Protein Kinase D Mediates Mitogenic Signaling By Gq-Coupled Receptors Through PKC-Independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

JAMES SINNETT-SMITH; RODRIGO JACAMO; ROBERT KUI; OSVALDO REY; RICHARD WALDRON; ENRIQUE ROZENGURT

Congreso; Digestive Diseaqses Week; 2009

AGA

Background: Protein kinase D (PKD) has been implicated in the regulation of multiple biological processes including DNA synthesis and cell proliferation in response to gastrointes- tinal peptides. Previous studies demonstrated the existence of a rapidly activated PKC-PKD protein kinase cascade(s), and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector. Recently, we extended previous models of PKD regulation by identifying a novel mechanism that leads to late, PKC-independ- ent PKD activation. However, the precise role of biphasic PKD activation in cell proliferation and ERK activation in response to GPCR agonists and other stimuli has not been elucidated. Aim: In order to elucidate the biological significance of biphasic PKD activation, we examined the mechanisms of early and late PKD activation in Swiss 3T3 cells, a well-established model system in which PKD signaling promotes long-term biological responses, including ERK signaling and mitogenesis. Results: Treatment of Swiss 3T3 cells overexpressing PKD with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (10 min) PKD catalytic activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 120 min). Similar results were obtained in cells stimulated with vasopressin, i.e. via a different Gq-coupled receptor agonist. These results imply that PKD activation induced via endogenously expressed bombesin receptors is medi- ated by early PKC-dependent and late PKC-independent mechanisms. To gain insight into the transphosphorylation and /or autophosphorylation mechanisms involved, we used West- ern blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser744 and Ser748. Our results indicate that transphosphorylation is a major mechanism targeting Ser744 and autophosphorylation is a predominant mechanism for Ser748 in cells stimulated with GPCR agonists. The pathways leading to the phosphoryl- ation of these residues depend on the time of GPCR stimulation. PKD phosphorylation on Ser744 depended on PKC at early times of bombesin stimulation but was PKC-independent at longer times of bombesin stimulation. Prolongation of ERK signaling and DNA synthesis in PKD overexpressing 3T3 cells were not diminished by treatment with PKC inhibitors, indicating that these events are facilitated by PKD through a PKC-independent pathway. Conclusion: The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation and mitogenic signaling.