PROTEIN-KINASE-D (PKD) ACTIVATION IN INTACT-CELLS THROUGH A PROTEIN-KINASE C-DEPENDENT SIGNAL-TRANSDUCTION PATHWAY

Protein kinase D (PKD) is a serine/threonine protein kinase that is di rectly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol es ters, membrane-permeant diacylglycerol and serum growth factors rapidl y induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 an d Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct . PKD activation was maintained during cell disruption and immunopurif ication and was associated with an electrophoretic mobility shift and enhanced P-32 incorporation into the enzyme, but was reversed by treat ment with alkaline phosphatase. PKD was activated, deactivated and rea ctivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Po 31-8220. In contrast, t hese compounds did not inhibit PKD activity when added directly in vit ro. Co-transfection of PKD with constitutively activated mutants of PK Cs showed that PKC epsilon and eta but not PKC zeta strongly induced P KD activation in COS-7 cells. Thus, our results indicate that PKD is a ctivated in living cells through a PKC-dependent signal transduction p athway.