Protein Kinase C of Human Platelets: Resolution of Ca2+-dependent and Independent Forms by Measuring Endogenous Phosphorylation

Abstract

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.