The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³⁵⁹ Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

Abstract

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr³¹³-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr³¹³-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser³⁵⁹. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser^359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser³⁵⁹ phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr³¹³ docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01436-14.

ACKNOWLEDGMENTS

This work was supported by HL77860 (S.F.S) and grants to the Johns Hopkins Innovation Proteomics Center in Heart Failure (HHSN268201000032C and NHLBI-HV-10-05 to J.E.V.E.). S.F.S. was supported by an award from the NIH (1DP2 CA186752-01) and by an AHA Scientist Development Grant (13SDG14270009).