Recombinant IκB kinases α and β are direct kinases of IκBα

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), catalyzes the phosphorylation of I kappa B. The two kinase components of the IKK complex, IKK alpha and IKK beta, were overexpressed in insect cells and purified to homogeneity. Both purified IKK alpha and IKK beta specifically catalyzed the phosphorylation of the regulatory serine residues of I kappa B alpha. Hence, IKK alpha and IKK beta were functional catalytic subunits of the IKK complex. Purified IKK alpha and IKK beta also preferentially phosphorylated serine as opposed to threonine residues of I kappa B alpha, consistent with the substrate preference 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 between 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 detected between IKK alpha and IKK beta. Thus, in vitro, IKK alpha and IKK beta are two independent kinases of I kappa B alpha.