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Molecular and Cellular Biology, February 1999, p. 1526-1538, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Ikappa B Kinase (IKK)-Associated Protein 1, a Common Component of the Heterogeneous IKK Complex

Frank Mercurio,1,* Brion W. Murray,1 Andrej Shevchenko,2 Brydon L. Bennett,1 David B. Young,1 Jian Wu Li,1 Gabriel Pascual,1 Aparna Motiwala,1 Hengyi Zhu,1 Matthias Mann,2 and Anthony M. Manning1

Signal Pharmaceuticals, Inc., San Diego, California 92121,1 and Center for Experimental Bioinformatics, Odense University, Odense, Denmark2

Received 7 July 1998/Returned for modification 7 August 1998/Accepted 9 October 1998

Activation of the transcription factor NF-kappa B is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, Ikappa B. We recently purified a large multiprotein complex, the Ikappa B kinase (IKK) signalsome, which contains two regulated Ikappa B kinases, IKK1 and IKK2, that can each phosphorylate Ikappa Balpha and Ikappa Bbeta . The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappa B signal transduction cascade in vivo. In this report, we demonstrate reconstitution of Ikappa B kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappa B signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappa B signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappa B activation.

* Corresponding author. Mailing address: Signal Pharmaceuticals, Inc., 5555 Oberlin Dr., San Diego, CA 92121. Phone: (619) 558-7500 ext. 8129. Fax: (619) 623-0870. E-mail: fmercuri{at}signalpharm.com.

Molecular and Cellular Biology, February 1999, p. 1526-1538, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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