G protein β-like (GβL) is a member of WD repeat-con-taining family which are involved in various intracellular signaling events. In our previous report, we demon-strated that GβL regulates TNFα-stimulated NF-κB signaling by interacting with and inhibiting phosphorylation of IκB kinase. However, GβL itself does not seem to regulate IKK directly, because it contains no functional domains except WD domains. Here, using immunoprecipitation and proteomic analyses, we identified protein phosphatase 4 as a new binding partner of GβL. We also found that GβL interacts with PP2A and PP6, other members of the same phosphatase family. By interacting with protein phosphatases, which do not directly bind to IKKβ, GβL mediates the association of phosphatases with IKKβ. Overexpression of protein phosphatases inhibited TNFκ-induced acti-vation of NF-κB signaling, which is an effect similar to that of GβL overexpression. Down-regulation of GβL by small interfering RNA diminished the inhibitory effect of phosphatases, resulting in restoration of NF-κB signaling. Thus, we propose that GβL functions as a negative regulator of NF-κB signaling by recruiting pro-tein phosphatases to the IKK complex.

Keywords: GβL, IκB kinase, NF-κB, phosphorylation, protein phosphatases