Transcription factor NF-kappaB governs the expression of multiple genes that mediate innate and adaptive immunity. The inappropriate, persistent activation of NF-kappaB underlies acute and chronic inflammatory diseases, rendering it an attractive target for therapeutic intervention. Biologic inducers of NF-kappaB include bacterial lipopolysaccharides, proinflammatory cytokines, antigen receptor agonists, and viral gene products such as the Tax oncoprotein of human T-cell leukemia virus type 1. Each of these signal-dependent responses is regulated by I-kappaB, a cytoplasmic inhibitor of NF-kappaB, and an inducible multicomponent I-kappaB kinase called IKK. Following cellular stimulation, the IKKbeta catalytic subunit of IKK phosphorylates I-kappaB, leading to degradation of the inhibitor and the release of NF-kappaB to its nuclear site of action. This grant application focuses on IKKgamma, a noncatalytic subunit of the same macromolecular enzyme complex. Mutations in the X-linked gene encoding IKKgamma can lead to skin inflammation or humeral immunodeficiencies. Although its mechanism of action remains unknown, IKKgamma is required for signal-dependent activation of IKKbeta. Studies in the applicant's laboratory have revealed that IKKgamma is subject to signal-dependent phosphorylation at multiple serine residues via a mechanism involving IKKbeta. This discovery highlights a previously unrecognized interplay between the two subunits that will be exploited to unravel the biochemical function of IKKgamma phosphorylation. The central hypothesis is that IKKbeta-mediated phosphorylation of IKKgamma affects the regulation of I-kappaB kinase activity, leading to altered gene expression and changes in the cellular responses under NF-kappaB control. To test this hypothesis, experiments are proposed to determine the IKKbeta-responsive sites in IKKgamma that are subject to signal-dependent phosphorylation (Specific Aim 1), the intracellular biochemical function of IKKgamma phosphorylation in NF-kappaB signal transduction (Specific Aim 2), and the in vivo phenotype of mice expressing phosphorylation-defective forms of IKKgamma (Specific Aim 3). The latter experiments will uncover the physiologic basis for IKKgamma phosphorylation in inflammation and immunity. Together, these proposed studies will significantly advance our currently limited knowledge about the molecular mechanism of IKKgamma action and provide new animal models to study NF-kappaB signaling in the immune system.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052379-04
Application #
7026473
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Mallia, Conrad M
Project Start
2003-03-01
Project End
2008-02-28
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
4
Fiscal Year
2006
Total Cost
$368,629
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Zhao, Yongge; Ma, Chi A; Wu, Liming et al. (2015) CYLD and the NEMO Zinc Finger Regulate Tumor Necrosis Factor Signaling and Early Embryogenesis. J Biol Chem 290:22076-84
Ni, Chang-Yuan; Wu, Zhao-Hui; Florence, William C et al. (2008) Cutting edge: K63-linked polyubiquitination of NEMO modulates TLR signaling and inflammation in vivo. J Immunol 180:7107-11
Zhao, Tiejun; Yang, Long; Sun, Qiang et al. (2007) The NEMO adaptor bridges the nuclear factor-kappaB and interferon regulatory factor signaling pathways. Nat Immunol 8:592-600
O'Donnell, Sean M; Holm, Geoffrey H; Pierce, Janene M et al. (2006) Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus. J Virol 80:1077-86
Kray, Arlene E; Carter, Robert S; Pennington, Kevin N et al. (2005) Positive regulation of IkappaB kinase signaling by protein serine/threonine phosphatase 2A. J Biol Chem 280:35974-82
Carter, Robert S; Pennington, Kevin N; Arrate, Pia et al. (2005) Site-specific monoubiquitination of IkappaB kinase IKKbeta regulates its phosphorylation and persistent activation. J Biol Chem 280:43272-9
Carter, Robert S; Pennington, Kevin N; Ungurait, Bradley J et al. (2003) In vivo identification of inducible phosphoacceptors in the IKKgamma/NEMO subunit of human IkappaB kinase. J Biol Chem 278:19642-8
Carter, Robert S; Pennington, Kevin N; Ungurait, Bradley J et al. (2003) Signal-induced ubiquitination of I kappaB Kinase-beta. J Biol Chem 278:48903-6