NF-kappaB is an ubiquitously expressed transcription factor that plays an important role in the immune, anti-apoptotic and inflammatory responses. NF-kappaB is normally sequestered in the cytoplasm by interacting with inhibitory IkappaB molecules. Upon stimulation, IkappaB is phosphorylated and subsequently degraded by the proteasome, allowing NF-kappaB to translocate into the nucleus where they regulate target gene expression. Two kinases, IKK-alpha and IKK-beta, which are responsible for IkappaB phosphorylation were recently identified. We have recently identified a cytokine inducible IKK-i, a kinase related to IKK-alpha and -beta. IKK-i significantly induced NF-kappaB activation upon over-expression, as did IKK-alpha and IKK-beta. Unlike IKK-alpha and IKK-beta, IKK-i phosphorylated Ser36 but not Ser32 in vitro, suggesting that IKK-i activates NF-kappaB by distinct mechanisms from the conventional IKKs.I-TRAF/TANK was isolated as a molecule that interacts specifically with inducible IkappaB 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-kappaB 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-beta. IKK-i over-expression also induced c-Jun N-terminal kinase. These results show that I-TRAF is a substrate of IKK-i. NF-kappaB activation by IKK-i may be mediated through phosphorylation of I-TRAF by IKK-i and subsequent liberation of TRAF2.These results indicate that NF-kappaB activation by IKK-i is mediated through phosphorylation of I-TRAF/TANK by IKK-i and subsequent liberation of TRAF2.