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

Protein kinase C-δ (PKCδ) is a signaling kinase that regulates many cellular responses. While most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation. This study uses MS-based methods to identify PKCδ phosphorylation at Thr 50 and Ser 645 (in resting and PMA-treated cardiomyocytes) as well as Thr 37 , Thr 38 , Ser 130 , Thr 164 , Thr 211 , Thr 215 , Ser 218 , Thr 295 , Ser 299 and Thr 656 (sites that increase with PMA). We focused on the consequences of phosphorylation at Ser 130 and Thr 141 (sites just N-terminal to the pseudosubstrate domain). We show that S130D and T141E substitutions cooperate to increase PKCδ’s lipid-independent activity and that Ser 130 /Thr 141 di-phosphorylation influences PKCδ’s substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor Ser residue and that this is due to constitutive phosphorylation at Ser 357 , an ATP-positioning G-loop site that limits PKCδ’s Thr kinase activity. This study shows that S130D and T141E substitutions increase PKCδ’s Thr kinase activity indirectly by decreasing G loop phosphorylation at Ser 357 . A S130F substitution (mimicking a SNP identified in some human populations) also increases PKCδ’s maximal lipid-dependent catalytic activity and confers Thr kinase activity. Finally, we show that Ser 130 /Thr 141 phosphorylations relieve autoinhibitory constraints that limit PKCδ’s activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity.