Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.     

NF-kappaB activation and IkappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells

This review will discuss the recent literature on the molecular mechanism of NF-kappaB activation, with special focus on IkappaB alpha dynamism involved in iNOS- and chemokine-induction in glial cells. NF-kappaB, a heterotrimer composed of p50, p65 (Rel A) and IkappaB alpha, has been shown to be activated by elimination of the regulatory subunit IkappaB alpha from the heterotrimer. The elimination of IkappaB alpha (formation of active NF-kappaB, p50-p65) is due to phosplorylation of serines 32 and 36 of IkappaB alpha, followed by polyubiquitination and 26S proteasomal degradation of IkappaB alpha. Experiments using stable clones of rat C6 glioma cells transfected with dominant negative IkappaB alpha (serines 32 and 36 replaced by alanine) suggest that NF-kappaB activation (phosphorylation of IkappaB alpha) is involved in LPS/IFNgamma- or IL-1beta/IFNgamma-induced iNOS expression. Furthermore, the time courses of phosphorylation, ubiquitination of IkappaB alpha and proteasome activity after IL-1beta treatment also suggest that 26S proteasomal degradation of IkappaB alpha is more crucial for chemokine expression in glial cells.     

Constitutive nuclear expression of the I kappa B kinase complex and its activation in human neutrophils

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus.