Ubiquitination of RIP is required for tumor necrosis factor α-induced NF-κB activation

Stimulation of cells with tumor necrosis factor (TNFα) triggers a recruitment of various signaling molecules, such as RIP, to the TNFα receptor 1 complex, leading 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 be determined whether the ubiquitination of RIP is required for TNFα-induced NF-κB activation. In this study, we have identified Lys³⁷⁷ of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-κB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFα receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation.