J Biol Chem. 2003 Nov 7;278(45):44338-47. Epub 2003 Aug 14.

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling.External

Harper, N., Hughes, M. A., Farrow, S. N., Cohen, G. M., MacFarlane, M.,
--- - Medical Research Council Toxicology Unit, University of Leicester, Hodgkin Bldg., P. O. Box 138, Lancaster Rd., Leicester LE1 9HN, United Kingdom.
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.
PMID: 12920112External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vitro interaction
  DD1 DD2 Reference
Family DD1 DD2 Method Species Region Expression Species Region Expression
DD DR4 Link FADD GST fusion protein pull-down Not specified 269-468 E.coli HeLa lysate 12920112
DD DR5 Link FADD GST fusion protein pull-down Not specified 209-411 E.coli HeLa lysate 12920112
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Functional Role
Family DD1 DD2 Method Death-related Death-unrelated Reference
DD DR4 Link FADD Annexin V staining Apoptosis (DISC) 12920112
DD DR5 Link FADD Annexin V staining Apoptosis (DISC) 12920112
(Link: click this icon to show interactions only between the two corresponding DDs)