Platelet ERK2 activation by thrombin is dependent on calcium and conventional protein kinases C but not Raf‐1 or B‐Raf

FEBS Letters

Nadal-Wollbold, Florence¹  Pawlowski, Marc¹  Lévy-Toledano, Sylviane¹  Rosa, Jean-Philippe¹  Berrou, Eliane¹  Bryckaert, Marijke¹ 

[1] U348 INSERM, IFR 6 Circulation Lariboisière, Hôpital Lariboisière, 41 Bvd de la Chapelle, 75475 Paris Cedex 10, France

关键词: Platelet;    Extracellular signal-regulated kinase-2;    Conventional protein kinase C;    Raf;    MAPK;    mitogen-activated protein kinase;    ERK;    extracellular signal-regulated kinase;    MEK;    MAPK/ERK kinase;    JNK;    C-Jun N-terminal kinase;    PKC;    protein kinase C;    RGDS;    Arg-Gly-Asp-Ser;    PMA;    phorbol myristate acetate;    TPO;    thrombopoietin;   

DOI  :  10.1016/S0014-5793(02)03587-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 μM) of RO318425, a specific inhibitor of conventional PKCs. Second, Ca²⁺, 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 μM to 25 μM 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.

【 授权许可】

Unknown