Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex*

Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCδ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCδ-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCδ expression using small interfering RNA. Ceramide induced the translocation of PKCδ to the Golgi complex and the concomitant activation of PKCδ via phosphorylation of Tyr311 and Tyr332 in the hinge region of the enzyme. Unphosphorylatable PKCδ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCδ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCδ to the Golgi complex and that PKCδ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCδ by small interfering RNA to study the significance of phosphorylation of Tyr311 and Tyr332 in PKCδ for ceramide-induced apoptosis and found that phosphorylation of Tyr311 and Tyr332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCδ, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

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