Phosphorylation of serine 68 in the IκB kinase (IKK)-binding domain of NEMO interferes with the structure of the IKK complex and tumor necrosis factor-α-induced NF-κB activity

The I kappa B-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the I kappa B proteins and thus controls the activity of the transcription factor NF-kappa B. Besides the two kinases IKK alpha and IKK beta, the IKK complex contains NEMO/IKK gamma, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKK beta-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKK beta phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser(68) phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKK beta-NEMO interaction. In contrast, the NEMO-IKK alpha interaction was only mildly affected by the phosphorylation of Ser68. However, functional analysis revealed that Ser68 phosphorylation primarily affects the activity of IKK alpha. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF-alpha-induced NF-kappa B activity, thus emphasizing the inhibitory role of the Ser68 phosphorylation on the signal-induced NF-kappa B activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser(68)-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex.