scholarly journals Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation.

1996 ◽  
Vol 16 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
S Sun ◽  
J Elwood ◽  
W C Greene
Keyword(s):  
Transcription Factor ◽  
Nuclear Translocation ◽  
Terminal Region ◽  
Dominant Negative ◽  
Tnf Alpha ◽  
Human Immune Deficiency Virus ◽  
Nf Kappa B ◽  
Inhibitory Protein ◽  
I Kappa B Alpha ◽  
Transcription Factor Complex

Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by an ankyrin-rich inhibitory protein termed I kappa B alpha. I kappa B alpha specifically binds to and masks the nuclear localization signal of the RelA subunit of NF-kappa B, thereby effectively sequestering this transcription factor complex in the cytoplasm. Specific cellular activation signals lead to the rapid proteolytic degradation of I kappa B alpha and the concomitant nuclear translocation of NF-kappa B. However, the precise biochemical mechanisms underlying the inhibitory effects of I kappa B alpha on RelA and its inducible pattern of degradation remain unclear. By using HeLa cells transfected with various cDNAs end-coding epitope-tagged mutants of I kappa B alpha, our studies demonstrate the following: (i) sequences within the 72-amino-acid N-terminal region of I kappa B alpha are required for tumor necrosis factor alpha (TNF-alpha)-induced degradation but are fully dispensable for I kappa B alpha binding to and inhibition of RelA; (ii) serine residues located at positions 32 and 36 within the N-terminal region of I kappa B alpha represent major sites of induced phosphorylation (substitution of these serine residues with alanine abrogates TNF-alpha-induced degradation of I kappa B alpha); (iii) the C-terminal 40 residues of I kappa B alpha (amino acids 277 to 317), which include a PEST-like domain, are entirely dispensable for TNF-alpha-induced degradation and inhibition of RelA; (iv) a glutamine- and leucine-rich (QL) region of I kappa B alpha located between residues 263 and 277 and overlapping with the sixth ankyrin repeat is required for both inducible degradation and inhibition of RelA function; (v) regulation of I kappa B alpha degradation by this QL-rich region appears to occur independently of phosphorylation at serines 32 and 36. These findings thus indicate that I kappa B alpha is generally organized within distinct modular domains displaying different functional and regulatory properties. These studies have also led to the identification of a novel class of dominant-negative I kappa B alpha molecules that retain full inhibitory function on NF-kappa B yet fail to undergo stimulus-induced degradation. These molecules, which lack N-terminal sequences, potently inhibit TNF-alpha-induced activation of the human immune deficiency virus type 1 kappa B enhancer, thus indicating their possible use as general inhibitors of NF-kappa B.

scholarly journals Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B.

1995 ◽  
Vol 15 (5) ◽  
pp. 2689-2696 ◽  
Author(s):  
F Arenzana-Seisdedos ◽  
J Thompson ◽  
M S Rodriguez ◽  
F Bachelerie ◽  
D Thomas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Regulatory Function ◽  
Primary Role ◽  
Nf Kappa B ◽  
Nuclear Expression ◽  
I Kappa B Alpha

The transcription factor NF-kappa B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein I kappa B alpha maintains NF-kappa B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, I kappa B alpha is rapidly degraded, leading to nuclear translocation of free NF-kappa B. However, NF-kappa B-dependent transcription of the I kappa B alpha gene leads to rapid resynthesis of the I kappa B alpha protein and inhibition of NF-kappa B-dependent transcription. Here we demonstrate a new regulatory function of I kappa B alpha exerted on NF-kappa B in the nuclear compartment. Although normally found in the cytoplasm, I kappa B alpha, newly synthesized in response to tumor necrosis factor or interleukin I, is transported to the nucleus. In the nucleus I kappa B alpha associates with the p50 and p65 subunits of NF-kappa B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of I kappa B alpha correlates with transcription termination of transfected NF-kappa B-dependent luciferase genes. Following the appearance of I kappa B alpha in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-kappa B-I kappa B alpha complexes are cleared from the nucleus.

scholarly journals Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression.

