An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, DifferentiallyMediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation

Abstract

NF-B is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKK␣ and IKK␤, and a noncatalytic subunit, IKK␥. IKK␥ is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKK␥ mRNA isoform, encoding an in-frame deletion of exon 5, termed IKK␥-⌬. Using a specific reverse transcription-PCR assay, we find that IKK␥-⌬ is widely expressed in cultured human cells and normal human tissues. Because IKK␥-⌬ protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax Nuclear factor-B (NF-B) is an inducible transcription factor that controls the expression of inducible inflammatory and antiapoptotic genes (49, 51). NF-B responds to a diverse series of inflammatory activators, including UV light, doublestranded RNA, cytokines, vasoactive peptides, and viral oncogenes through several distinct intracellular pathways (reviewed in reference 36). Because of its central role as an integrator of stress and inflammatory stimuli, the pathways controlling NF-B have been intensively investigated.Currently, it is thought that NF-B activation is controlled by two distinct pathways, termed the canonical (23) and noncanonical pathways (9, 43). The canonical pathway controls nuclear translocation of the prototypical NF-B complex, composed of 65-kDa Rel A-50-kDa NF-B1 heterodimers. Under normal conditions, the Rel A · NF-B1 complex is sequestered and inactivated in the cytoplasm by the IB inhibitors, proteins which inactivate Rel A DNA binding and nuclear translocation by masking its nuclear localization sequence (reviewed in reference 1). NF-B activators induce IB phosphorylation on serine residues 32 and 36 on its NH 2 -terminal regulatory domain (reviewed in reference 23). Phospho-IB is then specifically bound by the Skp1-Cullin-F-Box-type E3 ubiquitin ligase, E3RS, initiating IB ubiquitination and proteolysis through the proteasome (4, 23, 24) and calpain pathways (15). Nuclear translocated NF-B binds high-affinity chromatin sites and activates the expression of a diverse gene network (50) by inducing assembly of active promoters (2) and recruiting coactivators to target gene promoters (44).The cytoplasmic IB kinase (IKK), also known as the "signalsome," is the rate-limiting kinase responsible for inducible IB␣ phosphorylation (23) and is composed of core catalytic kinases and scaffolding proteins. The catalytic core contains the ubiquitous helix-loop-helix proteins IKK␣ and IKK␤ (33,54), associated with the noncatalytic regulatory protein, IKK␥, in a precise stoichiometric relationship of 2 catalytic subuni...

Involvement of a Novel Rac/RhoA Guanosine Triphosphatase-Nuclear Factor-κB Inducing Kinase Signaling Pathway Mediating Angiotensin II-Induced RelA Transactivation

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IκB kinase complexes: gateways to NF-κB activation and transcription

“…In NEMO, the kinase-binding region (KBD) has been mapped to various, in part non-overlapping N-terminal sequences between amino acids 50-93 (May et al, 2000), 105-200 (Poyet et al, 2000) or 1-196 and 65-196 for IKKa and IKKb, respectively (Tegethoff et al, 2003). A recently described frequent NEMO splice variant, IKKgD (Hai et al, 2006), lacking amino acids 174-224 (Figure 2), binds both IKK kinases, and thus would delimit the KBD to sequences N-terminal to residue 174. The different regions in NEMO required for binding to IKKa or IKKb suggest non-equivalent interactions.…”

Retinoic Acid-Inducible Gene I Mediates Early Antiviral Response and Toll-Like Receptor 3 Expression in Respiratory Syncytial Virus-Infected Airway Epithelial Cells

“…Nuclear extracts (NE) were prepared as described previously (17). A total of 5 g NE was incubated in DNA binding buffer (5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol, 5 mM MgCl 2 , 0.5 mM EDTA) with 1.5 g of poly(dA-dT) and 0.1 nM IRDye 700/IRDye 800-labeled ds oligonucleotide (Table 2) in a total volume of 10 l. Immunofluorescence microscopy.…”