Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity

The inhibitor of NF-kappaB (IkappaB) family of proteins is believed to regulate NF-kappaB activity by cytoplasmic sequestration. We show that in cells depleted of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-kappaB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins could be dispensable for cytoplasmic retention of NF-kappaB, they are essential for preventing NF-kappaB-dependent gene expression in the basal state. We also show that in the absence of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, cytoplasmic retention of NF-kappaB by other cellular proteins renders the pathway unresponsive to activation.     

Regulation of constitutive p50/c-Rel activity via proteasome inhibitor-resistant IkappaBalpha degradation in B cells

Constitutive NF-kappaB activity has emerged as an important cell survival component of physiological and pathological processes, including B-cell development. In B cells, constitutive NF-kappaB activity includes p50/c-Rel and p52/RelB heterodimers, both of which are critical for proper B-cell development. We previously reported that WEHI-231 B cells maintain constitutive p50/c-Rel activity via selective degradation of IkappaBalpha that is mediated by a proteasome inhibitor-resistant, now termed PIR, pathway. Here, we examined the mechanisms of PIR degradation by comparing it to the canonical pathway that involves IkappaB kinase-dependent phosphorylation and beta-TrCP-dependent ubiquitylation of the N-terminal signal response domain of IkappaBalpha. We found a distinct consensus sequence within this domain of IkappaBalpha for PIR degradation. Chimeric analyses of IkappaBalpha and IkappaBbeta further revealed that the ankyrin repeats of IkappaBalpha, but not IkappaBbeta, contained information necessary for PIR degradation, thereby explaining IkappaBalpha selectivity for the PIR pathway. Moreover, we found that PIR degradation of IkappaBalpha and constitutive p50/c-Rel activity in primary murine B cells were maintained in a manner different from B-cell-activating-factor-dependent p52/RelB regulation. Thus, our findings suggest that nonconventional PIR degradation of IkappaBalpha may play a physiological role in the development of B cells in vivo.     

Common pathway for the ubiquitination of IkappaBalpha, IkappaBbeta, and IkappaBepsilon mediated by the F-box protein FWD1.

FWD1 (the mouse homolog of Drosophila Slimb and Xenopus betaTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with IkappaBalpha and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to IkappaBbeta and IkappaBepsilon as well as to induce their ubiquitination and proteolysis. FWD1 was shown to recognize the conserved DSGPsiXS motif (where Psi represents the hydrophobic residue) present in the NH(2)-terminal regions of these three IkappaB proteins only when the component serine residues are phosphorylated. However, in contrast to IkappaBalpha and IkappaBbeta, the recognition site in IkappaBepsilon for FWD1 is not restricted to the DSGPsiXS motif; FWD1 also interacts with other sites in the NH(2)-terminal region of IkappaBepsilon. Substitution of the critical serine residues in the NH(2)-terminal regions of IkappaBalpha, IkappaBbeta, and IkappaBepsilon with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three IkappaB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF(FWD1) complex. FWD1 may thus play an important role in NF-kappaB signal transduction through regulation of the stability of multiple IkappaB proteins.     

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.     

Ikappakappa mediates NF-kappaB activation in human immunodeficiency virus-infected cells

Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-kappaB in the nucleus secondary to increased IkappaBalpha, IkappaBbeta, and IkappaBepsilon degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IkappaB, we have sought to identify the regulatory domains of IkappaBalpha targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IkappaBalpha, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IkappaBalpha. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IkappaBalpha degradation and NF-kappaB activation. Of the putative N-terminal IkappaBalpha kinases, we demonstrated that the Ikappakappa complex, but not p90(rsk), is activated by HIV infection and mediates HIV-dependent NF-kappaB activation. Analysis of viral replication in cells that constitutively express IkappaBalpha negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-kappaB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.     

Reductions in I kappa B epsilon and changes in NF-kappa B activity during B lymphocyte differentiation

The levels and stability of IkappaBepsilon have been examined in unstimulated and stimulated splenic B cells and compared with that of IkappaBalpha and IkappaBbeta. Primary murine splenic B cells but not T cells were found to contain high levels of IkappaBepsilon protein, equivalent to levels of the abundant IkappaBalpha. Most agents that activate IkappaBalpha and IkappaBbeta degradation do not induce rapid degradation of IkappaBepsilon. Interestingly, however, the levels of IkappaBepsilon, but not of IkappaBalpha or IkappaBbeta, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IkappaBepsilon exhibits substrate specificity for NF-kappaB Rel homodimers, this suggested the possibility that changes in NF-kappaB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-kappaB reporter construct sensitive to p65/RelA homodimers is activated at the time that IkappaBepsilon levels decline following B cell stimulation. In IgG(+) B cell lines, which contain low levels of IkappaBepsilon, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-kappaB-responsive genes in these IgG(+) B cell lines compared with their IgM(+) counterparts. From these data, we conclude that there are transient changes in NF-kappaB activity due to reductions in IkappaBepsilon, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IkappaBepsilon in the differential regulation of nuclear NF-kappaB activity in stimulated B cells.     

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.     

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.     

Retroviral oncoprotein Tax induces processing of NF-kappaB2/p100 in T cells: evidence for the involvement of IKKalpha.

IkappaB kinase (IKK) is a key mediator of NF-kappaB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF-kappaB, IkappaBalpha, which is responsible for the canonical NF-kappaB activation. Here, we show that in T cells infected with the human T-cell leukemia virus (HTLV), IKKalpha is targeted to a novel signaling pathway that mediates processing of the nfkappab2 precursor protein p100, resulting in active production of the NF-kappaB subunit, p52. This pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears to act by physically recruiting IKKalpha to p100, triggering phosphorylation-dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF-kappaB signaling pathway.