ACTIVATION OF PRK1 BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AND PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE - A COMPARISON WITH PROTEIN-KINASE-C ISOTYPES

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (beta(1), epsilon, zeta, eta) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bispho sphate (PtdIns 4,5-P-2) as well as phosphatidylinositol 3,4,5-trisphos phate (PtdIns-3,4,5-P-3) either as pure sonicated lipids or in deterge nt mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P-2 and PtdIns-3,4,5-P-3 were equipotent in activating PRK1, and, furtherm ore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (P tdSer) were equally effective. In detergent mixed micelles, PtdIns-4,B -P-2 and PtdIns-3,4,5-P-3 also showed a similar potency, but PtdIns an d PtdSer were 10-fold less effective in this assay. Similarly, PKC-bet a(1), -epsilon, and -eta were all activated by PtdIns-4,5-P-2 and PtdI ns-3,4,5-P-3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P, and PtdIns-3,4,5-P, were essentially the same for all t he kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P-2 and Ptd Ins-3,4,5-P-3 were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfat e. The similar responses of these two classes of lipid-activated prote in kinase to these phosphoinositides are discussed in light of their p otential roles as second messengers.