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

Protein kinase C-d (PKCσ) is a signalling kinase that regulates many cellular responses.Although most studies focus on allosteric mechanisms that activate PKCσ at membranes, PKCσ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35, 1727-1740]. The present study uses MSbased methods to identify PKCσ 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 Nterminal to the pseudosubstrate domain).We showthat S¹³⁰D and T¹⁴¹E substitutions co-operate to increase PKCσ's basal lipidindependent activity and that Ser¹³⁰/Thr¹⁴¹ di-phosphorylation influences PKCσ's substrate specificity. We recently reported that PKCσ 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 PKCσ's threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727-1740]. The present study shows that S¹³⁰D and T¹⁴¹E substitutions increase PKCσ's threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser³⁵⁷. A S¹³⁰F substitution [that mimics a S¹³⁰F singlent polymorphism (SNP) identified in some human populations] also increases PKCσ's maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser¹³⁰/Thr¹⁴¹ phosphorylations relieve auto-inhibitory constraints that limit PKCσ'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.