Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha...

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Bibliographic Details
Published in:The Journal of biological chemistry 1998-11, Vol.273 (46), p.30736-30741
Main Authors: Li, J, Peet, G W, Pullen, S S, Schembri-King, J, Warren, T C, Marcu, K B, Kehry, M R, Barton, R, Jakes, S
Format: Article
Language:English
Subjects:
Animals
Catalysis
DNA-Binding Proteins - metabolism
Humans
I-kappa B Kinase
I-kappa B Proteins
Jurkat Cells
Kinetics
NF-KappaB Inhibitor alpha
Phosphorylation
Protein-Serine-Threonine Kinases - metabolism
Recombinant Proteins - metabolism
Spodoptera
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Summary:Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.
ISSN:0021-9258