Complete reconstitution of human IκB kinase (IKK) complex in yeast -: Assessment of its stoichiometry and the role of IKKγ on the complex activity in the absence of stimulation

The I kappaB kinase (IKK) complex, composed of two catalytic subunits (IKK alpha and IKK beta) and a regulatory subunit (IKK gamma), 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 IKK alpha . beta . gamma complex is alpha (1)beta (1)gamma (2), and the core subunit composition of IKK beta . 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 IKK gamma, IKK beta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKK gamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKK beta or IKK alpha prevented IKK gamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKK gamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.