Molecular and biological studies of a novel regulatory component of the IkappaB kinase complex essential for the NF-kappaB signaling pathway

The family of NF-κB transcription factors play a central role in innate and adaptive immunity, apoptosis, development and cancer. The mechanism necessary to trigger NF-κB activation in response to a plethora of external signals involves phosphorylation, ubiquitination and degradation of the IκB proteins. Previously, purification of cytokine responsive IkappaB kinase (IKK) activity revealed a high molecular weight multi-protein complex responsible for phosphorylation of the IκB proteins. This complex was predicted to contain several subunits, including two catalytic kinases, IKK1/α (IKK1) and IKK2/β (IKK2) and an essential regulatory subunit, NEMO/IKKγ/IKKAP1 (NEMO). Here, we describe the characterization of a novel regulatory subunit, ELKS, that was co-purified during purification of the IKK complex. Co-immunoprecipitation studies confirm that ELKS is a core component of the high molecular weight IKK signalsome that associates directly with IKK1 and IKK2, but not with NEMO. Utilizing RNA interference (RNAi) to silence ELKS gene expression, we demonstrate that IKK activation is severely impaired in the absence of ELKS leading to a subsequent loss of NF-κB regulated gene expression in response to cytokines, and the inability to protect from programmed cell death. This demonstrates that ELKS is an indispensable component of the IKK complex playing a vital role in NF-κB activation.; We describe a role for ELKS in facilitating the recruitment of IκBα to the IKK complex. An amino-terminal deletion mutant of ELKS can elicit IKK and NF-κB activation in the absence of cytokine induction, advancing insight into IKK activation mechanisms. The regulatory subunits, NEMO and ELKS, are non-redundant essential components for IKK and NF-κB activation. The IKK complex was also found to be associated with the trafficking protein Rab6 through a common association with ELKS.; Finally, a novel member of the ELKS protein family (human) is described here, ELKS2. ELKS2 is strikingly similar to ELKS in its sequence (72% amino acid identity), IKK activating potential and interacting proteins. ELKS2 can influence expression of specific NF-κB target genes, TNFα. The striking difference between the two proteins is their tissue distribution. Future studies into this gene family will give valuable insight into the regulation of IKK and NF-κB transcription in diverse tissues.