IKKγ/NEMO facilitates the recruitment of the IκB proteins into the IκB kinase complex

IKK gamma /NEMO is an essential regulatory component of the I kappaB kinase complex that is required for NF-kappaB activation in response to various stimuli including tumor necrosis factor-alpha and interleukin-1 beta. To investigate the mechanism by which IKK gamma /NEMO regulates the IKK complex, we examined the ability of IKK gamma /NEMO to recruit the I kappaB proteins into this complex. IKK gamma /NEMO binding to wild-type, but not to a kinase-deficient IKK beta protein, facilitated the association of I kappaB alpha and I kappaB beta with the high molecular weight IKK complex. Following tumor necrosis factor-alpha treatment of HeLa cells, the majority of the phosphorylated form of endogenous I kappaB alpha was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKK gamma /NEMO-deficient cells. Finally, we demonstrate that IKK gamma /NEMO facilitates the association of the I kappaB proteins and IKK beta and leads to increases in IKK beta kinase activity. These results suggest that an important function of IKK gamma /NEMO is to facilitate the association of both IKK beta and I kappaB in the high molecular weight IKK complex to increase I kappaB phosphorylation.