Ro52-mediated Monoubiquitination of IKKβ Down-regulates NF-κB Signalling

Upon activation, NF-kappa B translocates into the nucleus and initiates biological events. This NF-kappa B signalling is mainly regulated by the protein kinase IKK beta. Early in this signalling pathway, IKK beta is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of HTLV-1. In cells infected by HTLV-1, IKK beta is persistently phosphorylated and conjugated with monoubiquitin due to Tax expression. Although this Tax-induced monoubiquitination appears to be an important regulation system for IKK beta, how the monoubiquitination occurs is unknown and its role in NF-kappa B signalling is still unclear. Here, we show that an E3-ubiquitin ligase Ro52 interacts weakly with wild-type IKK beta but strongly with a phosphomimetic mutant IKK beta to conjugate monoubiquitin in cooperation with an E2-ubiquitin-conjugating enzyme UbcH5B. These results suggest that the Tax-induced phosphorylation of IKK beta causes an interaction with Ro52 for the subsequent monoubiquitination. NF-kappa B reporter assays have shown that the IKK beta activity is suppressed by wild-type Ro52, but not by its inactive mutant. In addition, monoubiquitin fusion of IKK beta reduced its activity for NF-kappa B signalling. We also found that Ro52 dramatically reduces the level of Tax. These results suggest that Ro52 down-regulates Tax-induced NF-kappa B signalling by monoubiquitinating IKK beta and by reducing the level of Tax.