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

Jianli Gong, Ronald J. Holewinski, Jennifer E. Van Eyk, Susan F. Steinberg

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)311-320
Number of pages10
JournalBiochemical Journal
Volume473
Issue number3
DOIs
StatePublished - Feb 1 2016

Keywords

  • Phosphorylation
  • Protein kinase C-delta
  • Pseudosubstrate domain
  • Single nucleotide polymorphism

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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