IKKα and IKKβ serve as kinase-independent scaffolds that prevent proteasomal degradation of NEMO

NF-κB essential modulator (NEMO) is the critical scaffold protein of the canonical I kappa B kinase (IKK) complex. Whilst studying the contribution of IKKα and IKKβ to NF-κB signalling we noted that NEMO protein levels were reduced in IKK knockout (KO) HCT116 cells. Loss of NEMO was more pronounced in IKKα KO cells than IKKβ KO, whilst IKKα+IKKβ double knockout (DKO) cells exhibited a 90% reduction in NEMO abundance. In contrast, the abundance of IKKs was unaffected by NEMO KO, although this abolished canonical NF-κB activation. Loss of IKKs had no effect on NEMO mRNA levels but IKK DKO cells exhibited accelerated NEMO protein turnover that was reversed by the proteasome inhibitor, MG132, but not the NEDDylation inhibitor, MLN4924. TAK-243, a selective inhibitor of UBA1, the E1 ubiquitin-activating enzyme, increased basal NEMO levels in DKO cells but did not prevent NEMO turnover suggesting both ubiquitylation-dependent and independent pathways for degradation. Bioinformatic analysis identified substantial regions of intrinsic disorder within the N- and C-termini of NEMO. NEMO was rapidly degraded by isolated 20S proteasomes in vitro and this was inhibited by interaction with IKKα or IKKβ. Finally, re-expression of IKKα increased NEMO expression in IKK DKO cells and this required the C-terminal NEMO binding domain (NBD) but not a functional kinase domain. Thus the IKKs act as critical kinase-independent scaffolds that stabilise NEMO; loss of these interactions results in NEMO degradation via the proteasome. These results may be relevant to strategies for inhibiting NF-kB signalling by disrupting NEMO:IKK interactions.