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

Stimulation of cells with tumor necrosis factor (TNF alpha) triggers a recruitment of various signaling molecules, such as RIP, to the TNF alpha receptor 1 complex, leading to activation of NF-K B. Previous studies indicate that RIP plays an essential role for TNF alpha- induced NF-KB activation, but the molecular mechanism by which RIP mediates TNF alpha signals to activate NF-KB 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 TNF alpha-induced NF-KB activation. In this study, we have identified Lys(377) of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-K B activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNF alpha receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNF alpha-induced NF-KB activation.