Recombinant IκB Kinases α and β Are Direct Kinases of IκBα*

Activation of the transcription factor NF-κB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IκB. A large multiprotein complex, the IκB kinase (IKK), catalyzes the phosphorylation of IκB. The two kinase components of the IKK complex, IKKα and IKKβ, were overexpressed in insect cells and purified to homogeneity. Both purified IKKα and IKKβ specifically catalyzed the phosphorylation of the regulatory serine residues of IκBα. Hence, IKKα and IKKβ were functional catalytic subunits of the IKK complex. Purified IKKα and IKKβ also preferentially phosphorylated serine as opposed to threonine residues of IκBα, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKα and IKKβ revealed that the kinase activity of IKKβ on IκBα is 50–60-fold higher than that of IKKα. The primary difference between the two activities is the K m for IκBα. The kinetics of both IKKα and IKKβ followed a sequential Bi Bi mechanism. No synergistic effects on IκBα phosphorylation were detected between IKKα and IKKβ. Thus, in vitro, IKKα and IKKβ are two independent kinases of IκBα.

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