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

Abstract

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 PKC␦ 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 PKC␦-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKC␦ expression using small interfering RNA. Ceramide induced the translocation of PKC␦ to the Golgi complex and the concomitant activation of PKC␦ via phosphorylation of Tyr 311 and Tyr 332 in the hinge region of the enzyme. Unphosphorylatable PKC␦ (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 PKC␦ 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 PKC␦ to the Golgi complex and that PKC␦ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKC␦ by small interfering RNA to study the significance of phosphorylation of Tyr 311 and Tyr 332 in PKC␦ for ceramide-induced apoptosis and found that phosphorylation of Tyr 311 and Tyr 332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKC␦, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

“…Translocation of PKC␦ to the Golgi may prevent PKC␦ cleavage by caspase-3, although the mechanism involved in the activation of caspase-3 by PKC␦ is unclear. In agreement with this assumption, there are reports indicating that ceramide stimulation induces translocation of PKC␦ from the cytosol to the Golgi, whereas no significant degradation of PKC␦ was observed in HeLa cells treated with ceramide (20,43). It was also reported that a constitutively active PKC␦ mutant that was targeted to the cytosol and mitochondria underwent cleavage by caspase, whereas no cleavage was observed in the PKC␦ mutant that was targeted to the nucleus and endoplasmic reticulum (44).…”

Nanomolar concentration of NSC606985, a camptothecin analog, induces leukemic-cell apoptosis through protein kinase Cδ–dependent mechanisms

“…DeVries et al 53 showed that the import of PKC␦ into the nucleus was required for the initiation of etoposide-induced apoptosis, while the attachment of PKC␦ to membrane and translocation to the Golgi complex was required for the ultraviolet-and ceramide-induced apoptosis, respectively. 54,55 Here, we showed that NSC606985-induced PKC␦ cleavage simultaneously occurred in the cytoplasm and nuclei of both NB4 and U937 cells, although PKC␦ was differentially distributed within these cells. Unlike untreated U937 cells in which PKC␦ was present in the cytoplasm and nuclei, NB4 cells had undetectable level of cytoplasmic PKC␦.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…Phosphorylation at Tyr-334, rather than at Tyr-313, is required for the cleavage of PKC to the kinase domain fragment in TRAIL/cisplatin-treated cells [113]. In contrast, phosphorylation at Tyr-313 is required for the cleavage of PKC and apoptosis in response to oxidative stress [116]. These studies highlight the fact that the signals that activate PKC and regulate its ability to modulate apoptosis is cell type and stimulus specific.…”