Basal Phosphorylation of the PEST Domain in IκBβ Regulates Its Functional Interaction with the c-<i>rel</i> Proto-Oncogene Product

Chu, Zhi Liang; McKinsey, Timothy A.; Liu, L; Qi, Xiaowen; Ballard, Dean W.

doi:10.1128/mcb.16.11.5974

Cited by 81 publications

(93 citation statements)

References 73 publications

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“…We then examined the potential role of the Cterminal sequences in the degradation of IB␤. In this regard, the C-terminal portion of IB␤ is rich in serines and aspartic acids (12) and has recently been shown to contain the sites for constitutive phosphorylation (41). Consistent with this recent study, an IB␤ mutant lacking the C-terminal 51 amino acids {IB␤(1-308)} migrated on SDS-PAGE as a single band (lane 7), indicating the lack of constitutive phosphorylation.…”

Section: Tnf-␣ Promotes Calyculin A-induced Degradation Of Ib␤-supporting

confidence: 70%

The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ

Harhaj

Sun

1997

Journal of Biological Chemistry

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Signal-initiated activation of the transcription factor NF-B is mediated through proteolysis of its cytoplasmic inhibitory proteins IB␣ and IB␤. While most NF-B inducers trigger the degradation of IB␣, only certain stimuli are able to induce the degradation of IB␤. The degradation of IB␣ is targeted by its sitespecific phosphorylations, although the mechanism underlying the degradation of IB␤ remains elusive. In the present study, we have analyzed the effect of phosphatase inhibitors on the proteolysis of IB␤. We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of IB␤ in both human Jurkat T cells and the murine 70Z-3 preB cells, which is associated with the nuclear expression of active NF-B. The calyculin A-mediated degradation of IB␤ is further enhanced by the cytokine tumor necrosis factor-␣ (TNF-␣), although TNF-␣ alone is unable to induce the degradation of IB␤. Mutational analyses have revealed that the inducible degradation of IB␤ induced by calyculin A, and TNF-␣ requires two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites present in IB␣. Furthermore, the C-terminal 51 amino acid residues, which are rich in serines and aspartic acids, are also required for the inducible degradation of IB␤. These results suggest that the degradation signal of IB␤ may be controlled by the opposing actions of protein kinases and phosphatases and that both the Nand C-terminal sequences of IB␤ are required for the inducible degradation of this NF-B inhibitor.The NF-B/Rel family of transcription factors play a pivotal role in the regulation of various cellular genes involved in the immediate early processes of immune, acute phase, and inflammatory responses (1, 2). In addition, these cellular factors have also been implicated in the transcriptional activation of certain human viruses, most notably the type 1 human immune deficiency virus (3-7). The mammalian NF-B/Rel family is composed of at least five structurally related DNA-binding proteins, including p50, p52, RelA, RelB, and c-Rel, which bind to a target DNA sequence (B) as various heterodimers or homodimers (reviewed in Siebenlist et al. (8)). In most cell types, including resting T cells, the NF-B/Rel proteins are sequestered in the cytoplasmic compartment by physical association with inhibitory proteins that are characteristic of the presence of various numbers of ankyrin-like repeats (reviewed in Verma et al. (9)). The major cytoplasmic inhibitors include IB␣ (10, 11), IB␤ (12), and the precursor proteins of p50 and p52 (9). The IB molecules appear to bind to and mask the nuclear localization signal of NF-B/Rel, thereby preventing the nuclear translocation of these transcription factors (13-16).The latent cytoplasmic NF-B/Rel complexes can be activated by a variety of cellular stimuli, including the mitogen phorbol esters, cytokines such as tumor necrosis factor-␣ (TNF-␣), 1 and interleukin-1, the bacterial component lipopolysaccharide, serine/...

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Section: Tnf-␣ Promotes Calyculin A-induced Degradation Of Ib␤-supporting

confidence: 70%

The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ

Harhaj

Sun

1997

Journal of Biological Chemistry

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“…This change may coincide with either greater or lesser sensitivity to specific inducing agents. In addition, it has been reported that IB␣ is controlled under some circumstances by tyrosine phosphorylation, which results in dissociation but not degradation (13,26). As discussed above, this mechanism is more tenable if the affinity of the complex is relatively low.…”

Section: Discussionmentioning

confidence: 99%

Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation

Carlotti

Dower

Qwarnström

2000

Journal of Biological Chemistry

139

123

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Activation of the nuclear factor B (NFB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFB and the inhibitory subunit IB␣ between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IB␣ occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFB⅐IB complex rather than its direct nuclear import or degradation and resynthesis of IB␣. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFB is not complexed to IB␣ in a resting cell. A numerical model is presented to validate the proposed regulation of NFB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IB␣. We suggest that the non-saturated interaction of NFB with the inhibitor may enhance the specificity of action of IB proteins on different NFB dimers and allow additional modes of regulation of IB function. NFB1 is a central mediator of immune and inflammatory responses. It consists of homo-or heterodimers of a family of at least five related proteins characterized by the rel homology domain (1, 2). The known members of this family are c-REL, RELA, RELB, p50, and p52. Homodimers of p50 and p52 are generally considered to be inhibitory, because they lack activation domains, but they may be converted to activators by association with the Bcl3 protein (3, 4). NFB is regulated by a family of at least seven inhibitory proteins, IB, characterized by multiple ankyrin-type repeats (5). The expression of both NFB and IB proteins is cell type-and developmental stagespecific (2). In non-lymphomyeloid cells, the most prevalent NFB species are RELA-p50 heterodimers and p50 homodimers.The current model for NFB function postulates that the transcription factor is anchored in the cytoplasm by association with the inhibitor in unstimulated cells (6). Whereas both NFB and IB have nuclear localization signals, resulting in import of the free factors to the nucleus, these are mutually masked within the complex to abolish their nuclear import functions (7). A diverse range of stimuli, including inflammatory cytokines, bacterial lipopolysaccharide, and phorbol esters (8 -10), result in the serine phosphorylation of IB and its subsequent degradation (11, 12). Translocation of the free transcription factor to the nucleus and activation of genes bearing cognate binding sites ensues. Tyrosine phosphorylation of IB␣, without subsequent degradation, has also been reported to release active NFB into the nucleus (13). In addition to nuclear translocation, phosphorylation of NFB may be important for its full activation (14 -17). This phosphorylation may be induced by parallel pathways initiated by the same stimulus as that causing IB degradation.We have pre...

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“…PLC-g1 antibody was obtained from Transduction Laboratories and used at a 1 : 1000 dilution. The p50, p65 and c-rel antibodies, plus pCMV4-IkBa(DN37) were generous gifts from Dr Bean Ballard (Vanderbilt University, Nashville, TN) and were previously described (Brockman et al, 1995;Chu et al, 1996).…”

Section: Reagentsmentioning

confidence: 99%

Epidermal growth factor activation of NF-κB is mediated through IκBα degradation and intracellular free calcium

1998

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The transcription factor NF-kB is normally sequestered in the cytoplasm by its inhibitory subunit IkB. Most extracellular signals activate NF-kB through a mechanism involving the phosphorylation and proteasomedependent degradation of IkB. EGF activates NF-kB in A-431 carcinoma cells, which overexpress EGF receptors and in mouse embryo ®broblasts, which have a normal complement of receptors. Supershift experiments indicate that the NF-kB complexes induced by EGF are composed of p50/p50 homodimers and p65/p50 heterodimers, but not c-rel. EGF stimulation enhances the degradation of IkBa, but not IkBb nor an N-terminal deletion mutant of IkBa. Treatment of cells with a proteasome inhibitor, such as ALLN or MG132, blocks EGF-mediated NF-kB activation, indicating that EGFinduced NF-kB activation requires proteasome-dependent IkB degradation. Also, Bapta A/M (a cell-permeable chelator of intracellular calcium) blocks EGF-induced NF-kB activation and IkBa degradation, suggesting a requirement of intracellular free Ca 2+ for this growth factor response. Protein kinase C inhibition, in contrast, did not in¯uence EGF activation of NF-kB.

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Basal Phosphorylation of the PEST Domain in IκBβ Regulates Its Functional Interaction with the c-rel Proto-Oncogene Product

Cited by 81 publications

References 73 publications

The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ

The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ

Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation

Epidermal growth factor activation of NF-κB is mediated through IκBα degradation and intracellular free calcium

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