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

Abstract

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.

Hypericin in the Dark: Foe or Ally in Photodynamic Therapy?

“…The S645 is considered a priming phosphorylation site, and it has been suggested that this modification converts PKCδ to its mature form [28,42,43]. There was no difference in non-specific PKCδ staining.…”

Protein kinase C: perfectly balanced

“…Notably, Src-dependent phosphorylation at positions in the regulatory moiety, including the C2 domain, drives PKCδ into the nucleus, where it can be cleaved by caspases to commit to an irreversible activation (Humphries et al 2008). Additionally, this isozyme can be converted to a constitutively active enzyme following oxidative stress-induced phosphorylation of Tyr311 in the hinge region, forming a docking site for its C2 domain: interaction between the hinge and C2 not only disrupts autoinhibitory constraints, but promotes the dephosphorylation of Ser357 in the kinase domain, a residue whose phosphorylation state tunes selectivity between Ser and Thr (Gong et al 2016). Phosphorylation sites immediately preceding the pseudosubstrate of PKCδ have also been identified and shown to enhance lipid-independent activity (Gong et al 2016).…”

“…Additionally, this isozyme can be converted to a constitutively active enzyme following oxidative stress-induced phosphorylation of Tyr311 in the hinge region, forming a docking site for its C2 domain: interaction between the hinge and C2 not only disrupts autoinhibitory constraints, but promotes the dephosphorylation of Ser357 in the kinase domain, a residue whose phosphorylation state tunes selectivity between Ser and Thr (Gong et al 2016). Phosphorylation sites immediately preceding the pseudosubstrate of PKCδ have also been identified and shown to enhance lipid-independent activity (Gong et al 2016). Additionally, PKCβII has been shown to autophosphorylate at sites preceding its pseudosubstrate (Flint et al 1990), suggesting a general mechanism for PKC isozymes to sustain signaling.…”

Positive selection-driven fixation of a hominin-specific amino acid mutation related to dephosphorylation in IRF9

“…Interestingly, the alignment between IRF3 and IRF9 revealed that the IRF9 ancestral state Ser129 can be aligned to the IRF3 Ser123. In Eukaryotes, serine (S), threonine (T), and tyrosine (Y) residues are the most commonly used phosphorylation sites [ 26 , 37 , 38 ]. Through the linker region phosphorylation, the EBV-encoded kinase, BGLF4, can down-regulate the IRF3 transactivation [ 26 ].…”