Protein kinase D1 autophosphorylation via distinct mechanisms at Ser744/Ser748 and Ser916

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.