Protein Kinase C of Human Platelets: Resolution of Ca(2+)-dependent and Independent Forms by Measuring Endogenous Phosphorylation.
This study explores a kinetic approach to distinguish Ca(2+)-dependent and independent forms of PKC activity in a cell-free system of human platelets. Incorporation of (32)P from [γ-(32)P] ATP into total proteins, in the presence or in the absence of histone IIIS, at various combinations of added lipids (diolein and phosphatidyl serine) and Ca(2+), fail to distinguish PKC from other kinases. Phosphorylation of the 40 kDa protein, a major and specific platelet PKC substrate resolved by SDS-PAGE, is completely dependent on the added lipids, allowing the determination of PKC and its two sub-forms: dependent and independent Ca(2+). The activities of these forms and their ratio are characteristics for an individual, while the inter-individual difference is much greater, with an overall average ratio of 2:1 (n = 9). The forms differ in sensitivity to staurosporine with IC(50) = 14.6 and 4.3 nM for the Ca(2+)-dependent and independent forms, respectively. Furthermore, cAMP at 1 µM inhibits selectively the Ca(2+)-dependent form by 45%. Okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, enhances at 1 µM the activity of the Ca(2+)-dependent form. It is concluded that the two PKC forms that are determined in the crude cell free system of human platelets by measuring the endogenous phosphorylation have distinct properties.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
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Koslovsky
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Livne