Platelet-derived growth factor activates protein kinase Cε through redundant and independent signaling pathways involving phospholipase Cγ or phosphatidylinositol 3-kinase

Abstract

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKCε upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR 'add-back' mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3- kinase (PI 3-kinase) caused translocation of PKCε from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase Cγ (PLCγ), but not PI 3-kinase, also caused the PDGF- dependent translocation of PKCε. The translocation of PKCε upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKCε was further confirmed in terms of PKCε-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)luciferase reporter. Further, purified PKCε was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-tris-phosphate. These results clearly demonstrate that PKCε is activated through redundant and independent signaling pathways which most likely involve PLCγ or PI 3- kinase in vivo and that PKCε is one of the downstream mediators of PI 3- kinase whose downstream targets remain to be identified.