Mechanisms by which IκB proteins control NF-κB activity

Abstract

The biological activity of the transcription factor NF-B is differentially controlled by three IB proteins, IB␣, IB␤, and IB. We have examined the molecular basis for the differential inhibitory strengths of IB proteins by constructing hybrid IBs and found that the first ankyrin repeat of IB␣ is responsible for its strong inhibitory effect. Swapping a putative ␤-turn within the first ankyrin repeat between the strong IB␣ and the weak IB␤ inhibitors switches their in vivo inhibitory activity on NF-B. The qualitatively distinct contacts made by this ␤-turn in IB␣ and IB␤ with NF-B determine the efficiency by which IBs sequester NF-B to the cytoplasm, thus explaining their distinct effects on gene activity.The transcription factor NF-B orchestrates the activation of numerous genes involved in the control of cell activities in the immune system and is also vital for craniofacial, liver, and limb development in higher eukaryotic organisms. NF-B exists in virtually all cell types in the form of dimeric complexes consisting of different members of the Rel family of proteins. In mammals, there are five Rel proteins, p50, p52, p65, c-Rel, and RelB, all of which share an amino-terminal 300 amino acid conserved region known as Rel Homology Region. This region is responsible for DNA binding, dimerization, and nuclear localization. Unlike most transcriptional activators, NF-B resides in the cytoplasm and must therefore translocate to the nucleus to function. Association with the inhibitory IB proteins tightly regulates the activity of NF-B. These interactions have two functional consequences. First, NF-B͞IB complexes are sequestered in the cytoplasm, because IBs mask the nuclear localization signal (NLS) of NF-B, presumably by means of direct protein-protein interactions, and secondly, IBs can inhibit NF-B DNA binding. In response to a large variety of extracellular stimuli, the IB proteins, while still bound to NF-B, are phosphorylated, ubiquitinated, and finally degraded by the proteasome. The free NF-B translocates to the nucleus, where it activates gene transcription (reviewed in refs. 1-5).The IB family consists of three members, IB␣, IB␤, and IB (6-10). Importantly, the carboxyl-terminal regions of the precursors for p50 and p52, p105, and p100, respectively, can also function as IBs. Each member of the family contains six copies of a 33 amino acid module known as ankyrin repeat, which functions as a protein-protein interaction domain. The region carboxyl-terminal to the ankyrin repeats contains a proline (P), glutamate (E), serine (S), and threonine (T) (PEST) sequence regulating basal level protein turnover and is also required for inhibition of DNA binding, whereas the amino-terminal region is the signal responsive domain (2, 5). Despite their extensive structural similarities, IB␣, IB␤, and IB exhibit substantial differences in vivo (10-13). Depending on the cell type and on the stimulus, IBs respond differentially to NF-B-inducing signals. In general, IB␣ is rapidly degraded, whereas IB␤ and IB are degrade...

Vesnarinone Suppresses TNF-Induced Activation of NF-κB, c-Jun Kinase, and Apoptosis

“…Thus, it is possible that vesnarinone inhibited I B␣ phosphorylation by inhibiting the activity of MEKK1. TNF-induced NF-B activation involves the sequential interaction of TNF receptor with TRADD, TRAF2, and NIK, which then activates IKK (48,49). Our findings that vesnarinone blocks NF-B-dependent reporter gene expression induced by TNFR1, TRAF2, and NIK, but not by p65, also suggests that vesnarinone acts at a step downstream from NIK.…”

“…TNF-induced NF-B activation is mediated through sequential interaction of the TNF receptor with TNF receptor associated death domain (TRADD), TRAF2, NIK, and IKK-␤, resulting in phosphorylation of I B␣ (48,49). To delineate the site of action of vesnarinone in the TNF-signaling pathway leading to NF-B activation, cells were transfected with TRAF2, NIK, and p65 plasmids, and then NF-B-dependent SEAP expression was monitored in untreated and vesnarinone-treated cells.…”

Targeting Nuclear Factor-κB Activation Pathway by Thymoquinone: Role in Suppression of Antiapoptotic Gene Products and Enhancement of Apoptosis

“…We next investigated where TQ acts in the sequence of TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-h recruitment that characterizes TNF-induced NF-nB activation (42). In cells transfected with TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, IKK-h, and p65 plasmids, NF-nB -dependent secretory alkaline phosphatase (SEAP) expression was induced; TQ substantially suppressed NF-nB -dependent SEAP expression in all cases (Fig.…”

Simvastatin Potentiates TNF-α-Induced Apoptosis through the Down-Regulation of NF-κB-Dependent Antiapoptotic Gene Products: Role of IκBα Kinase and TGF-β-Activated Kinase-1

“…We investigated where simvastatin acts in the sequence of TNFR1, TRADD, TRAF2, NF-B-inducing kinase (NIK), and IKK recruitment that characterizes TNF-␣-induced NF-B activation (47,48). In cells transfected with TNFR1, TRADD, TRAF2, NIK, IKK␤, and p65 plasmids, NF-B-dependent SEAP expression was induced; simvastatin substantially suppressed NF-B-dependent SEAP expression in all cells except those transfected with p65 (Fig.…”