Reduced CaM/FLIP binding by a single point mutation in c-FLIP(L) modulates Fas-mediated apoptosis and decreases tumorigenesis.

Abstract

We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIP(L) in a Ca(2+)-dependent manner. Deletion of the CaM-binding region (amino acid 197-213) results in reduced CaM binding, and increased Fas-mediated apoptosis and decreased tumorigenesis of cholangiocarcinoma cells. The present studies were designed to identify the precise amino acids between 197 and 213 that are responsible for CaM/FLIP binding, and their roles in mediating the anti-apoptotic function of c-FLIP(L). Sequence analysis of the CaM-binding region at 197-213 predicted three unique positively charged residues at 204, 207 and 209, which might be responsible for the CaM/FLIP binding. A point mutation at H204 of c-FLIP(L) was found to markedly reduce CaM binding, whereas point mutation at R207 or K209 did not affect c-FLIP(L) binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIP(L) mutant. Reduced CaM binding by the H204 mutant resulted in increased sensitivity to Fas-mediated apoptosis and inhibited tumor growth in mice compared with wild-type c-FLIP(L). Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIP(L) recruited into the DISC. Concurrently, increased caspase 8 was recruited to the DISC, which resulted in increased cleavage and activation of caspase 8, activation of downstream caspase 3 and increased apoptosis. Therefore, these results demonstrate that the H204 residue is responsible for c-FLIP(L) binding to CaM, which mediates the anti-apoptotic function of c-FLIP(L), most likely through affecting recruitment of caspase 8 into the DISC and thus caspase 8 activation. These studies further characterized CaM/FLIP interaction and its function in regulating Fas-mediated apoptosis and tumorigenesis, which may provide new therapeutic targets for cancer therapy.

• Authors: Gu Jing, Kaiyu Yuan, Qiuli Liang, Yong Sun, Xia Mao, Jay M McDonald, Yabing Chen
• Journal: Laboratory investigation; a journal of technical methods and pathology (Lab Invest) Vol. 92 Issue 1 Pg. 82-90 (Jan 2012) ISSN: 1530-0307 [Electronic] United States
• PMID: 21912376 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• CASP8 and FADD-Like Apoptosis Regulating Protein
• CFLAR protein, human
• Calmodulin
• fas Receptor
• Caspases

Topics

• Animals
• Apoptosis
• Bile Duct Neoplasms (prevention & control)
• Bile Ducts, Intrahepatic
• CASP8 and FADD-Like Apoptosis Regulating Protein (physiology)
• Calmodulin (metabolism)
• Caspases (metabolism)
• Cell Line, Tumor
• Cell Transformation, Neoplastic (metabolism)
• Cholangiocarcinoma (metabolism, pathology, prevention & control)
• Humans
• Male
• Mice
• Point Mutation
• fas Receptor (physiology)