Regulation of protein kinase C δ by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes

Abstract

Protein kinase C (PKC) delta; is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCδ for activation, it could be regulatory. We examined the regulation of PKCδ in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCδ(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCδ to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCδ subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, ∼25% of PKCδ lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCδ activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCδ translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCδ and the increase in phosphatidylserine/PMA- independent activity persisted after PKCδ retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCδ becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.