A nuclear export signal in the N-terminal regulatory domain of IκBα controls cytoplasmic localization of inactive NF-κB/IκBα complexes

Abstract

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...

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CRM1/Ran-Mediated Nuclear Export of p27Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

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