title: 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.affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Gu
authors.initials: G
authors.lastname: Jing
authors.forename: Kaiyu
authors.initials: K
authors.lastname: Yuan
authors.forename: Qiuli
authors.initials: Q
authors.lastname: Liang
authors.forename: Yong
authors.initials: Y
authors.lastname: Sun
authors.forename: Xia
authors.initials: X
authors.lastname: Mao
authors.forename: Jay M
authors.initials: JM
authors.lastname: McDonald
authors.forename: Yabing
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D017209:Apoptosis; D001650:Bile Duct Neoplasms / Q000517:prevention & control*; D001653:Bile Ducts, Intrahepatic; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D018281:Cholangiocarcinoma / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D006801:Humans; D008297:Male; D051379:Mice; D017354:Point Mutation; D019014:fas Receptor / Q000502:physiology*
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002147:Calmodulin; D019014:fas Receptor; D020169:Caspases