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The IKK␤ and NEMO/IKK␥ subunits of the NF-B-activating signalsome complex are known to be essential for activating NF-B by inflammatory and other stresslike stimuli. However, the IKK␣ subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-B-dependent and -independent functions. In contrast to this generally accepted view of IKK␣'s physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKK␤ and NEMO/IKK␥, IKK␣ is also a global regulator of tumor necrosis factor ␣-and IL-1-responsive IKK signalsome-dependent target genes including many known NF-B targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, ␤ 2 -microglobulin, IL-1␣, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-B-dependent target genes were preferentially dependent on IKK␣ or IKK␤. Constitutive expression of a trans-dominant I B␣ superrepressor (I B␣SR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-B. A subset of NF-B target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-B in established MEFs. Overall, a sizable number of novel NF-B/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-␤ and -␦, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (␣ and ␤) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-B/IKK-induced gene, suggesting that IKKmediated NF-B activation can coordinately illicit negative effects on STAT signaling.The NF-B transcription factors are pivotal regulators of gene expression programs culminating in stress-like responses and the genesis of innate and acquired immunity (reviewed in Refs. 1-4). A host of extracellular stimuli including inflammatory cytokines, viral and bacterial infections, oxidative and DNA-damaging agents, UV light, and osmotic shock can all result in NF-B activation (1, 3-5). NF-B transcription factors bind to DNA as hetero-or homodimers that are selectively derived from five possible subunits (RelA/p65, c-Rel, RelB, p50, and p52) with each binding to half of a conserved 10-base pair consensus sequence (GGGRNWTYCC) (1, 5). Whereas the RelA/p65 and p50 subunits are ubiquitously expressed, the p52, c-Rel, and RelB subunits are more functionally important in specific differentiated cell types (1, 6). Cytoplasmic p50/p65 heterodimers, c-Rel homodimers, and RelB are bound to I Bs (inhibitors of NF-B...

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Activation of the transcription factor NF-B is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IB. A large multiprotein complex, the IB kinase (IKK), catalyzes the phosphorylation of IB. The two kinase components of the IKK complex, IKK␣ and IKK␤, were overexpressed in insect cells and purified to homogeneity. Both purified IKK␣ and IKK␤ specifically catalyzed the phosphorylation of the regulatory serine residues of IB␣. Hence, IKK␣ and IKK␤ were functional catalytic subunits of the IKK complex. Purified IKK␣ and IKK␤ also preferentially phosphorylated serine as opposed to threonine residues of IB␣, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKK␣ and IKK␤ revealed that the kinase activity of IKK␤ on IB␣ is 50 -60-fold higher than that of IKK␣. The primary difference between the two activities is the K m for IB␣. The kinetics of both IKK␣ and IKK␤ followed a sequential Bi Bi mechanism. No synergistic effects on IB␣ phosphorylation were detected between IKK␣ and IKK␤. Thus, in vitro, IKK␣ and IKK␤ are two independent kinases of IB␣.The transcription factor NF-B plays a critical role in immune and inflammatory responses. It is regulated by the signaling of receptors for inflammatory cytokines such as TNF-␣, 1 IL-1, or other external stimuli (1). In resting cells, NF-B is sequestered in the cytoplasm through its association with inhibitory proteins termed IB. Two IB proteins, IB␣ and IB␤, are rapidly phosphorylated at Ser residues in the Nterminal region upon stimulation by TNF-␣ and IL-1. The regulated phosphorylation is at Ser-32 and Ser-36 of IB␣ and, correspondingly, Ser-19 and Ser-23 of IB␤ (2, 3). The more recently cloned IB isoform, IB⑀, also contains the two conserved Ser residues at the N terminus for signal-induced degradation (4). Phosphorylated IB␣ and IB␤ are subsequently ubiquitinated and undergo ubiquitin-dependent degradation by the 26 S proteasome (3, 5). Degradation of IB results in the release of NF-B, which then translocates to the nucleus, where it up-regulates the transcription of its target genes (1).A 500 -900-kDa protein complex that contains the TNF-␣-induced IB kinase (IKK) has been purified and characterized independently by two groups (6, 7). The IKK complex phosphorylates IB␣ at the specific Ser residues that target the protein for ubiquitination and degradation. Two kinase subunits of the IKK complex, denoted IKK␣ and IKK␤, have been cloned (6 -10). IKK␣ or IKK␤ overexpressed in mammalian cells specifically phosphorylates IB␣ and IB␤ after immunoprecipitation, and their kinase activities can be induced by TNF-␣ or IL-1 (6 -10). In HeLa cells, expression of antisense IKK␣ inhibited NF-B activation by TNF-␣ or IL-1 (6). Furthermore, overexpression of dominant-negative mutants of either IKK␣ or IKK␤ blocked TNF-␣/IL-1-induced NF-B activation (8, 10). Thus, both IKK␣ and IKK␤ contribute to the activity of the IKK complex and are involved in NF-B activation. An additional protein kinase, NF-B-inducing kina...