Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1β-converting enzyme]-like inhibitory protein}. A Ca²⁺-dependent direct interaction between CaM and FLIP_L, but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3±5.7% increase (n=6, P=0.001) in CaM–FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca²⁺ that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM–FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP_L was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197–213 of FLIP_L. Compared with overexpression of wild-type FLIP_L that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP_L with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM–FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.