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 (Tyr⁴³², Tyr⁴⁶³, and Tyr⁵⁰²), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr⁴⁶³) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr⁴⁶³ in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr⁴³² and Tyr⁵⁰², 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.