Activation of the nuclear factor B (NFB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFB and the inhibitory subunit IB␣ between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IB␣ occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFB⅐IB complex rather than its direct nuclear import or degradation and resynthesis of IB␣. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFB is not complexed to IB␣ in a resting cell. A numerical model is presented to validate the proposed regulation of NFB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IB␣. We suggest that the non-saturated interaction of NFB with the inhibitor may enhance the specificity of action of IB proteins on different NFB dimers and allow additional modes of regulation of IB function. NFB1 is a central mediator of immune and inflammatory responses. It consists of homo-or heterodimers of a family of at least five related proteins characterized by the rel homology domain (1, 2). The known members of this family are c-REL, RELA, RELB, p50, and p52. Homodimers of p50 and p52 are generally considered to be inhibitory, because they lack activation domains, but they may be converted to activators by association with the Bcl3 protein (3, 4). NFB is regulated by a family of at least seven inhibitory proteins, IB, characterized by multiple ankyrin-type repeats (5). The expression of both NFB and IB proteins is cell type-and developmental stagespecific (2). In non-lymphomyeloid cells, the most prevalent NFB species are RELA-p50 heterodimers and p50 homodimers.The current model for NFB function postulates that the transcription factor is anchored in the cytoplasm by association with the inhibitor in unstimulated cells (6). Whereas both NFB and IB have nuclear localization signals, resulting in import of the free factors to the nucleus, these are mutually masked within the complex to abolish their nuclear import functions (7). A diverse range of stimuli, including inflammatory cytokines, bacterial lipopolysaccharide, and phorbol esters (8 -10), result in the serine phosphorylation of IB and its subsequent degradation (11, 12). Translocation of the free transcription factor to the nucleus and activation of genes bearing cognate binding sites ensues. Tyrosine phosphorylation of IB␣, without subsequent degradation, has also been reported to release active NFB into the nucleus (13). In addition to nuclear translocation, phosphorylation of NFB may be important for its full activation (14 -17). This phosphorylation may be induced by parallel pathways initiated by the same stimulus as that causing IB degradation.We have pre...

We have studied the dynamics of nuclear translocation during nuclear factor B activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1␤ caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1␤ was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1␤ and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1␤ suggesting that the pathway is limited by incoming signals. The response to IL-1␤ was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFP-RELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.Inflammatory responses involve the rapid and coordinated activation of a diverse set of genes. This activation is initiated by a variety of agonists including bacterial lipopolysaccharide, phorbol esters, IL-1, 1 and tumor necrosis factor-␣ (1-6) and in addition by stress (7,8) and cell adhesion (9, 10). These agents dynamically regulate cytoplasmic signal transduction networks dependent on developmental stage-and cell type-specific expression of extracellular receptors and intracellular adaptor proteins. A major downstream target of these networks is the NF-B family of transcription factors (11-15) that has been shown to possess both pro-and anti-apoptotic functions dependent on cell type (16, 17).NF-B transcription factors are hetero-or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains (p65-RELA, RELB, and c-REL) and those lacking activation domains (p50, p52) (18). The prototypical NF-B is a heterodimer of p65 (RELA) and p50 (NF-B1). Additionally, a family of at least seven inhibitory subunits, IB, characterized by multiple ankyrin-like repeats has been described (19,20). The paradigm for NF-B activation is nuclear translocation of p50 (NF-B1)/p65 (RELA) heterodimers following IB␣ degradation. Specifically, IB␣ binds to these dimers, thereby masking their nuclear localization signals and preventing nuclear import. Activation of cytoplasmic signal transduction pathways results in the phosphorylation and then ubiquitination and subsequent degradation of IB␣, allowing the dimers to move to the nucleus w...

The Tat transactivator protein of human immunodeficiency virus type 1 contains a highly conserved cysteine-rich region, containing seven cysteines from residues 22 through 37. To investigate the importance of noncysteine residues in this region of the Tat protein, we have carried out a mutational analysis, in most cases substituting a single alanine for the wild-type noncysteine residue. Alanine substitution of residue 23, 24, 46, or 47 had no effect on Tat activity in plasmid transfection assays. In contrast, alanine substitutions of all eight noncysteines analyzed, from residues 26 through 41, significantly reduced the activity of the Tat protein, in some cases as drastically as mutations in cysteine residues. The results demonstrate that the precise sequence of the cysteine-rich region is crucial for a fully functional Tat protein.

Background:Anthracycline is an antitumor agent of the topoisomerase inhibitor family. Results: Doxorubicin inhibits the expression of hypoxia-inducible genes, suppresses HIF-dependent migration of target tumors, and dampens angiogenic response of the host heart. Conclusion: Doxorubicin blocks recruitment of HIF heterodimers to the enhancer and inhibits hypoxia response. Significance: The pleiotropic effect of doxorubicin on HIF signaling provides a clue for understanding efficacy and toxicity of cancer chemotherapy.