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

Abstract

As potential targets for polyphosphoinositides, activation 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 activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) either as pure sonicated lipids or in detergent 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, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 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-P 2 and PtdIns-3,4,5-P 3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P 2 and PtdIns-3,4,5-P 3 were essentially 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-P 2 and PtdIns-3,4,5-P 3 were found to be inhibited at 10 mM Mg 2؉ and mimicked by high concentrations of inositol hexaphosphate 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.The phosphatidylinositol 3-kinase family of lipid kinases are responsible for the phosphorylation of inositol lipids at the 3-OH position (reviewed in Ref. 1). In response to various agonists, phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) 1 accumulates and labeling studies suggest that this is the primary product and as such is the most likely second messenger candidate (2). While this remains an attractive hypothesis, in the absence of a defined intracellular target(s), the operation of the "PtdIns 3-kinase signaling pathway" will remain enigmatic.By their nature, lipid-dependent protein kinases are attractive candidates as targets for the postulated role of PtdIns-3,4,5-P 3 as a second messenger. Protein kinase C (PKC) isotypes constitute the major group of such enzymes, but several recent reports have indicated the existence of an additional class of lipid-activated protein kinases. These kinases, termed PKC-related kinases (PRKs) are closely homologous to PKC isotypes in the catalytic domain while retaining a distinct amino-terminal regulatory domain ((3, 4) see Fig. 6A). Unlike the PKCs which characteristically retain a cysteine-rich C1 domain responsible for effector binding, the PRKs do not encode a C1 domain but show two distinct conserved regulatory domains termed HR1 and HR2 (3). Consistent with the lack of a C1 domain, it has become clear that while the PRKs resemble PKCs in being activated by proteolysis, they differ from PKCs in not being activated by phorbol esters. However, like PKC, various fatty acids and phospholipids have bee...

Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

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Phosphatidylinositol 3-kinase-γ activates Bruton’s tyrosine kinase in concert with Src family kinases

“…It is conceivable that a PDK1-related enzyme could control Btk activity in an analogous manner. PI 3-kinase also activates a number of PKC isoforms in vitro (41)(42)(43)(44). Isoforms of PKC bind the Btk PH domain with high affinity (45,46).…”

Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation

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