scholarly journals Arkadia Activates Smad3/Smad4-Dependent Transcription by Triggering Signal-Induced SnoN Degradation

2007 ◽  
Vol 27 (17) ◽  
pp. 6068-6083 ◽  
Author(s):  
Laurence Levy ◽  
Michael Howell ◽  
Debipriya Das ◽  
Sean Harkin ◽  
Vasso Episkopou ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Transforming Growth Factor ◽  
E3 Ubiquitin Ligase ◽  
Cancer Cell Line ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Esophageal Cancer Cell ◽  
Esophageal Cancer Cell Line

ABSTRACT E3 ubiquitin ligases play important roles in regulating transforming growth factor β (TGF-β)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-β induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-β response. Knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-β-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-β/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-β-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer.

scholarly journals HIV-1 Vpr Induces the K48-Linked Polyubiquitination and Proteasomal Degradation of Target Cellular Proteins To Activate ATR and Promote G2 Arrest

2010 ◽  
Vol 84 (7) ◽  
pp. 3320-3330 ◽  
Author(s):  
Jean-Philippe Belzile ◽  
Jonathan Richard ◽  
Nicole Rougeau ◽  
Yong Xiao ◽  
Éric A. Cohen
Keyword(s):  
Ubiquitin Ligase ◽  
Affinity Purification ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Proteasomal Degradation ◽  
Dominant Negative ◽  
Cellular Proteins ◽  
Dominant Negative Mutant ◽  
M Phase ◽  
Hiv 1

ABSTRACT HIV-1 viral protein R (Vpr) induces cell cycle arrest at the G2/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G2 arrest. While attractive, this model is based solely on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G2 arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that the depletion of VPRBP by RNA interference or the overexpression of a dominant negative mutant of CUL4A decreased this association. Importantly, G2-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed a decreased association with ubiquitinated proteins. We also found that the inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, the inhibition of K48 polyubiquitination specifically impaired the Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that the association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of as-yet-unknown cellular proteins, resulting in their proteasomal degradation and ultimately leading to the activation of ATR and G2 arrest.

scholarly journals RB1CC1 Protein Positively Regulates Transforming Growth Factor-β Signaling through the Modulation of Arkadia E3 Ubiquitin Ligase Activity

2011 ◽  
Vol 286 (37) ◽  
pp. 32502-32512 ◽  
Author(s):  
Daizo Koinuma ◽  
Masahiko Shinozaki ◽  
Yoshiko Nagano ◽  
Hiroaki Ikushima ◽  
Kana Horiguchi ◽  
...  
Keyword(s):  
Growth Factor ◽  
Ubiquitin Ligase ◽  
Transforming Growth Factor ◽  
E3 Ubiquitin Ligase ◽  
Transforming Growth Factor Β ◽  
Ubiquitin Ligase Activity

scholarly journals Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-β autocrine regulatory loop

2004 ◽  
Vol 381 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Séverine ALLEGRA ◽  
Jacques Yuan LI ◽  
José Maria SAEZ ◽  
Dominique LANGLOIS
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Terminal Differentiation ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Time Interval ◽  
Tgfβ Signalling ◽  
Regulatory Loop ◽  
Myod Expression

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the βMHC (β-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFβ (transforming growth factor β) on βMHC expression in Sol 8 cells. TGFβ1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited βMHC expression in Sol 8 cells. This inhibition of βMHC expression was progressively lost when TGFβ1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFβ1 did not inhibit βMHC accumulation any longer. Two independent approaches showed that a TGFβ autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFβs released by the cells into DM increased βMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFβ type II receptor was overexpressed in Sol 8 cells under the control of the βMHC promoter. Both the dominant-negative receptor and the βMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFβ signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote βMHC expression. To conclude, TGFβ inhibits Sol 8 cell terminal differentiation within a narrow time interval (24–34 h) that coincides with the onset of βMHC expression.

