Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity

• Arfaptin 2 is a novel IKKbeta and NEMO-binding protein.
• Arfaptin 2 is able to negatively regulate NF-kappaB signaling.
• Dimerization of arfaptin 2 is essential to inhibit NF-kappaB activation through multimodal interaction with IKKbeta/NEMO.

IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner. In this study, arfaptin 2 was identified as an IKKβ-specific binding partner. Interestingly, arfaptin 2 also interacted with NEMO. Domain mapping studies revealed that the C-terminal region, including the IKKβ HLH domain and the first coiled-coil NEMO region were respectively required for interactions with the arfaptin 2 N-terminal flexible region. Overexpression of arfaptin 2 inhibited tumor necrosis factor (TNF)-α-stimulated nuclear factor-κB (NF-κB) signaling, whereas downregulation of arfaptin 2 by small interfering RNA enhanced NF-κB activity. Dimerization of arfaptin 2 through the Bin–Amphiphysin–Rvs domain may be essential to inhibit activation of NF-κB through multimodal interactions with IKKβs or IKKβ/NEMO, as ectopic expression of the arfaptin 2 fragment responsible for IKK interactions did not change TNFα-stimulated NF-κB activation. These data indicate that arfaptin 2 is the first molecule to regulate NF-κB signaling by interacting with the functional IKK complex but not by direct inhibiting IKKβ phosphorylation.