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Caspase-8 and caspase-10 activate NF-kappaB through RIP, NIK and IKKalpha kinases
Yoshiaki Shikama1, Masao Yamada, Toshiyuki Miyashita
1Department of Genetics, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan.
Abstract:
NF-kappaB regulates the expression of various genes involved in cell growth and differentiation, immune response and inhibition of apoptosis. Recently, some death effector domain (DED)-containing proteins, such as FADD and c-FLIP were reported to activate NF-kappaB. We previously reported that the prodomain-only isoforms of caspase-8 and -10 (PDCasp8/10), containing two DED motifs, could inhibit Fas-mediated apoptosis. Here, we demonstrate that these isoforms also activate NF-kappaB, implying this to be one of the mechanisms by which these polypeptides inhibit apoptosis. The GST pull-down assay revealed that, among upstream kinases that activate NF-kappaB, only NIK and RIP, but not RICK or IKKalpha/beta, could directly bind to PDCasp8/10. In addition, both modules ofDED in PDCasp8/10 were required for these interactions as well as NF-kappaB activation. Experiments using a kinase-dead mutant of IKKalpha and an RIP mutant lacking a kinase domain, both of which function as dominant-negative mutants for their wild-type counterparts, blocked PDCasp8/10-mediated NF-kappaB activation. Using small interfering RNA technology, we further demonstrate that the down-regulation of IKKalpha but not IKKbeta significantly inhibits PDCasp8-mediated NF-kappaB activation. Taken together, these results suggest that caspase-8 and -10 have roles in a non- or anti-apoptotic signaling pathway leading to NF-kappaB activation through RIP, NIK and IKKalpha.
Insights
Prodomain-only caspase-8/10 isoforms activate NF-kappaB, a pathway involving RIP, NIK, and IKKalpha. This highlights a non-apoptotic role for these caspase isoforms in regulating gene expression.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Apoptosis Regulation
Background:
- NF-kappaB is crucial for cell growth, immune response, and apoptosis inhibition.
- Death effector domain (DED)-containing proteins like FADD and c-FLIP can activate NF-kappaB.
- Prodomain-only caspase-8 and -10 (PDCasp8/10) inhibit Fas-mediated apoptosis.
Purpose of the Study:
- To investigate if PDCasp8/10 activate NF-kappaB.
- To elucidate the mechanism of NF-kappaB activation by PDCasp8/10.
- To identify upstream kinases involved in this pathway.
Main Methods:
- GST pull-down assays to identify binding partners.
- Use of dominant-negative mutants for IKKalpha and RIP.
- Small interfering RNA (siRNA) to down-regulate IKKalpha and IKKbeta.
- Analysis of NF-kappaB activation.
Main Results:
- PDCasp8/10 were found to activate NF-kappaB.
- Only NIK and RIP directly bound to PDCasp8/10, requiring both DED motifs.
- IKKalpha, but not IKKbeta, was essential for PDCasp8/10-mediated NF-kappaB activation.
- Dominant-negative mutants of IKKalpha and RIP blocked this activation.
Conclusions:
- PDCasp8/10 activate NF-kappaB, contributing to their anti-apoptotic function.
- The pathway involves RIP, NIK, and IKKalpha.
- Caspase-8 and -10 participate in a non-apoptotic signaling cascade activating NF-kappaB.