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

ABSTRACT: 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 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) (as 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 co-operate to increase PKC?'s basal lipid-independent activity and that Ser(130)/Thr(141) 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 Ser(357), 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 Ser(357). A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKC?'s maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser(130)/Thr(141) phosphorylations relieve auto-inhibitory constraints that limit PKC?'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.

SUBMITTER: Gong J 

PROVIDER: S-EPMC5022567 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature