title: Complete reconstitution of human IkappaB kinase (IKK) complex in yeast. Assessment of its stoichiometry and the role of IKKgamma on the complex activity in the absence of stimulation.
abstract: The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB). To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK. For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells. We show that there is one regulatory subunit per kinase subunit. Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2). The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells. In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKKgamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.
authors.affiliation: Department of Molecular Microbiology and Immunology and Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9176, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Miller
authors.forename: E
authors.initials: E
authors.lastname: Zandi
mesh_terms: D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004789:Enzyme Activation; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human