scholarly journals Identification of lysine residues critical for the transcriptional activity and polyubiquitination of the NF-κB family member RelB

2008 ◽  
Vol 416 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Julia Leidner ◽  
Lysann Palkowitsch ◽  
Uta Marienfeld ◽  
Dietmar Fischer ◽  
Ralf Marienfeld
Keyword(s):  
Dna Methyltransferase ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Nuclear Factor Κb ◽  
Death Domain ◽  
293 Cells ◽  
Lysine Residues ◽  
Negative Effect ◽  
The Stability

RelB is the key component of the alternative NF-κB (nuclear factor κB) signalling pathway. However, RelB exerts also a negative effect via the recruitment of a DNMT1 (DNA methyltransferase 1)–Daxx (death domain-associated protein) complex to NF-κB target genes. Importantly, the molecular mechanisms which determine the functions of RelB are still largely unknown. In the present study, we aimed to analyse whether ubiquitination of RelB might be involved in the regulation of RelB. Indeed, RelB is constitutively polyubiquitinated in the B-cell lines Namalwa and 70Z/3. Although a PMA+ionomycin-induced augmentation of RelB polyubiquitination was linked to its proteasomal degradation in B-cells, the constitutive RelB polyubiquitination seems to affect non-proteasomal functions. Consistently, a significant RelB polyubiquitination in HEK (human embryonic kidney)-293 cells correlated with an augmentation of the transcriptional activity of RelB. Yet, neither nuclear localization nor DNA binding was enhanced by RelB polyubiquitination. Interestingly, basal RelB polyubiquitination depends neither on Lys48 nor on Lys63 conjugates, but might involve unconventional ubiquitin conjugates. Mapping of the ubiquitination target sites in RelB revealed the existence of various lysine residues, which serve as ubiquitination acceptors. However, only the substitution of Lys273/274 and Lys305/308 significantly decreased the basal RelB activity and the ubiquitin-induced augmentation of the RelB activity. Collectively, these results imply a dual role of RelB polyubiquitination for the stability and activity of this transcription factor.

scholarly journals Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

2003 ◽  
Vol 23 (22) ◽  
pp. 8295-8305 ◽  
Author(s):  
Keizo Nishikawa ◽  
Makoto Kobayashi ◽  
Atsuko Masumi ◽  
Susan E. Lyons ◽  
Brant M. Weinstein ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Germ Line ◽  
Zebra Fish ◽  
Wild Type ◽  
Lysine Residues ◽  
Self Association ◽  
Gata Motif

ABSTRACT Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.

scholarly journals Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Sabine Probst ◽  
Florian Riese ◽  
Larissa Kägi ◽  
Maik Krüger ◽  
Natalie Russi ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Adaptor Protein ◽  
Proteolytic Processing ◽  
Class Iii ◽  
Lysine Deacetylase ◽  
Lysine Residues ◽  
Lysine Acetyltransferase ◽  
Complex Forms ◽  
Spot Formation

AbstractProteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 to glutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs at different lysine residues, regulates stability of Fe65. This is the first report describing a role for acetylation in the regulation of Fe65 transcriptional activity, with Tip60 being the only KAT tested that supports AFT spot formation.

Calcineurin regulates the stability and activity of estrogen receptor α

2021 ◽  
Vol 118 (44) ◽  
pp. e2114258118
Author(s):  
Takahiro Masaki ◽  
Makoto Habara ◽  
Yuki Sato ◽  
Takahiro Goshima ◽  
Keisuke Maeda ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Breast Cancer Cells ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
The Stability ◽  
Positive Breast Cancer

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser294 in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser294 and Ser118. Finally, the expression of the calcineurin A–α gene (PPP3CA) was associated with poor prognosis in ER-α–positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α–positive breast cancer.

scholarly journals Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

2015 ◽  
Vol 112 (51) ◽  
pp. 15755-15760 ◽  
Author(s):  
Miyeon Choi ◽  
Seung Hoon Lee ◽  
Sung Eun Wang ◽  
Seung Yeon Ko ◽  
Mihee Song ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Export ◽  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Calmodulin Kinase ◽  
Defective Mutant ◽  
Antidepressant Effects

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine.

scholarly journals Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

2014 ◽  
Vol 289 (44) ◽  
pp. 30680-30689 ◽  
Author(s):  
Sunhee Lee ◽  
Madhavi Challa-Malladi ◽  
Shawn B. Bratton ◽  
Casey W. Wright
Keyword(s):  
Target Genes ◽  
Tumor Necrosis Factor Receptor ◽  
Nuclear Factor Κb ◽  
Inhibitor Of Apoptosis ◽  
Binding Motif ◽  
The Novel ◽  
Amino Terminal ◽  
Close Proximity ◽  
The Stability ◽  
And Function

Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.

