Effective Of The Interaction Between CK8 And NEMO/IKKγ On NF-κB Signal Pathway

Nuclear factor-κB (NF-κB) is a ubiquitously expressed family of transcription factors ,play a centrol role in regulating the expression of a wide variety of genes involved in diverse physiological and pathological processes, including cell growth/death,immune/inflammatory response, stress responses, and oncogenesis. A key event leading connecting extracellular stimuli to NF-κB activation is the regulation of IKK(Inhibitior of kappaB kinase) activity.In some cases, the dysregulation of this pathways could result in certain human diseases, especially in liver. For example, hepatitis, liver fibrosis, hepatocirrhosis and even liver cancer were already proved to be concerned with NF-κB signaling pathway. The IKK complex contains two highly homologous kinase subunits,IKK1/IKKαand IKK2/IKKβ,and a regulatory subunit NEMO/ IKKγ,Therefore, we identified CK8 as NEMO/ IKKγ-interacting partner by yeast two-hybrid screen of fetal liver cDNA library using NEMO/ IKKγas a bait .CK8 is typeⅡcytokeratin which primarily expressed in epithelial cells and many epithelial cell-derived neoplasms. CK8/18 are essential for maintaining structural integrity, they also exert non-mechanical functions, modulate hepatic cell adhesion, size and G1/S transition.This paper tested the unknown interaction between CK8 and NEMO/IKKγ. Firstly,we confirmed the physiological interaction between CK8 and NEMO/IKKγby extragenous and endogenous co-immunoprecipitation,GST-pull down and fluorescence co-localization,and mapped CK8-NEMO/IKKγinteracting domains,we found NEMO/ IKKγ(1-229aa)interacted with CK8,and CK8(139-235aa),CK8(253-402aa) interacted with NEMO/ IKKγ.Then ,to test a possible involvement of CK8 in the NF-κB activation pathway, in reporter gene assay, we found overexpressed CK8 inhibition basal NF-κB activation in a dose-dependent mannar,overexpression of CK8 inhibited TNFα-induced NF-κB activity as well as its basal transcriptional activity;CK8 inhibited TRAF2 (TNFR-associated factor 2)- mediated NF-κB activation, did not inhibit TRAF6- mediated NF-κB activation; CK8 inhibited NF-κB activation by overexpression of IKKα,IKKβand IKKγ,this suggested that NEMO/ IKKγmight be the target molecule upon which CK8 acts. Finally ,to explore the mechanism by which the interactions of CK8 with NEMO/IKKγdown-regulate NF-κB signaling, we found CK8, TRAF2 and NEMO/IKKγwere present in the same complex,by In Vivo Ubiquitination and De-ubiquitination Assays we found CK8 Inhibits NF-κB by De-ubiquitinating TRAF2 and NEMO/IKKγin a dose-dependent mannar ,we also fonud CK8 enhanced the stability of NEMO/ IKKγand IκBα,CK8 overexpression decreased significantly the nuclear translocation of NF-κB P65.These results indicated that the inhibition of NF-κB activation by CK8 is mediated, at least in part, by the deubiquitination and inactivation of TRAF2, and following deubiquitination of NEMO/IKKγ.In addition, CK8 indeed suppressed expression of NF-κB target genes such as Flips and XIAP which can inhibit apoptosis,but the relationship of CK8 with apoptosis remains to be elucidated.In conclusion, the interaction between CK8 and NEMO/IKKγplay an important role in the NF-κB signaling, and these findings may help us to explore a new regulatory mechanism in NF-κB pathway.