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3-kinase (5 microM to 25 microM and 50 nM, respectively). This suggests that PI3-kinase was not necessary for ERK2 activation and therefore, that PI3-kinase-dependent atypical PKCs were not involved. Surprisingly, in contrast to proliferative cells, we found that the serine/threonine kinases Raf-1 and B-Raf were not an intermediate kinase between conventional PKCs and MEK1/2. After immunoprecipitation of Raf-1 and B-Raf, the basal glutathione S-transferase-MEK1 phosphorylation observed in resting platelets was not upregulated by thrombin and was still observed in the absence of anti-Raf-1 or anti-B-Raf antibodies. In these conditions, the in vitro cascade kinase assay did not detect any MEK activity. Thus in platelets, thrombin-induced ERK2 activation is activated by conventional PKCs independently of Raf-1 and B-Raf activation.
authors.affiliation: U348 INSERM, IFR 6 Circulation Lariboisière, Hôpital Lariboisière, 41 Bvd de la Chapelle, 75475 Cedex 10, Paris, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Nadal-Wollbold
authors.forename: Marc
authors.initials: M
authors.lastname: Pawlowski
authors.forename: Sylviane
authors.initials: S
authors.lastname: Lévy-Toledano
authors.forename: Eliane
authors.initials: E
authors.lastname: Berrou
authors.forename: Jean Philippe
authors.initials: JP
authors.lastname: Rosa
authors.forename: Marijke
authors.initials: M
authors.lastname: Bryckaert
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002417:Cattle; D004789:Enzyme Activation; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D013917:Thrombin