Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling.
Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling.
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DOI：
10.1042/bj20071507
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发表时间：
2008-06
期刊：
影响因子：
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通讯作者：
Pritish Pawar;Keith J. Micoli;Haitao Ding;W. Cook;J. Kappes;Yabing Chen;J. McDonald
中科院分区：
文献类型：
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作者：
Pritish Pawar;Keith J. Micoli;Haitao Ding;W. Cook;J. Kappes;Yabing Chen;J. McDonald
我们和其他人已经证明Fas介导的细胞凋亡是胆管癌的潜在治疗靶点。以前，我们报道了钙调素（calmodulin）拮抗剂通过Fas相关机制诱导胆管癌细胞凋亡。此外，我们确定了一个直接的相互作用之间的钙调素和Fas与招聘钙调素到Fas介导的DISC（死亡诱导信号复合物），这表明一个新的作用钙调素Fas信号。因此，我们表征了CaM与招募到Fas介导的DISC中的蛋白质的相互作用，包括含有FADD（Fas相关死亡结构域）的蛋白、半胱天冬酶8和c-FLIP {细胞FLICE [FADD（Fas相关死亡结构域）样白细胞介素1 β转化酶]样抑制蛋白}。CaM和FLIP（L）之间存在Ca（2+）依赖的直接相互作用，而FADD或caspase 8则不存在。此外，在Fas刺激后30 min，观察到CaM-FLIP结合增加37.3+/-5.7%（n=6，P=0.001），60 min后恢复到基线水平，并与Fas诱导的胆管癌细胞内Ca（2+）增加相关，在30 min时达到峰值，60 min后逐渐下降。钙调素拮抗剂TFP（三氟拉嗪）抑制Fas诱导的CaM-FLIP结合增加，同时抑制ERK（细胞外信号调节激酶）磷酸化，FLIP的下游信号。使用重组蛋白证明了CaM和FLIP（L）之间的直接结合，并且在FLIP（L）的氨基酸197-213中鉴定了CaM结合区域。与野生型FLIP（L）过表达导致自发性和Fas诱导的凋亡减少相比，缺失CaM结合区的突变型FLIP（L）导致胆管癌细胞自发性和Fas诱导的凋亡增加。了解CaM-FLIP结合的生物学可能为胆管癌和其他癌症提供新的治疗靶点。
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.