scholarly journals Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2341
Author(s):  
Tiit Örd ◽  
Daima Örd ◽  
Minna U. Kaikkonen ◽  
Tõnis Örd
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Proteasome Inhibitor ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Cell Resistance ◽  
Human Hepatoma Cells

The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib.

scholarly journals PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2

2015 ◽  
Vol 308 (2) ◽  
pp. F140-F148 ◽  
Author(s):  
Hong Wang ◽  
Joan D. Ferraris ◽  
Janet D. Klein ◽  
Jeff M. Sands ◽  
Maurice B. Burg ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Renal Medulla ◽  
Protein Abundance ◽  
Activated T Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Src Homology ◽  
Src Homology 2 Domain

High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser591 on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591.

scholarly journals SUMO modification regulates the transcriptional activity of XBP1

2010 ◽  
Vol 429 (1) ◽  
pp. 95-102 ◽  
Author(s):  
Hui Chen ◽  
Ling Qi
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Protein Accumulation ◽  
Transactivation Domain ◽  
Signal Transducer ◽  
Defence Mechanism ◽  
Unfolded Protein ◽  
Lysine Residues ◽  
Protein Response ◽  
Cellular Defence

The UPR (unfolded protein response), a cellular defence mechanism against misfolded protein accumulation in the ER (endoplasmic reticulum), is associated with many human diseases such as aging, cancer and diabetes. XBP1 (X-box-binding protein 1), a key transcription factor of the UPR, is critical in maintaining ER homoeostasis. Nevertheless, the mechanism by which XBP1 transcriptional activity is regulated remains unexplored. In the present study we show that XBP1s, the active spliced form of XBP1 protein, is SUMOylated, mainly by PIAS2 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 2] at two lysine residues located in the C-terminal transactivation domain. Ablation of these SUMOylation events significantly enhances the transcriptional activity of XBP1s towards UPR target genes. Thus our results reveal an unexpected role for SUMO (small ubiquitin-related modifier) in the regulation of UPR activation and ER homoeostasis.

scholarly journals Identification of Target Genes Involved in the Antiproliferative Effect of Glucocorticoids Reveals a Role for Nuclear Factor-κB Repression

2005 ◽  
Vol 19 (3) ◽  
pp. 632-643 ◽  
Author(s):  
Lars-Göran Bladh ◽  
Johan Lidén ◽  
Ahmad Pazirandeh ◽  
Ingalill Rafter ◽  
Karin Dahlman-Wright ◽  
...  
Keyword(s):  
Target Genes ◽  
Nuclear Factor Κb ◽  
Antiproliferative Effect ◽  
Nuclear Factor ◽  
Activator Protein 1 ◽  
Human Embryonic Kidney ◽  
Inhibitory Protein ◽  
293 Cells ◽  
M Phase ◽  
Responsive Element

Abstract Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-κB (NF-κB) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-κB signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-κB-inhibitory protein IκBα, in line with repression of NF-κB signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of IκBα was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of IκBα in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-κB is an important mechanism in the antiproliferative response to GCs.

scholarly journals Mediator Complex Recruits Epigenetic Regulators via Its Two Cyclin-dependent Kinase Subunits to Repress Transcription of Immune Response Genes

2013 ◽  
Vol 288 (29) ◽  
pp. 20955-20965 ◽  
Author(s):  
Taiki Tsutsui ◽  
Rikiya Fukasawa ◽  
Kaori Shinmyouzu ◽  
Reiko Nakagawa ◽  
Kazuyuki Tobe ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Proximal Region ◽  
Mediator Complex ◽  
Cyclin Dependent Kinase ◽  
Histone H4 ◽  
Promoter Regions

The Mediator complex (Mediator) plays pivotal roles in activating transcription by RNA polymerase II, but relatively little is known about its roles in repression. Here, we identified the histone arginine methyltransferase PRMT5 and WD repeat protein 77/methylosome protein 50 (WDR77/MEP50) as Mediator cyclin-dependent kinase (CDK)-interacting proteins and studied the roles of PRMT5 in the transcriptional regulation of CCAAT enhancer-binding protein (C/EBP) β target genes. First, we purified CDK8- and CDK19-containing complexes from HeLa nuclear extracts and subjected these purified complexes to mass spectrometric analyses. These experiments revealed that two Mediator CDKs, CDK8 and CDK19, individually interact with PRMT5 and WDR77, and their interactions with PRMT5 cause transcriptional repression of C/EBPβ target genes by regulating symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) in the promoter regions of those genes. Furthermore, the recruitment of the DNA methyltransferase DNMT3A correlated with H4R3 dimethylation potentially leading to DNA methylation at the promoter proximal region and tight inhibition of preinitiation complex formation. In vertebrates, C/EBPβ regulates many genes involved in immune responses and cell differentiation. These findings shed light on the molecular mechanisms of the repressive roles of Mediator CDKs in transcription of C/EBPβ target genes and might provide clues that enable future studies of the functional associations between Mediators and epigenetic regulation.

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