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Molecular and Cellular Biology, February 1999, p. 1526-1538, Vol. 19, No. 2
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-
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
I
B Kinase (IKK)-Associated Protein 1, a Common
Component of the Heterogeneous IKK Complex
B is controlled by the
sequential phosphorylation, ubiquitination, and degradation of its
inhibitory subunit, I
B. We recently purified a large multiprotein complex, the I
B kinase (IKK) signalsome, which contains two
regulated I
B kinases, IKK1 and IKK2, that can each phosphorylate
I
B
and I
B
. The IKK signalsome contains several additional
proteins presumably required for the regulation of the NF
B signal
transduction cascade in vivo. In this report, we demonstrate
reconstitution of I
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-
B
signal transduction pathway. IKKAP1 therefore appears to mediate an
essential step of the NF-
B signal transduction cascade.
Heterogeneity of IKK complexes in vivo may provide a mechanism for
differential regulation of NF-
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.
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