The IkappaB-Kinase (IKK) complex is a multisubunit protein complex crucial for 
signal-induced phosphorylation of the IkappaB proteins and thus controls the 
activity of the transcription factor NF-kappaB. Besides the two kinases IKKalpha 
and IKKbeta, the IKK complex contains NEMO/IKKgamma, 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 IKKbeta-mediated phosphorylation of the IKK-binding domain of NEMO. 
In vitro, IKKbeta 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 IKKbeta-NEMO interaction. In contrast, the NEMO-IKKalpha interaction 
was only mildly affected by the phosphorylation of Ser(68). However, functional 
analysis revealed that Ser(68) phosphorylation primarily affects the activity of 
IKKalpha. Furthermore, in complementation experiments of NEMO-deficient murine 
embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant 
decreased, TNF-alpha-induced NF-kappaB activity, thus emphasizing the inhibitory 
role of the Ser(68) phosphorylation on the signal-induced NF-kappaB 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.