Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity

The NF-kappa B pathway is regulated by multiple post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. Many of these modifications act on the natural inhibitor I kappa B alpha modulating its capacity to control signalmediated NF-kappa B activity. While the canonical pathway involving the phosphorylation and polyubiquitylation of I kappa B alpha has been well characterized, the role of these post-translational modifications in the control of basal NF-kappa B activity has not been deeply explored. Using the recently developed Tandem-repeated Ubiquitin Binding Entities (also known as ubiquitin traps) to capture ubiquitylated proteins, we identified monoubiquitylated forms of I kappa B alpha from multiple rat organs and cell types. The identification of these forms was demonstrated through different procedures such as immunoprecipitations with specific ubiquitin antibodies or His6-Ubiquitin pull downs. Monoubiquitylated forms of I kappa B alpha are resistant to TNF alpha-mediated degradation and can be captured using TUBEs, even after proteasome inhibitors treatment. As it occurs for monoSUMOylation, monoubiquitylation is not dependent of the phosphorylation of I kappa B alpha on the serines 32/36 and is not optimally degraded after TNF alpha stimulation. A ubiquitin-I kappa B alpha fusion exhibits phosphorylation defects and resistance to TNF alpha mediated degradation similar to the ones observed for endogenous monoubiquitylated I kappa B alpha. The N-terminal attachment of a single ubiquitin moiety on the I kappa B alpha fusion results in a deficient binding to the IKK beta kinase and recruitment of the SCF ligase component beta TrCP, promoting a negative impact on the NF-kappa B activity. Altogether, our results suggest the existence of a reservoir of monoubiquitylated I kappa B alpha resistant to TNF alpha-induced proteolysis, which is able to interact and repress DNA binding and NF-kappa B transcriptional activity. Such pool of I kappa B alpha may play an important role in the control of basal and signal-mediated NF-kappa B activity.