Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling
| Autor: | William J. Cook, Jay M. McDonald, Pritish Pawar, John C. Kappes, Keith J. Micoli, Yabing Chen, Haitao Ding |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
MAPK/ERK pathway
Caspase 8 Binding Sites Calmodulin biology Kinase CASP8 and FADD-Like Apoptosis Regulating Protein Apoptosis Cell Biology Biochemistry Cell biology Cell Line Tumor biology.protein Humans Calcium Death effector domain fas Receptor FADD Molecular Biology Signal Transduction Death domain |
| Zdroj: | Biochemical Journal. 412:459-468 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj20071507 |
| Popis: | 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 1β-converting enzyme]-like inhibitory protein}. A Ca2+-dependent direct interaction between CaM and FLIPL, 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 Ca2+ 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 FLIPL was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197–213 of FLIPL. Compared with overexpression of wild-type FLIPL that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIPL 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. Abbreviations: AM, acetoxymethyl ester; CaM, calmodulin; CaMS, CaM–Sepharose; c-FLIP, cellular FLICE-like inhibitory protein; Co-IP, co-immunoprecipitation; CS, control Sepharose; DD, death domain; DED, death effector domain; DISC, death-inducing signalling complex; ERK, extracellular-signal-regulated kinase; FADD, Fas-associated DD; FLICE, FADD-like interleukin 1β-converting enzyme; GST, glutathione transferase; HBSS, Hanks balanced salt solution; HRP, horseradish peroxidase; IPTG, isopropyl β-D-thiogalactoside; TFP, trifluoperazine; TNF, tumour necrosis factor; TNF-R1, TNF receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R1, TRAIL receptor 1; WT, wild-type |
| Databáze: | OpenAIRE |
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