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 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged 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 were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.
Zhukova
E
E
Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA.
Sinnett-Smith
J
J
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D001839:Bombesin / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D004247:DNA / Q000096:biosynthesis; D051379:Mice; D009479:Neuropeptides / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000819:agonists; D011994:Recombinant Proteins / Q000096:biosynthesis; D014667:Vasopressins / Q000494:pharmacology*
D018797:Cell Cycle Proteins; D009479:Neuropeptides; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014667:Vasopressins; D015240:Phorbol 12,13-Dibutyrate; D004247:DNA; C089212:protein kinase D; D011493:Protein Kinase C; D001839:Bombesin