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 Tyr(313)-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 Tyr(313)-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 Ser(359). 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 Ser(359) 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-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ

“…Figure 4 shows that WTPKCδ is detected as a Ser 357 phosphorylated enzyme in resting HEK293 cells. Whereas the stoichiometry of PKCδ-Ser 357 phosphorylation was not quantified in the previous study performed in cardiomyocytes [5], Figure 4 shows that WT-PKCδ-Ser 357 phosphorylation increases in response to treatment of HEK293 cells with PMA, indicating that HEK293 cells must contain a pool of enzyme that lacks Ser 357 phosphorylation. The PMA-dependent increase in WT-PKCδ-Ser 357 phosphorylation is prevented by the PKC inhibitor GF109203X (Figure 4B), indicating that this is mediated by a PKC-dependent (possibly autocatalytic) reaction.…”

“…This unbiased phosphoproteomic analysis, which was performed on relatively complex whole-cell lysate samples, identified 17 phosphorylation sites on PKCδ; almost all of the PKCδ phosphorylation sites detected in previous studies were captured in this analysis (Figure 1, the data supplement; Table 1 and references cited therein). However, the Ser 357 phosphorylation site identified in our previous more targeted MS study ([5] which used in-gel trypsin digestion of the immunoprecipitated PKCδ band followed by a more targeted multiple reaction monitoring MS approach to specifically measure phosphorylated and unphosphorylated Ser 357 peptide species) was not captured in the present study. The failure to detect the Ser 357 phosphorylation site in this set of experiments could be due to differences in MS methods/instrumentation, differences in sample complexity (whole cell lysate compared with in-gel digestion of only the PKCδ band) or the different database search engines used for the analysis.…”

“…We recently showed that PKCδ is recovered from cardiomyocytes as a G loop Ser 357 -phosphorylated enzyme and that Ser 357 phosphorylation limits PKCδ’s threonine kinase activity [5]. We used an immunoblotting approach with a PKCδ-pSer 357 PSSA to determine whether the S130D and T141E substitutions increase PKCδ’s threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser 357 .…”

“…Recent studies implicate another phosphorylation site at Ser 357 at the tip of PKCδ’s glycine-rich ATP-positioning loop (G-loop, also known as the phosphate-binding P-loop) as another modification that dynamically regulates catalytic activity [5]. Ser 357 sits in a highly conserved phosphorylation motif at the entrance to the ATP-binding cleft in close proximity to the P-site on substrates.…”

“…Our recent studies indicate that PKCδ is recovered from unstimulated cardiomyocytes as a Ser 357 -phosphorylated enzyme and that Ser 357 phosphorylation limits PKCδ’s threonine kinase activity (i.e. the Ser 357 -phosphorylated enzyme shows a strong preference for substrates with a serine residue at the P-site [5]). Oxidative stress triggers a tyrosine phosphorylation-dependent (C2 domain-driven) mechanism that results in Ser 357 dephosphorylation.…”