Regulation of protein kinase D by multisite phosphorylation - Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) vi a a phorbol ester/PKC-dependent pathway involves phosphorylation events. Th e present study identifies five in vivo phosphorylation sites by mass spect rometry, and the role of four of them was investigated by site-directed mut agenesis. Four sites are autophosphorylation sites, the first of which (Ser (916)) is located in the C terminus; its phosphorylation modifies the confo rmation of the kinase and influences duration of kinase activation but is n ot required for phorbol ester-mediated activation of PKD. The second autoph osphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3 tau. The last two autophosphorylatio n sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the ful l-length PKD; they may affect enzyme catalysis but are not involved in the activation of wildtype PKD by phorbol ester. We also present evidence for p roteolytic activation of PKD. The fifth site (Ser(255)) is transphosphoryla ted downstream of a PKC-dependent pathway after in vivo stimulation with ph orbol ester, In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very dis tinct ways.