Heterologous SUMO-2/3-Ubiquitin Chains Optimize IκBα Degradation and NF-κB Activity

The NF-kappa B pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule I kappa B alpha, the IKK subunit gamma/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of I kappa B alpha and activation of NF-kappa B mediated by TNF alpha. We found that under conditions of deficient SUMOylation, an important delay in both TNF alpha-mediated proteolysis of I kappa B alpha and NF-kappa B dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on I kappa B alpha containing SUMO-2/3 and ubiquitin after TNF alpha stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on I kappa B alpha after activation of the TNF alpha signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of I kappa B alpha by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of I kappa B alpha modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified I kappa B alpha to the action of 26S proteasome, contributing to the optimal control of NF-kappa B activity after TNF alpha-stimulation.