Activation of PRK1 by Phosphatidylinositol 4,5-Bisphosphate and Phosphatidylinositol 3,4,5-Trisphosphate

Abstract

As potential targets for polyphosphoinositides, activa- tion of protein kinase C (PKC) isotypes ( 1, , , ) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be acti- vated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) as well as phosphatidylinositol 3,4,5- trisphosphate (PtdIns-3,4,5-P3) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equipotent in activating PRK1, and, furthermore, soni- cated phosphatidylinositol (PtdIns) and phosphatidyl- serine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC- 1,- , and - were all activated by PtdIns-4,5-P2 and PtdIns- 3,4,5-P3 in detergent mixed micelles. The activation con- stants for PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were essen- tially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P2 and PtdIns- 3,4,5-P3 were found to be inhibited at 10 mM Mg2 and mimicked by high concentrations of inositol hexaphos- phate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.