Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cell s potentiates the proliferative response to either the G protein-coupled re ceptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cell s stably overexpressing PKD, cultures of these cells were infected with ret rovirus encoding murine PKD and green fluorescent protein (GFP) expressed a s two separate proteins translated from the same mRNA. GFP was used as a ma rker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 ce lls was dramatically activated by cell treatment with bombesin or PDBu as j udged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation wer e prevented by exposure of the cells to protein kinase C-specific inhibitor s. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cell s overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulat ion of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results d emonstrate that overexpression of PKD selectively potentiates mitogenesis i nduced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.