Role of IKKγ/NEMO in assembly of the IκB kinase complex

IKK gamma /NEMO is a protein that is critical for the assembly of the high molecular weight I kappaB kinase (IKK) complex. To investigate the role of IKK gamma /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 IKK gamma /NEMO and the IKKs were examined. When expressed alone following transfection, IKK alpha and IKK beta were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKK gamma /NEMO, both IKK alpha and IKK beta migrated at similar to 600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-alpha. When either IKK alpha or IKK beta was expressed alone with IKK gamma /NEMO, IKK beta but not IKK alpha: migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKK beta were both incorporated into the high molecular weight IKK complex in the presence of IKK gamma /NEMO. The amino-terminal region of IKK gamma /NEMO, which interacts directly with IKK beta, was required for formation of the high molecular weight IKK complex and for stimulation of IKK beta kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKK gamma /NEMO is a crucial step involved in IKK function.