title: Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling.
abstract: We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Hodgkin Bldg., P. O. Box 138, Lancaster Rd., Leicester LE1 9HN, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Harper
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Hughes
authors.forename: Stuart N
authors.initials: SN
authors.lastname: Farrow
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Cohen
authors.forename: Marion
authors.initials: M
authors.lastname: MacFarlane
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008928:Mitochondria / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D020672:I-kappa B Proteins; D007211:Indoles; D008301:Maleimides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D010718:Phosphatidylserines; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D045304:Cytochromes c; D011493:Protein Kinase C; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate