p105.Ikappa Bgamma and prototypical Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF-kappa B

p105, also known as NF-kappaB1, is an atypical IkappaB molecule with a multi-domain organization distinct from other prototypical IkappaBs, like IkappaBalpha and IkappaBbeta. To understand the mechanism by which p105 binds and inhibits NF-kappaB, we have used both p105 and its C-terminal inhibitory segment known as IkappaBgamma for our study. We show here that one IkappaBgamma molecule binds to NF-kappaB dimers wherein at least one NF-kappaB subunit is p50. We suggest that the obligatory p50 subunit in IkappaBgamma.NF-kappaB complexes is equivalent to the N-terminal p50 segment in all p105.NF-kappaB complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by IkappaBgamma, whereas the NLS of the nonobligatory NF-kappaB subunit is exposed. Thus, the global binding mode of all IkappaB.NF-kappaB complexes seems to be similar where one obligatory (or specific) NF-kappaB subunit makes intimate contact with IkappaB and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case of IkappaBalpha and IkappaBbeta, the specific NF-kappaB subunit in the complex is p65. In contrast to IkappaBalpha.NF-kappaB complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105.NF-kappaB and IkappaBgamma.NF-kappaB complexes are cytoplasmic. We show that the death domain of p105 (also of IkappaBgamma) is essential for the cytoplasmic sequestration of NF-kappaB by p105 and IkappaBgamma. However, the death domain does not mask the exposed NLS of the complex. We also demonstrate that the death domain alone is not sufficient for cytoplasmic retention and instead functions only in conjunction with other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-kappaB dimer. We speculate that additional cytoplasmic protein(s) may sequester the entire p105.NF-kappaB complex by binding through the death domain and other segments, including the exposed NLS.     

Induced fit, folding, and recognition of the NF-kappaB-nuclear localization signals by IkappaBalpha and IkappaBbeta

Protein structure prediction codes based on the associative memory Hamiltonian were used to probe the binding modes between the nuclear localization signal (NLS) polypeptide of NF-kappaB and the inhibitors IkappaBalpha and IkappaBbeta. Experimentally, it is known that the NLS polypeptide is unstructured in the NF-kappaB complex with DNA but it forms an extended helical structure with the NLS (residues 301-304) between the two helices in the NF-kappaB/IkappaBalpha complex. The simulations included the NF-kappaB(p65) and (p50) NLS polypeptides and various mutants alone and in the presence of IkappaBalpha and IkappaBbeta. The simulations predict that the NLS polypeptide by itself binds tightly to IkappaBalpha and IkappaBbeta. In the NF-kappaB (p50/p65) heterodimer, the p50 NLS is predicted to remain free to bind to importin alpha. In the interaction with IkappaBalpha, both p65 NLSs are predicted to be bound. In IkappaBbeta, the NLS polypeptide binds to two binding sites, as seen in the crystal structure, with one site heavily favored for stable binding.     

IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.     

RelA/p65 regulation of IkappaBbeta

IkappaB inhibitor proteins are the primary regulators of NF-kappaB. In contrast to the defined regulatory interplay between NF-kappaB and IkappaBalpha, much less is known regarding the regulation of IkappaBbeta by NF-kappaB. Here, we describe in detail the regulation of IkappaBbeta by RelA/p65. Using p65(-/-) fibroblasts, we show that IkappaBbeta is profoundly reduced in these cells, but not in other NF-kappaB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, IkappaBalpha is also reduced, but not nearly to the same extent as IkappaBbeta, thus highlighting the degree to which IkappaBbeta is dependent on p65. This dependence is based on the ability of p65 to stabilize IkappaBbeta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IkappaBbeta was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65(-/-) fibroblasts, expression of a proteolysis-resistant form of IkappaBbeta, but not IkappaBalpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IkappaBbeta protein by p65 is necessary for the maintenance of cellular homeostasis.     

Increased p50/p50 NF-κB Activation in Human Papillomavirus Type 6- or Type 11-Induced Laryngeal Papilloma Tissue

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.     

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.     

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.     

Cell-Specific Association and Shuttling of IκBα Provides a Mechanism for Nuclear NF-κB in B Lymphocytes

Mature B lymphocytes are unique in containing nuclear Rel proteins prior to cell stimulation. This activity consists largely of p50-c-Rel heterodimers, and its importance for B-cell function is exemplified by reduced B-cell viability in several genetically altered mouse strains. Here we suggest a mechanism for the cell specificity and the subunit composition of constitutive B-cell NF-kappaB based on the observed properties of Rel homo- and heterodimers and IkappaBalpha. We show that c-Rel lacks a nuclear export sequence, making the removal of c-Rel-containing complexes from the nucleus less efficient than removal of p65-containing complexes. Second, the nuclear import potential of p65 and c-Rel homodimers but not p50-associated heterodimers was attenuated when they were complexed to IkappaBalpha, leading to a greater propensity of heterodimers to be nuclear. We propose that subunit composition of B-cell NF-kappaB reflects the inefficient retrieval of p50-c-Rel heterodimers from the nucleus. Cell specificity may be a consequence of c-Rel-IkappaBalpha complexes being present only in mature B cells, which leads to nuclear c-Rel due to IkappaBalpha turnover and shuttling of the complex.     

IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain.

Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.