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 W WM) of RO318425, a speci¢c 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 signi¢cant e¡ect on ERK2 activation, even at concentrations that inhibit phosphatidylinositol (PI)3-kinase (5 W WM to 25 W WM 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. ß

Thrombin Induces Nitric-oxide Synthase via Gα12/13-coupled Protein Kinase C-dependent I-κBα Phosphorylation and JNK-mediated I-κBα Degradation

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Platelet MEKK3 regulates arterial thrombosis and myocardial infarct expansion in mice

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Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways

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