The role of RIP ubiquitination in the TNFalpha signaling pathway
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Stimulation of cells by TNFα triggers recruitment of various signaling molecules, including RIP, to the TNFα receptor 1 complex, which leads to activation of NF-κB. Previous studies indicate that RIP plays an essential role for TNFα-induced NF-κB activation, but the molecular mechanism by which RIP mediates TNFα signals to activate NF-κB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to determine whether the ubiquitination of RIP is required for TNFα-induced NF-κB activation. In this study, we have identified K377 of RIP as the functional ubiquitination site, since mutating this residue to arginine completely abolished ligand-dependent ubiquitination of RIP and its function for NF-κB activation in the TNFα pathway. The defect of K377R mutation of RIP is because this mutant fails to recruit its downstream signaling component, TAK1, into the TNFR1 complex, which is needed for the activation of TAK1 and the phosphorylation of IKKα/β after TNFα stimulation. The lipid raft recruitment of NEMO, another subunit of the IKK complex is RIP-independent. This study provides the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation by inducing the phosphorylation of the IKKα/β complex. In the last part of our study, we show that cIAPs, instead of TRAF2, might be the E3 ligase responsible for the ubiquitination of RIP and NEMO. Thus, it is possible that cIAPs are involved in the TNFα signaling pathway by activating the IKK complex through RIP-dependent IKKα/β phosphorylation and RIP-independent NEMO ubiquitination.