IKKγ/NEMO Facilitates the Recruitment of the IκB Proteins into the IκB Kinase Complex

Abstract

IKK␥/NEMO is an essential regulatory component of the IB kinase complex that is required for NF-B activation in response to various stimuli including tumor necrosis factor-␣ and interleukin-1␤. To investigate the mechanism by which IKK␥/NEMO regulates the IKK complex, we examined the ability of IKK␥/NEMO to recruit the IB proteins into this complex. IKK␥/NEMO binding to wild-type, but not to a kinase-deficient IKK␤ protein, facilitated the association of IB␣ and IB␤ with the high molecular weight IKK complex. Following tumor necrosis factor-␣ treatment of HeLa cells, the majority of the phosphorylated form of endogenous IB␣ was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKK␥/NEMO-deficient cells. Finally, we demonstrate that IKK␥/NEMO facilitates the association of the IB proteins and IKK␤ and leads to increases in IKK␤ kinase activity. These results suggest that an important function of IKK␥/NEMO is to facilitate the association of both IKK␤ and IB in the high molecular weight IKK complex to increase IB phosphorylation.The NF-B proteins are critical for activating the expression of cellular genes that are involved in the control of the immune and inflammatory response and in protecting cells from apoptosis in response to a variety of stress stimuli (1-4). NF-B is sequestered in the cytoplasm in most cells, where it is bound to a family of inhibitory proteins known as IB (2,5,6). A variety of stimuli including the cytokines TNF␣ 1 and interleukin-1, double-stranded RNA, and the viral transactivator Tax activate the NF-B pathway (4, 7-11). These stimuli increase the activity of two related kinases, IKK␣ and IKK␤, to result in the phosphorylation of the IB proteins (9,(12)(13)(14)(15)(16). A variety of studies using IKK␣ and IKK␤ knock-out mice indicate that IKK␤ is critical for NF-B activation in response to cytokine treatment, whereas IKK␣ is not required for this function (17)(18)(19)(20)(21)(22). The IB␣ protein is phosphorylated on serine residues 32 and 36, while IB␤ is phosphorylated on serine residues 19 and 23, and this leads to their ubiquitination and degradation by the proteasome (10,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). IB mutants in which these serine residues are changed to alanine are resistant to proteasome-mediated degradation and thus prevent the nuclear translocation of the NF-B proteins (33).IKK␥/NEMO was initially identified in a genetic complementation assay as a factor that could restore NF-␤ activation in cells that were resistant to a variety of stimuli that normally induce the NF-B pathway (34). IKK␥/NEMO was also identified independently in biochemical studies as an essential component of the high molecular weight IKK complex (35, 36). Finally, this factor was characterized as a factor known as FIP-3 that bound to the adenovirus E3 protein and could inhibit TNF␣-induced apoptosis (37). IKK␥/NEMO in conjunction with IKK␣ and IKK␤ is a component of the high molecular weight IKK complex, which migrates between 60...

Characterization of the IκB-kinase NEMO Binding Domain

“…This reasoning is based upon the observations that NF-B cannot be activated in NEMO-deficient cells (24 -26) and overexpression of NEMO can activate IKK in mammalian and yeast systems (30,34,38). In contrast, some workers have described NEMO as an inhibitor of IKK function (29,41), and others have demonstrated that variations in the cellular concentration of NEMO dictate, whether it functions to activate or inhibit NF-B (32).…”

“…It has been proposed that NEMO plays a regulatory role by recruiting the IKK complex to ligated cytokine receptors, where IKKs may be activated by as yet poorly defined mechanisms that possibly include oligomerization, conformational changes, or proximityinduced transphosphorylation events (29 -33). Additionally or alternatively, NEMO may facilitate the recruitment of upstream IKK activators such as kinases that specifically target the activation loops within the catalytic domains of the IKK subunits or enable the interaction of the IKKs with IB proteins (22,34).…”

Nuclear Role of IκB Kinase-γ/NF-κB Essential Modulator (IKKγ/NEMO) in NF-κB-dependent Gene Expression

“…Plasmid Constructs-The pCMV5 expression vectors encoding FLAG-or Myc-tagged IKK␣, IKK␤, IKK␥, or p65 or HA-tagged CBP were described previously (32,40,41). Gal4-CBP constructs were kindly provided by Dr. Tucker Collins, and fusions of the VP16 activation domain with IKK␣, IKK␤, or IKK␥/NEMO were constructed in pCMV5 as described previously (32).…”

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

“…NEMO has multiple protein domains, which are responsible for integrating various signals and binding to adaptor molecules or kinases (IKKα/β) [2]. In addition, NEMO is post-translationally modified by ubiquitination, phosphorylation and SUMOylation in various contexts depending on cell type and stimulus [3].…”