Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser(744)/Ser(748) and Ser(916)

作者：Rybin Vitalyi O; Guo Jianfen; Steinberg Susan F^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2009, 284(4): 2332-2343.

DOI：10.1074/jbc.M806381200

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 Ser(744) and Ser(748) followed by PKD1 autophosphorylation at Ser(916). Although PKD-Ser(916) 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 Go6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser(916)-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser(916) 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 Ser(744) and Ser(748) are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser(916)-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser(744). However, the S916A substitution leads to a Ser(748) phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser(744) phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser(916) autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser(916) is required for subsequent autophosphorylation at Ser(748). Finally, this study exposes a novel role for Ser(916) and/or Ser(748) autophosphorylation to terminate the cellular PKD1 signaling response.

• 出版日期2009-1-23