1996 ◽  
Vol 133 (5) ◽  
pp. 1083-1093 ◽  
Author(s):  
C Kretz-Remy ◽  
P Mehlen ◽  
M E Mirault ◽  
A P Arrigo
Keyword(s):  
Glutathione Peroxidase ◽  
Nuclear Translocation ◽  
T47d Cell ◽  
Time Correlation ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
T47d Cells ◽  
I Kappa B Alpha ◽  
Intracellular Ros ◽  
Gshpx Activity

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

scholarly journals Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

1996 ◽  
Vol 16 (4) ◽  
pp. 1842-1850 ◽  
Author(s):  
G Baier-Bitterlich ◽  
F Uberall ◽  
B Bauer ◽  
F Fresser ◽  
H Wachter ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Alpha ◽  
Transcriptional Complex ◽  
Induced Signal ◽  
Transcription Factor Complex

T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-O K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-enRasS17N completely inhibited in the PKC-O A148E-induced signal, identifying PKC-theta as a specific constituent upstream of or parallel to Ras in the signaling cascade leading to AP transcriptional activation.

Involvement of a NF-kappa B-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines

1990 ◽  
Vol 10 (2) ◽  
pp. 561-568
Author(s):  
H Shimizu ◽  
K Mitomo ◽  
T Watanabe ◽  
S Okamoto ◽  
K Yamamoto
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Interleukin 6 ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Nuclear Factor ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Mediators Of Inflammation

Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-alpha despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-kappa B transcription factor-binding motifs and binds an IL-1-TNF-alpha-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular localizations of this factor are indistinguishable from those of NF-kappa B. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-alpha in vivo. These results indicate that a nuclear factor indistinguishable from NF-kappa B is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-alpha.

scholarly journals Requirement for Transcription Factor NFAT in Interleukin-2 Expression

1999 ◽  
Vol 19 (3) ◽  
pp. 2300-2307 ◽  
Author(s):  
Chi-Wing Chow ◽  
Mercedes Rincón ◽  
Roger J. Davis
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Nuclear Translocation ◽  
Interleukin 2 ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Activated T Cells ◽  
Nfat Activation ◽  
Nfat Transcription Factor

ABSTRACT The nuclear factor of activated T cells (NFAT) transcription factor is implicated in expression of the cytokine interleukin-2 (IL-2). Binding sites for NFAT are located in the IL-2 promoter. Furthermore, pharmacological studies demonstrate that the drug cyclosporin A inhibits both NFAT activation and IL-2 expression. However, targeted disruption of the NFAT1 and NFAT2 genes in mice does not cause decreased IL-2 secretion. The role of NFAT in IL-2 gene expression is therefore unclear. Here we report the construction of a dominant-negative NFAT mutant (dnNFAT) that selectively inhibits NFAT-mediated gene expression. The inhibitory effect of dnNFAT is mediated by suppression of activation-induced nuclear translocation of NFAT. Expression of dnNFAT in cultured T cells caused inhibition of IL-2 promoter activity and decreased expression of IL-2 protein. Similarly, expression of dnNFAT in transgenic mice also caused decreased IL-2 gene expression. These data demonstrate that NFAT is a critical component of the signaling pathway that regulates IL-2 expression.

scholarly journals A CCAAT/Enhancer Binding Protein β Isoform, Liver-Enriched Inhibitory Protein, Regulates Commitment of Osteoblasts and Adipocytes

2005 ◽  
Vol 25 (5) ◽  
pp. 1971-1979 ◽  
Author(s):  
Kenji Hata ◽  
Riko Nishimura ◽  
Mio Ueda ◽  
Fumiyo Ikeda ◽  
Takuma Matsubara ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Osteoblast Differentiation ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Mesenchymal Cells ◽  
Dominant Negative ◽  
Inhibitory Protein ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

ABSTRACT Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein β (C/EBPβ), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPβ, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPβ, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPβ isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

scholarly journals N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity.

1995 ◽  
Vol 15 (10) ◽  
pp. 5339-5345 ◽  
Author(s):  
S T Whiteside ◽  
M K Ernst ◽  
O LeBail ◽  
C Laurent-Winter ◽  
N Rice ◽  
...  
Keyword(s):  
Phorbol Myristate Acetate ◽  
26S Proteasome ◽  
Proteolytic Degradation ◽  
Myristate Acetate ◽  
Nf Kappa B ◽  
Inhibitory Protein ◽  
Stable Transfectants ◽  
I Kappa B Alpha ◽  
C Terminus ◽  
Mutant Forms

The proteolytic degradation of the inhibitory protein MAD3/I kappa B alpha in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kappa B. Analysis of the expression of human I kappa B alpha protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous I kappa B alpha is degraded in response to inducers of NF-kappa B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human I kappa B alpha protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of I kappa B alpha. Our results show that deletion of the C terminus of the I kappa B alpha molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of I kappa B alpha maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-kappa B to translocate to the nucleus and to activate gene expression.

