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

Academic Article

Abstract

  • 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 FLIPL, 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 FLIPL 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 FLIPL that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIPL 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. © The Authors.
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    Digital Object Identifier (doi)

    Author List

  • Pawar PS; Micoli KJ; Ding H; Cook WJ; Kappes JC; Chen Y; McDonald JM
  • Start Page

  • 459
  • End Page

  • 468
  • Volume

  • 412
  • Issue

  • 3