No abstract

The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma, the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets.

The transcription factor NF-kappaB modulates apoptotic responses induced by genotoxic stress. We show that NF-kappaB essential modulator (NEMO), the regulatory subunit of IkappaB kinase (IKK) (which phosphorylates the NF-kappaB inhibitor IkappaB), associates with activated ataxia telangiectasia mutated (ATM) after the induction of DNA double-strand breaks. ATM phosphorylates serine-85 of NEMO to promote its ubiquitin-dependent nuclear export. ATM is also exported in a NEMO-dependent manner to the cytoplasm, where it associates with and causes the activation of IKK in a manner dependent on another IKK regulator, a protein rich in glutamate, leucine, lysine, and serine (ELKS). Thus, regulated nuclear shuttling of NEMO links two signaling kinases, ATM and IKK, to activate NF-kappaB by genotoxic signals.

Transcription factor NF-.cB regulates the expression of a plethora of genes. The activity of NF-ucB proteins is regulated by IKB proteins. We report that induction of IcBa, a member of the IKB family of proteins, is preceded by activation of NF-ucB complex. Nuclear factor NF-KB was identified as a lymphoid-specific heterodimeric protein complex that binds to a decameric sequence motif located in the immunoglobulin K light chain enhancer (1). Subsequently, it was shown that the NF-KB transcription factor participates in the regulation and transcriptional activities of many cellular and viral genes (2-4). Molecular cloning of the p5O and p65 subunits, the two components of the NF-KB complex, revealed a region in the N-terminal half of these proteins that shares extensive sequence homology with protooncogene rel and the Drosophila morphogen dorsal (5-9). The Rel homology region contains the DNA binding and the dimerization domain (4, 9).

Appropriate subcellular localization is crucial for regulation of NF-B function. Herein, we show that latent NF-B complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-B͞IB␣ complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45-54 of IB␣ that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-B nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-B͞IB␣ complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-B activation without affecting the subcellular localization of upstream kinases IKK␤ and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-B activation by extracellular signals. R egulatory pathways can be modulated by the subcellular compartmentalization of individual components. A prototypic example is signal-induced activation of the transcription factor NF-B that plays an important role in immune and inflammatory responses and the regulation of apoptosis (1, 2). In unstimulated cells, inactive NF-B preexists in the cytoplasm associated with its inhibitor IB (3). On exposure to extracellular signals, a series of biochemical events targets the inhibitor protein for degradation, allowing the NF-B to migrate into the nucleus to regulate gene expression.Cis-acting elements of IB govern its protein stability and subcellular localization (1, 2). IB␣, the most studied IB family member, is composed minimally of three domains: an Nterminal regulatory domain that controls signal-dependent degradation, a central ankyrin repeat domain (ARD) that is necessary for NF-B binding, and a C-terminal region rich in proline, glutamate/aspartate, serine, and threonine regulating basal turnover. An additional sequence, a leucine-rich nuclear export sequence (NES) within the last ankyrin repeat, is postulated to function during the termination of NF-B activity (4). Activated NF-B stimulates the synthesis of IB␣ mRNA (5, 6), and newly synthesized IB␣ proteins can enter the nucleus to bind to and remove NF-B from gene promoters (7). It is believed that the C-terminal NES (C-NES) of IB␣ can actively export these IB␣͞NF-B complexes out to the cytoplasm to restore the preinduction state of the complexes, a process known as postinduction repression (4).The leucine-rich NES is a highly conserved sequence use...

Summary The dimeric transcription factor nuclear factor κB (NF-κB) functions broadly in coordinating cellular responses during inflammation and immune reactions, and its importance in the pathogenesis of cancer is increasingly recognized. Many of the signal transduction pathways that trigger activation of cytoplasmic NF-κB in response to a broad array of immune and inflammatory stimuli have been elaborated in great detail. NF-κB can also be activated by DNA damage, though relatively less is known about the signal transduction mechanisms that link DNA damage in the nucleus with activation of NF-κB in the cytoplasm. Here, we focus on the conserved signaling pathway that has emerged that promotes NF-κB activation following DNA damage. Post-translational modification of NF-κB essential modulator (NEMO) plays a central role in linking the cellular DNA damage response to NF-κB via the ataxia telangiectasia mutated (ATM) kinase. Accumulating evidence suggests that DNA damage-dependent NF-κB activation may play significant biological roles, particularly during lymphocyte differentiation and progression of human malignancies.

Treatment with acetylcholine (ACh) of a b-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca 2+ concentration was clamped at 1 mM. The AChinduced Ca 2+ sensitization of myo®laments was signi®cantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca 2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was signi®cantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca 2+ sensitization of myo®laments.

A large body of literature describes elaborate NF-κB signaling networks induced by inflammatory and immune signals. Decades of research has revealed that transcriptionally functional NF-κB dimers are activated by two major pathways, canonical and non-canonical. Both pathways involve the release of NF-κB dimers from inactive cytoplasmic complexes to cause their nuclear translocation to modulate gene expression programs and biological responses. NF-κB is also responsive to genotoxic agents; however, signal communication networks that are initiated in the nucleus following DNA damage induction are less defined. Evidence in the literature supports the presence of such signaling pathways induced by multiple distinct genotoxic agents, resulting in the activation of cytoplasmic IKK complex. An example is a pathway that involves the DNA damage-responsive kinase ataxia telangiectasia mutated (ATM) and a series of post-translational modifications of NF-κB essential modulator (NEMO) in the nucleus of a genotoxinexposed cell. Recent evidence also suggests that this nuclear-initiated NF-κB signaling pathway plays significant physiological and pathological roles, particularly in lymphocyte development and human cancer progression. This review will summarize these new developments, while identifying significant unanswered questions and providing new hypotheses that may be addressed in future studies.