RECOMBINANT I-KAPPA-B KINASE-ALPHA AND KINASE-BETA ARE DIRECT KINASESOF I-KAPPA-B-ALPHA

Activation of the transcription factor NF-kappa B is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, I kappa B. A large multiprotein complex, the I kappa B kinase (IKK), c atalyzes the phosphorylation of I kappa B. The two kinase components o f the IKK complex, IKK alpha and IKK beta, were overexpressed in insec t cells and purified to homogeneity. Both purified IKK alpha and IKK b eta specifically catalyzed the phosphorylation of the regulatory serin e residues of I kappa B alpha. Hence, IKK alpha and IKK beta were func tional catalytic subunits of the IKK complex. Purified IKK alpha and I KK beta also preferentially phosphorylated serine as opposed to threon ine residues of I kappa B alpha, consistent with the substrate prefere nce of the IKK complex. Kinetic analysis of purified IKK alpha and IKK beta revealed that the kinase activity of IKK beta on I kappa B alpha is 50-60-fold higher than that of IKK alpha. The primary difference b etween the two activities is the K-m for I kappa B alpha. The kinetics of both IKK alpha and IKK beta followed a sequential Bi Bi mechanism. No synergistic effects on I kappa B alpha phosphorylation were detect ed between IKK alpha and IKK beta. Thus, in vitro, IKK alpha and IKK b eta are two independent kinases of I kappa B alpha.