The transcription factor NF-B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein IB␣ maintains NF-B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, IB␣ is rapidly degraded, leading to nuclear translocation of free NF-B. However, NF-B-dependent transcription of the IB␣ gene leads to rapid resynthesis of the IB␣ protein and inhibition of NF-B-dependent transcription. Here we demonstrate a new regulatory function of IB␣ exerted on NF-B in the nuclear compartment. Although normally found in the cytoplasm, IB␣, newly synthesized in response to tumor necrosis factor or interleukin 1, is transported to the nucleus. In the nucleus IB␣ associates with the p50 and p65 subunits of NF-B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of IB␣ correlates with transcription termination of transfected NF-B-dependent luciferase genes. Following the appearance of IB␣ in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-B-IB␣ complexes are cleared from the nucleus.Nuclear factor B (NF-B) is a sequence-specific DNAbinding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus (HIV), and a wide variety of cellular genes, particularly those involved in immune and inflammatory responses (for a review, see reference 6). NF-B is composed of two polypeptide species with molecular weights of 50,000 (p50) and 65,000 (p65) (7,27). Cloning of the p50 (21, 29) and p65 (40, 44) subunits of the NF-B heterodimer showed that they belong to a multigene family (the rel family) of proteins, all of which are implicated in transcriptionally regulated processes, such as the expression of cytokines, acute-phase response genes, and the determination of the dorsoventral polarity in early insect embryos (for a review, see reference 22).A major component of the regulation of p50-p65 complex activity is the control of the intracellular localization of the heterodimer. Indeed, in most cell types, NF-B is retained in an inactive form in the cytoplasm by the inhibitor protein IB␣ (for a review, see reference 6). Induction of NF-B by extracellular or intracellular stimuli has been demonstrated in a large number of cell types. Thus, it has been shown that following cell stimulation by a number of inducers, such as specific antigen recognition, tumor necrosis factor (TNF), interleukin 1 (IL-1), UV light, phorbol esters, bacterial lipids, oxygen radicals, or DNA and RNA virus infections (for a review, see reference 6), the inhibitory capacity of IB␣ is lost and transcriptionally active NF-B is translocated into the nucleus. IB␣, or MAD-3 (23), belongs to a family of proteins including IB␤ (50), Bcl-3 (41), cactus (18, 28), and the carboxy-terminal region of p105 IB␥ (11,26,33,43), which are c...

Recent studies have indicated a critical role for STIM (stromal interacting molecule) proteins in the regulation of the store-operated mode of receptor-activated Ca 2+ entry. Current models emphasize the role of STIM located in the endoplasmic reticulum membrane, where a Ca 2+ -binding EF-hand domain within the N-terminal of the protein lies within the lumen and is thought to represent the sensor for the depletion of intracellular Ca 2+ stores. Dissociation of Ca 2+from this domain induces the aggregation of STIM to regions of the ER immediately adjacent to the plasma membrane where it acts to regulate the activity of store-operated Ca 2+ channels. However, the possible effects of STIM on other modes of receptor-activated Ca 2+ entry have not been examined. Here we show that STIM1 also regulates the arachidonic-acid-regulated Ca 2+ -selective (ARC) channels -receptor-activated Ca 2+ entry channels whose activation is entirely independent of store depletion. Regulation of the ARC channels by STIM1 does not involve dissociation of Ca 2+ from the EF-hand, or any translocation of STIM1. Instead, a critical role of STIM1 resident in the plasma membrane is indicated. Thus, exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1 inhibits ARC channel activity without significantly affecting the store-operated channels. A similar specific inhibition of the ARC channels is seen in cells expressing a STIM1 construct in which the N -linked glycosylation sites essential for the constitutive cell surface expression of STIM1, were mutated. We conclude that, in contrast to store-operated channels, regulation of ARC channels by STIM1 depends exclusively on the pool of STIM1 constitutively residing in the plasma membrane. These data demonstrate that STIM1 is a more universal regulator of Ca 2+ entry pathways than previously thought, and appears to have multiple modes of action.

Transcriptional activation of nuclear factor B (NF-B) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IB␣. However, NF-B activation induces rapid resynthesis of IB␣, which is responsible for post-induction repression of transcription. Newly synthesized IB␣ translocates to the nucleus, where it dissociates NF-B from DNA and transports NF-B from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-B-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-B-dependent transcriptional activation mediated by interleukin 1␤ or tumor necrosis factor ␣. This is a consequence of the inhibition of signal-induced degradation of IB␣. Although LMB treatment does not affect the signal transduction pathway leading to IB␣ degradation, it blocks IB␣ nuclear export. IB␣ is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IB␣ is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IB␣ is therefore essential for maintaining a low level of IB␣ in the nucleus and allowing NF-B to be transcriptionally active upon cell stimulation.The NF-B 1 /Rel family of transcription factors is implicated in regulation of the expression of a number of cellular genes involved in immune responses, inflammation ,and apoptosis (for recent reviews, see Refs. 1-3). In vertebrates, the NF-B family of proteins is composed of transcriptionally active p65/ Rel A (4, 5), c-Rel (6), or Rel B (7) and transcriptionally silent p50/NF-B1 (8, 9) or p52/NF-B2 (10 -12). All NF-B proteins share a conserved region known as the Rel homology domain that contains the nuclear localization signal as well as the dimerization and DNA binding functions. The NF-B form activated by extracellular signals is composed of p50 and p65. NF-B transcriptional activity is controlled by inhibitor IB proteins that contain ankyrin repeat domains. Association of p50/p65 with IB not only occludes the nuclear localization sequence of p50 and p65, leading to cytoplasmic sequestration, but also prevents NF-B DNA binding activity. Several IBs have been described including IB␣ (13), IB␤ (14), IB⑀ (15), and Bcl-3 (16). Additionally, the precursors of p50 (p105) and p52 (p100) possess inhibitory ankyrin repeat domains that in isolation are known as IB␥ (17-19) and IB␦ (20, 21), respectively.IB␣ is organized in three domains: (a) an unstructured amino-terminal (aa 1-72) signal response domain, (b) a central region (aa 73-242) consisting of five ankyrin repeat domains, and (c) a carboxyl-terminal region (aa 243-317) containing a highly acidic domain (aa 276 -317) that is bound to the ankyrin repeat domain by a protease-sensitive linker (aa 243-275) and is protected by bound p65 (22)....

Agonist-activated Ca2+ entry plays a critical role in Ca 2+ signalling in non-excitable cells. One mode of such entry is activated as a consequence of the depletion of intracellular Ca 2+ stores. This depletion is sensed by the protein STIM1 in the endoplasmic reticulum, which then translocates to regions close to the plasma membrane where it induces the activation of store-operated conductances. The most thoroughly studied of these conductances are the Ca 2+ release-activated Ca 2+ (CRAC) channels, and recent studies have identified the protein Orai1 as comprising the essential pore-forming subunit of these channels. Although evidence suggests that Orai1 can assemble as homomultimers, whether this assembly is necessary for the formation of functional CRAC channels and, if so, their relevant stoichiometry is unknown. To examine this, we have used an approach involving the expression of preassembled tandem Orai1 multimers comprising different numbers of subunits into cells stably overexpressing STIM1, followed by the recording of maximally activated CRAC channel currents. In each case, any necessity for recruitment of additional Orai1 units to these preassembled multimers in order to form functional channels was evaluated by coexpression with a dominant-negative Orai1 mutant. In this way we were able to demonstrate, for the first time, that the functional CRAC channel pore is formed by a tetrameric assembly of Orai1 subunits.

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.