Abstract
RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFα-induced NF-κB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-κB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-κB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-κB signaling pathway activated by TNFα dependent on its kinase activity and NF-κB signaling pathway activated by caspase-10 independent of its kinase activity.
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Acknowledgments
We thank Dr. Karen Dawson for proofreading the manuscript. This study was supported by the Chinese 863 program (2006AA02A310) and Chinese Human Liver Proteome Project (2004BA711A19) to K. Huo.
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Supplementary Fig.1
Interaction between RIOK3 and caspase-10 in yeast two-hybrid system. Plasmids were cotransformed into yeast Y190 cells and plated on an SD/-Leu-Trp plate (upper) or an SD/-Leu-Trp-His plate with 30 mM 3-AT (middle). Blue colour due to expression of β-gal protein is indication of positive protein–protein interaction (bottom). I, pGBKT7-RIOK3+pGADT7 II, pGBKT7-RIOK3+pACT2-caspase-10 III, pGBKT7+pACT2-caspase-10. Supplementary material 2 (TIFF 3474 kb)
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Shan, J., Wang, P., Zhou, J. et al. RIOK3 interacts with caspase-10 and negatively regulates the NF-κB signaling pathway. Mol Cell Biochem 332, 113–120 (2009). https://doi.org/10.1007/s11010-009-0180-8
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DOI: https://doi.org/10.1007/s11010-009-0180-8