(R3794) Investigation of Biochemical Characterization of NEMO and Its Interaction with IKK/NF-kB

Daniel Hwang (University of California, San Diego, University of Pittsburgh)| Myung Ko (University of California, San Diego)

The transcriptional activity of the NF-kB family of protein factors in response to cellular signals is primarily regulated by two other families of proteins- the IkB inhibitors and IkB kinases (IKK). Two distinct signaling pathways activate IKK kinases leading to the degradation of specific IkB inhibitors and release of specific NF-kB factors. The canonical signaling pathway activates IKK2 and the non-canonical, IKK1. The activation of IKK2 is strongly dependent on an adapter protein, NEMO (NF-kB essential modulator), whereas IKK1 activation also requires NF-kB inducing kinase (NIK). In a resting cell, NF-kB remains inactive by association of the inhibitory IkB proteins. Upon upstream stimulation, NEMO promotes IKK2 phosphorylation that induces IkBα leading to its ubiquitination and consequent degradation by proteasome. Free NF-kB can transloca­te in the nucleus and bind to specific sites of gene promoters to induce target gene expressions. IKK complex is an uncharacterized heterotrimer of three proteins, IKK1, IKK2 and NEMO. The kinase domain of IKK2 (and also IKK1) contains a pair of serines within the kinase ‘activation loop’ that undergoes phosphorylation in response to canonical signaling pathway marking IKK2 activation. NEMO is a IKK regulatory subunit and leads to IKK activity. If cells do not activate NEMO, NF-kB will not be functioned through the main pathway. Disease-causing NEMO mutations indicate an impairment of NF-kB activation. The gene encoding NEMO is in the X chromosome. NEMO has two types of disorders by hemizygous or heterozygous NEMO mutations. Thus, NEMO plays a specific protective role in genetic diseases, which provides molecular targets for new therapies. However, there is little information about the NEMO disease and more research is needed to help further studies. To this end, we will first investigate biochemical characterization of NEMO wild-type (WT) and its mutant. Full-length human NEMO WT has already been subcloned into the pET15b vector in frame with an N-terminal hexahistidine tag. A disease-causing NEMO mutant will be prepared by the Quickchange mutagenesis protocol with base changes incorporated in the oligonucleotide primers. His-tagged NEMO proteins will be expressed in BL21 (DE3) cells and then the soluble proteins will be purified by Ni affinity chromatography. Eluted fractions will be collected and loaded on SDS-PAGE gels, and protein concentrations will be determined by Bradford assay. After that, we will compare the degree of self-association between WT and mutant using gel filtration on Fast protein liquid chromatography (FPLC). Since the IKK complex serves a central role in maintaining the NF-kB pathway, further study will involve NEMO:IKK2(or IKK1) interaction in order to provide more information about disease-causing NEMO mutants and their regulation in IKK/NF-kB activation. This would be helpful for future applications to other studies regarding its role in other associated diseases.