Regulation of nuclear factor kappaB subunit c-Rel through phosphorylation by two IKK-related kinases, IKKepsilon and TBK-1

The nuclear factor κB (NF-κB) transcription factors are key regulators of immunomodulatory genes regulation. NF-κB activity is regulated through the phosphorylation of inhibitory proteins (IκBs) by the IκB kinase (IKK) complex (IKK α/β/γ), leading to IκB degradation and NF-κB translocation to the nucleus where they promote transcription of immunoregulatory genes. Moreover, cRel and p65 activities are also regulated by direct phosphorylation of their transactivation domain. Recently, two IKK non-canonical homologues, IKKϵ and TBK-1 (TANK binding kinase-1) have been identified with functions distinct from the classical IKKα/IKKβ. TBK-1/IKKϵ trigger antiviral immunity through direct phosphorylation of the IRF3/IRF7 transcription factors, which are key regulators of the interferon response. Since IKKϵ modulates the activity of IRF3/IRF7, it is of interest to assess whether IKKϵ/TBK-1 also regulates the transactivation activity of NF-κB. Our hypothesis was that IKKϵ/TBK-1 modulates the activity of cRel by direct phosphorylation of its transactivation domain (TD). In this study, we demonstrate that IKKϵ and TBK-1 directly phosphorylate cRel in vitro and in vivo. Two of the three consensus sequences recognized by IKKϵ/TBK-1 in the cRel TD are directly phosphorylated by IKKϵ. cRel was translocated to the nucleus in cells expressing wild type versus kinase dead variant. The expression of IKKϵ increases c-Rel transactivation in reporter gene assays. Serine to alanine mutation was further used to characterize the function of this phosphorylation at the level of nuclear translocation and transactivation potential using immunofluorescence and reporter gene assay. Furthermore, co-expression studies revealed that IKKϵ and not the kinase dead variant is responsible for cRel degradation in a dose-dependent manner and this effect is partially reverted by proteasome inhibition. These results suggest a new level of regulation for cRel by direct phosphorylation by the IKK-related kinases IKKϵ/TBK-1.