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Molecular and Cellular Biology, April 2000, p. 2635-2649, Vol. 20, No. 8
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Functional Isoforms of IB Kinase  (IKK) Lacking Leucine Zipper and Helix-Loop-Helix Domains Reveal that IKK and IKK Have Different Activation Requirements

Fergus R. McKenzie,^1, Margery A. Connelly,^1, Darlene Balzarano,¹ Jurgen R. Müller,^1,§ Romas Geleziunas,² and Kenneth B. Marcu^1,*

Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215,¹ and Gladstone Institute of Virology and Immunology, San Francisco, California 94141-9100²

Received 7 September 1999/Returned for modification 13 October 1999/Accepted 18 January 2000

The activity of the NF-B family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IB subunits. Site-specific serine phosphorylation of IBs by two IB kinases (IKK [also known as CHUK] and IKK) targets them for proteolysis. IKK and - have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic -helical domains. Here, we describe the properties of two novel cellular isoforms of IKK: IKK-H and IKK-LH. IKK-H and IKK-LH are differentially spliced isoforms of the IKK mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKK is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKK-H and IKK-LH, like IKK, respond to tumor necrosis factor alpha stimulation to potentiate NF-B activation in HEK293 cells. A mutant, catalytically inactive form of IKK blocked IKK-, IKK-H-, and IKK-LH-mediated NF-B activation. Akin to IKK, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-B-inducing kinase). In contrast to IKK, IKK-LH failed to associate with either itself, IKK, IKK, or NEMO-IKK-IKKAP1, while IKK-H complexed with IKK and IKK but not with NEMO. Interestingly, each IKK isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKK could not. IKK-Cm, a recombinant mutant of IKK structurally akin to IKK-LH, was equally functional in these assays, but in sharp contrast, IKK-Cm, a structurally analogous mutant of IKK, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKK and IKK need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKK-H and IKK-LH isoforms illustrates potential modes of NF-B activation, which are not subject to the same in vivo regulatory constraints as either IKK or IKK.

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^* Corresponding author. Mailing address: Department of Biochemistry and Cell Biology, SUNY at Stony Brook, Stony Brook, NY 11794-5215. Phone: (516) 632-8553. Fax: (516) 632-9730. E-mail: kmarcu{at}ms.cc.sunysb.edu.

Present address: CNRS-UMR 6543, 06189 Nice cédex, France.

Present address: Dept. of Pharmacology, SUNY at Stony Brook, Stony Brook, NY, 11794.

^§ Present address: NIH, NIAID, Bldg. 10, Room 11B16, Bethesda, MD 20892-1876.

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Molecular and Cellular Biology, April 2000, p. 2635-2649, Vol. 20, No. 8
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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