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Characterization of protein kinases in platelets Samiei, Mitra

Abstract

Numerous studies of cell division control have demonstrated the importance of cell cycleregulated protein kinase networks that are modulated by altering phosphorylation state. To determine whether such kinase networks only function in cell cycle regulation or also function in terminally differentiated cells, studies were undertaken to identify and characterize protein kinases of blood platelets and to monitor kinase activation in platelets activated by phorbol myristate acetate (PMA), platelet activation factor (PAF) or thrombin. Protein kinase C (PKC) was reported to lose calcium- and phospholipid-dependence as a result of proteolysis. The activation state of platelet PKC was studied by anion exchange chromatography of the extracts of PMA-treated platelets. Length of PMA exposure correlated with the disappearance of peak I and II PKC forms and the appearance of new PKC species (peaks III and IV) with lower calcium and lipid dependence in histone HI phosphorylation assays. Alkaline phosphatase treatment of peak IV/V PKC forms led to the appearance of the peak I and III forms, implicating protein phosphorylation in the interconversion of PKC forms. A 32 kDa, cdc-2-like protein kinase in platelet cytosol extracts was identified by its affinity for p13[sub suc1] and its immunoreactivity with anti-PSTAIRE antibody; it associated with cyclin A. PAF or thrombin augmented the kinase activity of the p34[sub cdc2]-cyclin A complex. The stimulation of p34[sup cdc2] was near maximal within one minute exposure to either agonist. Column chromatography and immunoblotting studies revealed both p42[sup erk2] and p44[sup erk1] -like MAP kinases, but only p42eHc2 underwent tyrosyl phosphorylation. The S6 kinases p90[sup rsk] and p70[sup sk6] were found in fractions containing elevated S6 phosphotransferase activity. PMA-activation of MBP and S6 kinases occurred through a PKC-dependent pathway of MAP kinase activation. However, activation by PAF utilized both PKC-dependent and independent pathways. Further upstream, MAP kinase kinase (MEK1) was stimulated by PMA, or thrombin. Subsequently, Rafl, an upstream component of MEK1 signalling, was identified. Using immunoprecipitation and affinity chromotography, interaction and complex formation was detected among Rafl, MEK1, ERK1 and ERK2. These data were the first to demonstrate a complex MAP kinase-dependent signalling network in platelets. These findings provide evidence to support the hypothesis that a protein kinase cascade that has been strongly linked to the control of cell cycle progression in somatic cells has become adapted for alternative functions in terminally differentiated cells.

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