Regulation of IκB Kinase Complex by Phosphorylation of γ-Binding Domain of IκB Kinase β by Polo-like Kinase 1

I kappa B kinase (IKK) complex is a key regulator of NF-kappa B pathways. Signal-induced interaction of the IKK gamma ( NEMO) subunit with the C-terminal IKK gamma/NEMO-binding domain (gamma BD) of IKK beta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gamma BD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gamma BD has not been identified. In this study, we used a C-terminal fragment of IKK beta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gamma BD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gamma BD of IKK beta in vitro. Phosphorylating gamma BD with Plk1 decreased its affinity for IKK gamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gamma BD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous I kappa B alpha and reduced NF-kappa B activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gamma BD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNF alpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gamma BD of IKK beta with IKK gamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gamma BD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNF alpha can provide a potential mechanism for an involvement of TNF alpha in inflammation-induced cancer.