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

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 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-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 Ca(2+) 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.

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

“…Consistent with the NMR studies of FasDD, we used the numbering of FasDD amino acids as described (3,29 15 N-and/or 13 C-labeled proteins, respectively. Cells were induced with isopropyl ␤-D-1-thiogalactopyranoside, grown at 37°C for ϳ12 h, spun down, and stored overnight at Ϫ80°C.…”

“…The binding sites in CaM and FasDD can be identified through NMR resonance perturbations as detected in two-dimensional 1 H, 15 N HSQC (heteronuclear single quantum coherence) spectra. These experiments allow for identification of residues that are involved in the interaction and/or accompanying conformational changes and provide an effective method for examining the folding of the protein.…”

“…Asp-244 is located at the center of ␣3 (Fig. 1). 1 H, 13 C, and 15 N NMR signals of Fas m have been assigned using standard triple resonance methods (see "Experimental Procedures"). NMR chemical shifts of Fas m were very similar to those described for Fas K247A (48) except for residues in the proximity of the mutation site.…”

“…To identify the binding interface, we first conducted NMR titrations on a uniformly 15 N-labeled Fas m (100 M) as a function of added unlabeled CaM (Fig. 5).…”

“…Previous studies have shown that calmodulin (CaM) is recruited into DISC in cholangiocarcinoma (11)(12)(13)(14)(15)(16)(17) and pancreatic cancer cells (18), suggesting a novel role of CaM in Fas signaling. It has been hypothesized that Fas-CaM interaction may affect Fas-FADD interaction and thus regulates DISC assembly and inhibits apoptosis in cholangiocarcinoma cells (14).…”