scholarly journals The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
A. L. Furfaro ◽  
N. Traverso ◽  
C. Domenicotti ◽  
S. Piras ◽  
L. Moretta ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nuclear Translocation ◽  
Tumour Progression ◽  
Redox Homeostasis ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Inhibitory Protein ◽  
Cancer Cell Growth ◽  
Promising Target ◽  
Anticancer Treatment

The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), acts as a sensor of oxidative or electrophilic stresses and plays a pivotal role in redox homeostasis. Oxidative or electrophilic agents cause a conformational change in the Nrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which, through its binding to the antioxidant/electrophilic response element (ARE/EpRE), regulates the expression of antioxidant and detoxifying genes such as heme oxygenase 1 (HO-1). Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumour progression, aggressiveness, resistance to therapy, and poor prognosis. This review focuses on the Nrf2/HO-1 stress response mechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies.

A Pedigree Harboring a Germ-Line N-Terminal C/EBPα Mutation and Development of Acute Myeloblastic Leukemia with a Somatic C-Terminal C/EBPα Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1916-1916 ◽  
Author(s):  
Tomoko Nanri ◽  
Naokuni Uike ◽  
Toshiro Kawakita ◽  
Eisaku Iwanaga ◽  
Koyu Hoshino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Germ Line ◽  
Terminal Region ◽  
Dominant Negative ◽  
Acute Myeloblastic Leukemia ◽  
Peripheral Blood Cells ◽  
Insertion Mutations

Abstract Transcription factors involved in myeloid cell differentiation are frequent targets of chromosomal translocations and point mutations in patients with acute myeloblastic leukemia (AML). Familial AML harboring a mutation in a transcription factor should provide an association of clinical features with functions of the transcription factor. Recently, two pedigrees of AML carrying a germ-line mutation in the CEBPA, a gene encoding transcription factor C/EBPα have been reported. As C/EBPα-null mice lack of mature neutrophil and eosinophil granulocytes, C/EBPα is thought to play a central role in regulating the differentiation of granulocytes. We here present clinical and molecular features of a Japanese family in which two individuals developing AML and one healthy individual had an identical CEBPA mutation. Father had received a diagnosis of AML M2 in 1988 at the age of 39 years. Following a relapse 7 years after his first complete remission (CR), he received autologous stem cell transplantation, after which he has been in a lasting CR. His elderly son was diagnosed to have M2Eo in 2004 at the age of 26 years. He has achieved a continuous CR. Bone marrow cells at the time of diagnosis and peripheral blood cells obtained during CR in both patients showed a 4-base pair insertion in the N-terminal region of the CEBPA (350_351insCTAC). The corresponding protein is predicted to terminate prematurely at codon 107 (I68fsX107). Therefore, this heterozygous mutation causes truncation of the 42-kD C/EBPα protein and overproduction of a 30-kD isoform, which lacks a transactivation domain and functions in a dominant negative fashion, causing a decrease in C/EBPα activity. His younger son unaffected by AML aged 21 years also had the same mutation. The N-terminal C/EBPα mutations in sporadic AML patients are associated with FAB M1/M2 subtypes, presence of Auer rods, CD7 expression, normal karyotype, and a favorable prognosis. Familial AMLs with an N-terminal C/EBPα mutation demonstrate links of these unique features to a dominant negative C/EBPα mutation. It is of note that different in-frame insertion mutations in the C-terminal region of C/EBPα were identified in both patients at the time of diagnosis. As these C-terminal CEBPA mutations were not found in peripheral blood cells during CR in both patients, these in-frame insertion mutations in the C/EBPα basic region leucine zipper DNA-binding domain should be somatic mutations. Interestingly, father showed different in-frame insertion mutations in the C-terminal CEBPA at the time of diagnosis and relapse. These N- and C-terminal biallelic mutations in the CEBPA are thought to result in complete loss-of-function of C/EBPα. A recent study using conditional Cebpa knockout mice showed that C/EBPα deficiency in adult mice leads to persistence of immature myeloid blasts in the bone marrow without developing AML. Although the mechanism underlying the development of AML is yet unclear, this study indicates that the N-terminal CEBPA mutation appears to confer an increased risk occurring C-terminal C/EBPα mutations and other mutations as a second and further genetic hits, generating undifferentiated myeloid cells and eventually leading to AML over a long latency.

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