Tyrosine Phosphorylation of Protein Kinase D in the Pleckstrin Homology Domain Leads to Activation

Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCμ, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr 432 , Tyr 463 , and Tyr 502 ), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr 463 ) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr 463 in vitro , and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr 432 and Tyr 502 , had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.