Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase Cdelta in the Golgi complex.
Protein kinase C (PKC), a Ca(2+)/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 PKCdelta 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 PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (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 PKCdelta 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 PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta 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 PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.
Kajimoto
Taketoshi
T
Laboratories of Molecular Pharmacology and Biochemistry, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Shirai
Yasuhito
Y
Sakai
Norio
N
Yamamoto
Toshiyoshi
T
Matsuzaki
Hidenori
H
Kikkawa
Ushio
U
Saito
Naoaki
N
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001578:Benzopyrans / Q000494:pharmacology; D002518:Ceramides / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D000098:Acetophenones; D001578:Benzopyrans; D002518:Ceramides; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D012313:RNA; D006861:Hydrogen Peroxide; C085746:rottlerin; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta