Abstract
IKKgamma/NEMO is a protein that is critical for the assembly of the high molecular weight IkappaB kinase (IKK) complex. To investigate the role of IKKgamma/NEMO in the assembly of the IKK complex, we conducted a series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged IKKgamma/NEMO and the IKKs were examined. When expressed alone following transfection, IKKalpha and IKKbeta were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKKgamma/NEMO, both IKKalpha and IKKbeta migrated at approximately 600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-alpha. When either IKKalpha or IKKbeta was expressed alone with IKKgamma/NEMO, IKKbeta but not IKKalpha migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKbeta were both incorporated into the high molecular weight IKK complex in the presence of IKKgamma/NEMO. The amino-terminal region of IKKgamma/NEMO, which interacts directly with IKKbeta, was required for formation of the high molecular weight IKK complex and for stimulation of IKKbeta kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKKgamma/NEMO is a crucial step involved in IKK function.
Highlights
The NF-B proteins are a family of transcription factors that regulate the expression of a variety of cellular genes involved in the control of the immune and the inflammatory response [1,2,3,4]
Cytoplasmic extracts were subjected to gel filtration, and the column fractions were analyzed by Western blot analysis and by IKK kinase activity assay (Fig. 2)
Similar to the results obtained with wild-type IKK␥/NEMO shown in Fig. 2A, the mutant lacking the carboxyl-terminal portion of IKK␥/NEMO was able to recruit the majority of the epitopetagged IKK protein and kinase activity into the high molecular weight IKK complex (Fig. 5A)
Summary
TNF␣, tumor necrosis factor-␣; IKK, IB kinase; PCR, polymerase chain reaction; GST, glutathione S-transferase; CMV, cytomegalovirus; HA, hemagglutinin; aa, amino acid; CREB, cAMP-responsive element-binding protein. Proteins translocate from the cytoplasm to the nucleus where they activate the expression of specific cellular genes [8] Both IKK␣ and IKK are components of a high molecular weight complex migrating between 600 and 900 kDa that phosphorylates the IB proteins [12, 14, 23, 27,28,29,30]. IKK␥/NEMO was isolated independently as a component of the high molecular weight IKK complex [29, 30] and as a factor, designated FIP-3, that binds to the adenovirus E3 protein and inhibits the cytolytic effects of TNF␣ [40]. These results further establish that IKK␥/NEMO association with the IKK complex is critical for stimulation of IKK kinase activity
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