Taketoshi Kajimoto

Atypical protein kinase C (aPKC) isozymes play opposing roles in oncogenic and tumor-suppressive signaling pathways. These kinases are activated by phosphoinositide-dependent kinase 1 (PDK1) as well as by direct binding of the lipid second messenger sphingosine 1-phosphate (S1P). Here, we found that S1P and PDK1 provide mutually exclusive activation of aPKC by a common switch mechanism. In silico docking, molecular dynamics, and biochemical approaches revealed that the phosphate on S1P occupied the same position as the phosphate of the PDK1-phosphorylated Thr residue in the activation loop to structure the active site for catalysis. The interaction of either phosphate with the activation loop was coordinated by conserved Arg and Lys residues. When aPKC was phosphorylated, these residues positioned the activation loop in a manner that made the binding of S1P energetically prohibitive. Cellular studies revealed that S1P sustained basal signaling of aPKC, which suppressed apoptosis. In contrast, PDK1-dependent phosphorylation promoted apoptosis. These data identify a regulatory mechanism by which a ligand mimics a posttranslational modification and reveal that aPKC is at the crossroads of lipid second messenger signaling through cytosolic S1P pathways and canonical PDK1 signaling.

Protein kinase C (PKC) alters its localization upon various stimuli and exerts its function at the localized sites, which is called PKC translocation. We found that propofol, an intravenous anesthetic, induced PKC translocation and activated PKC. However, it is not clear which domains of PKC are important for propofol-induced PKC translocation. In this study, we generated mutants lacking each domain of PKC to elucidate the mechanism of propofol-induced PKC translocation and identified the PKC domains involved in this phenomenon.We used PKCα of conventional PKC and PKCδ of novel PKC. The regulatory region of PKCα consists of C1A and C1B lipid-binding domains and a calcium-binding C2 domain, while PKCδ consists of C1A and C1B domains. GFP-tagged Wild-type (WT) or domain-deficient mutants PKC were expressed in HeLa cells and HUVECs. WT PKCα was persistently translocated to the plasma membrane (PM) by propofol; deletion of either C1A or C1B or both reduced the duration time of PM translocation. Deletion of all C1A, C1B, and C2 domains abolished translocation to the PM, and PKC was translocated into nucleus. These results suggest that C1A, C1B, and C2 domains are involved in propofol-induced PKCα translocation. WT PKCδ was once accumulated to the Golgi apparatus and then translocated to the PM. Deletion of C1B domain abolished PKCδ translocation to the Golgi and PM, suggesting the involvement of the C1B domain in the propofol-induced translocation of PKCδ. These results reveal that the domains involved in propofol-induced PKC translocation are subtype-specific.

The sphingolipid S1P allosterically activates atypical protein kinase C to promote cell survival.

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