TY - JOUR
T1 - Reduced CaM/FLIP binding by a single point mutation in c-FLIPL modulates Fas-mediated apoptosis and decreases tumorigenesis
AU - Jing, Gu
AU - Yuan, Kaiyu
AU - Liang, Qiuli
AU - Sun, Yong
AU - Mao, Xia
AU - McDonald, Jay M.
AU - Chen, Yabing
PY - 2012/1
Y1 - 2012/1
N2 - We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIPL in a Ca2+-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-FLIPL binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIPL 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-FLIPL. Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIPL 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-FLIPL binding to CaM, which mediates the anti-apoptotic function of c-FLIPL, 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.
AB - We have previously demonstrated that calmodulin (CaM) binds directly to c-FLIPL in a Ca2+-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-FLIPL binding to CaM. Decreased CaM/FLIP binding was confirmed in cholangiocarcinoma cells overexpressing the H204 c-FLIPL 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-FLIPL. Death-inducing signaling complex (DISC) analysis showed that the reduced CaM binding to H204 mutant resulted in less c-FLIPL 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-FLIPL binding to CaM, which mediates the anti-apoptotic function of c-FLIPL, 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.
KW - FLICE-like inhibitory protein (FLIP)
KW - apoptosis
KW - calmodulin
KW - cholangiocarcinoma
KW - death-inducing signaling complex (DISC)
KW - point mutation
KW - tumorigenesis
UR - http://www.scopus.com/inward/record.url?scp=84855251212&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84855251212&partnerID=8YFLogxK
U2 - 10.1038/labinvest.2011.131
DO - 10.1038/labinvest.2011.131
M3 - Article
C2 - 21912376
AN - SCOPUS:84855251212
SN - 0023-6837
VL - 92
SP - 82
EP - 90
JO - Laboratory Investigation
JF - Laboratory Investigation
IS - 1
ER -