Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.
We have reported that ceramide mediates binding of atypical protein kinase C (PKC) zeta to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCzeta.PAR-4 complex. Ceramide and its analogs activated PKCzeta prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCzeta and glycogen synthase kinase-3beta and emergence of a PAR-4-to-phosphorylated PKCzeta fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCzeta increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCzeta counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-kappaB unless PAR-4-dependent inhibition of PKCzeta suppresses NF-kappaB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCzeta. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3beta and NF-kappaB.
Wang
Guanghu
G
Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta 30912, USA.
Silva
Jeane
J
Krishnamurthy
Kannan
K
Tran
Eric
E
Condie
Brian G
BG
Bieberich
Erhard
E
D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism; D002845:Chromatography; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013234:Stem Cells; D013997:Time Factors; D014162:Transfection; D000099221:Protein Kinase C zeta
D000199:Actins; D051017:Apoptosis Regulatory Proteins; D002518:Ceramides; D018076:DNA, Complementary; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D047908:Intracellular Signaling Peptides and Proteins; D008055:Lipids; D008156:Luciferases; D016328:NF-kappa B; D010718:Phosphatidylserines; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta; C101296:prostate apoptosis response-4 protein; C000605506:GSK3B protein, human; C000605507:Gsk3b protein, mouse