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

Protein kinase C- (PKC) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKC at membranes, PKC also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study uses MS-based methods to identify PKC phosphorylation at Thr50 and Ser645 (in resting and PMA-treated cardiomyocytes) as well as Thr37, Thr38, Ser130, Thr164, Thr211, Thr215, Ser218, Thr295, Ser299 and Thr656 (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser130 and Thr141 (sites just N-terminal to the pseudosubstrate domain). We show that S130D and T141E substitutions co-operate to increase PKC’s basal lipid-independent activity and that Ser130/Thr141 di-phosphorylation influences PKC’s substrate specificity. We recently reported that PKC preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser357, an ATP-positioning G-loop site that limits PKC’s threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study shows that S130D and T141E substitutions increase PKC’s threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser357. A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKC’s maximal lipid-...
Source: Biochemical Journal - Category: Biochemistry Authors: Tags: Signalling Research articles Source Type: research