Regulation of IκB Kinase (IKK)γ/NEMO Function by IKKβ-mediated Phosphorylation

Abstract The IκB kinase (IKK) complex includes the catalytic components IKKα and IKKβ in addition to the scaffold protein IKKγ/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IκB and the activation of the NF-κB pathway. Recent data indicate that the constitutive activation of the NF-κB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKγ/NEMO by IKKβ. To address further the significance of IKKβ-mediated phosphorylation of IKKγ/NEMO, we determined the sites in IKKγ/NEMO that were phosphorylated by IKKβ, and we assayed whether IKKγ/NEMO phosphorylation was involved in modulating IKKβ activity. IKKγ/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-α and interleukin-1 that activate the NF-κB pathway. By using both in vitro and in vivo assays, IKKβ was found to phosphorylate IKKγ/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKγ/NEMO to stimulate IKKβ kinase activity. These results indicate that the differential phosphorylation of IKKγ/NEMO by IKKβ and perhaps other kinases may be important in regulating IKK activity.