BackgroundNF‐κB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti‐apoptotic and inflammatory responses. NF‐κB is normally sequestered in the cytoplasm by interacting with inhibitory IκB molecules. Upon stimulation, IκB is phosphorylated and subsequently degraded by the proteasome, allowing NF‐κB to translocate into the nucleus where they regulate target gene expression. Two kinases, IKK‐α and IKK‐β, which are responsible for IκB phosphorylation were recently identified. We have recently identified a cytokine inducible IKK‐i, a kinase related to IKK‐α and ‐β. IKK‐i significantly induced NF‐κB activation upon over‐expression, as did IKK‐α and IKK‐β. Unlike IKK‐α and IKK‐β, IKK‐i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK‐i activates NF‐κB by distinct mechanisms from the conventional IKKs.ResultsI‐TRAF/TANK was isolated as a molecule that interacts specifically with inducible IκB kinase (IKK‐i) by the yeast two‐hybrid screening procedure. The association of IKK‐i and I‐TRAF is mediated via the interaction between the N‐terminal domain of I‐TRAF and the C‐terminal portion of IKK‐i. In vitro kinase assays demonstrate that IKK‐i phosphorylates I‐TRAF in the middle portion that associates with TRAF2. Interestingly, TRAF2 is freed from the I‐TRAF/TRAF2 complex after I‐TRAF phosphorylation. NF‐κB activation by IKK‐i is significantly blocked by coexpression of the N‐terminal domain of I‐TRAF, dominant negative TRAF2, and dominant negative NIK and IKK‐β. IKK‐i over‐expression also induced c‐Jun N‐terminal kinase. These results show that I‐TRAF is a substrate of IKK‐i. NF‐κB activation by IKK‐i may be mediated through phosphorylation of I‐TRAF by IKK‐i and subsequent liberation of TRAF2.ConclusionThese results indicate that NF‐κB activation by IKK‐i is mediated through phosphorylation of I‐TRAF/TANK by IKK‐i and subsequent liberation of TRAF2.