Shigeki Miyamoto

The growth, differentiation, and development of an organism is orchestrated by the choreographed expression of a wide array of genes. The switching "on" and "off" of gene expression is the province of transcription factors, which operate singly or in association with other proteins. Usually transcription factors form families, whereas individual members perform specific, distinct, or similar tasks. One such family includes the R ~~/ N F -K B proteins (NF-KB), which have the unique property of being sequestered in the cytoplasm in association with inhibitory proteins called IKB.

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-kappa B regulates the expression of a plethora of genes. The activity of NF-kappa B proteins is regulated by I kappa B proteins. We report that induction of I kappa B alpha, a member of the I kappa B family of proteins, is preceded by activation of NF-kappa B complex. The promoter of the I kappa B alpha gene contains a kappa B site that is directly involved in its induction by the NF-kappa B complex. Degradation of I kappa B alpha protein precedes activation of NF-kappa B DNA binding activity, whereas newly synthesized I kappa B alpha protein inhibits NF-kappa B activity. If the degradation of I kappa B alpha is prevented, the induction of DNA binding activity of NF-kappa B complex is severely curtailed. These data suggest the existence of an autoregulatory loop whereby I kappa B alpha regulates the activity of transcription factor NF-kappa B, which in turn regulates the I kappa B alpha activity.

Appropriate subcellular localization is crucial for regulation of NF-kappaB function. Herein, we show that latent NF-kappaB complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-kappaB/IkappaBalpha complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45-54 of IkappaBalpha 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-kappaB nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-kappaB/IkappaBalpha 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-kappaB activation without affecting the subcellular localization of upstream kinases IKKbeta 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-kappaB activation by extracellular signals.