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

Biochemical Journal

Susan F. Steinberg¹  Jennifer E. Van Eyk¹  Jianli Gong¹  Ronald J. Holewinski¹ 

DOI  :  10.1042/BJ20150812

来源: Portland Press Limited

【 摘 要 】

Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ 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 PKCδ 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 PKCδ’s basal lipid-independent activity and that Ser¹³⁰/Thr¹⁴¹ di-phosphorylation influences PKCδ’s substrate specificity. We recently reported that PKCδ 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 PKCδ’s threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35, 1727–1740]. The present study shows that S130D and T141E substitutions increase PKCδ’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 PKCδ’s maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser¹³⁰/Thr¹⁴¹ phosphorylations relieve auto-inhibitory 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.

【 授权许可】

Unknown