Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets.
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.
Börsch-Haubold
A G
AG
Department of Pharmacology, University of Oxford, UK.
Kramer
R M
RM
Watson
S P
SP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003094:Collagen / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007476:Ionophores; D047428:Protein Kinase Inhibitors; D017366:Serotonin Receptor Agonists; D012701:Serotonin; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D000255:Adenosine Triphosphate; D003094:Collagen; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D007213:Indomethacin