Activation of TGF-β-Smad signaling pathway following polyamine depletion in intestinal epithelial cells

2003 ◽  
Vol 285 (5) ◽  
pp. G1056-G1067 ◽  
Author(s):  
Lan Liu ◽  
Rachel Santora ◽  
Jaladanki N. Rao ◽  
Xin Guo ◽  
Tongtong Zou ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
Transcription Activation ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Receptor Signaling ◽  
Β Receptor

Smad proteins are transcription activators that are critical for transmitting transforming growth factor-β (TGF-β) superfamily signals from the cell surface receptors to the nucleus. Our previous studies have shown that cellular polyamines are essential for normal intestinal mucosal growth and that a decreased level of polyamines inhibits intestinal epithelial cell proliferation, at least partially, by increasing expression of TGF-β/TGF-β receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-β/TGF-β receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by α-difluoromethylornithine (DFMO) increased basal levels of Smad3 and Smad4 proteins, induced their nuclear translocation, and stimulated Smad sequence-specific DNA-binding activity. Polyamine depletion-induced Smads were also associated with a significant increase in transcription activation as measured by luciferase reporter gene activity of Smad-dependent promoters. Inhibition of Smads by a dominant-negative mutant Smad4 in the DFMO-treated cells prevented the increased Smad transcription activation. Polyamine-deficient cells highly expressed TGF-β and were growth-arrested at the G1 phase. Inhibition of TGF-β by treatment with either immunoneutralizing anti-TGF-β antibody or TGF-β antisense oligodeoxyribonucleotides not only blocked the induction of Smad activity but also decreased the Smad-mediated transcriptional activation in polyamine-depleted cells. These findings suggest that Smads are involved in the downstream cellular processes mediated by cellular polyamines and that increased TGF-β/TGF-β receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad target gene expression.

Abstract 1457: Cbl, E3 Ubiquitin Ligase, is a Regulator of Focal Adhesion Protein Proteasomal Degradation during Cardiomyocyte Anoikis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Khadija Rafiq ◽  
Rachid Seqqat ◽  
Marie Hanscom ◽  
Steven R Houser ◽  
Abdelkarim Sabri
Keyword(s):  
Dilated Cardiomyopathy ◽  
Protein Degradation ◽  
Focal Adhesion ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Cathepsin G ◽  
Dominant Negative Mutant ◽  
Myocyte Apoptosis

Proteasome degradation of ubiquitin-targeted proteins is an important mechanism that negatively controls activated signaling pathways. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitination and down-regulation. We have shown previously that neutrophil derived protease cathepsin G (Cat.G) induced focal adhesion (FA) protein degradation and myocyte apoptosis by anoikis. We hypothesized that Cbl is involved in myocyte apoptosis in response to Cat.G through ubiquitination and downregulation of FA proteins. Results : Immunoprecitpitation and western blot studies showed increased Cbl tyrosine phosphorylation in response to Cat.G that was associated with its interaction with FA proteins, FAK and paxillin. This led to an increase in FAK and Paxillin ubiquitination and degradation at 2 hrs after Cat.G addition. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced FAK and paxillin degradation. In contrast, adenoviral expression of wild type Cbl enhanced FAK and paxillin ubiquitination and degradation in response to Cat.G. Interestingly, Cbl activation was involved in Cat.G-induced myocyte apoptosis as overexpression of dominant negative Cbl significantly reduced caspase-3 activation and DNA fragmentation induced by Cat.G. Concomitant with these changes in vitro, heart tissue samples from patients with ischemic or dilated cardiomyopathy showed a marked increase in Cbl accumulation and FAK and paxillin degradation compared to heart controls. Conclusion : These studies show that Cbl abundance is increased in human ischemic and dilated cardiomyopathy and that activation of Cbl increases FA protein degradation and myocyte apoptosis. These results indicate that Cbl is a positive regulator of FA protein degradation and myocyte apoptosis and may play an important role in mediating the effect of neutrophil derived proteases during the progression of congestive heart failure.

scholarly journals LncRNA SNHG16 promotes pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Panpan Liu ◽  
Lei Zhao ◽  
Yuxia Gu ◽  
Meilan Zhang ◽  
Hongchang Gao ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Fibrosis ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Interstitial Lung Diseases ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation ◽  
And Migration

Abstract Background Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung diseases with a poor prognosis. Long non-coding RNAs (lncRNAs) have been reported to be involved in IPF in several studies. However, the role of lncRNA SNHG16 in IPF is largely unknown. Methods Firstly, experimental pulmonary fibrosis model was established by using bleomycin (BML). Histology and Western blotting assays were used to determine the different stages of fibrosis and expression of several fibrosis biomarkers. The expression of SNHG16 was detected by quantitative real-time polymerase chain reaction (qRT‐PCR). EdU staining and wound-healing assay were utilized to analyze proliferation and migration of lung fibroblast cells. Molecular mechanism of SNHG16 was explored by bioinformatics, dual-luciferase reporter assay, RNA immunoprecipitation assay (RIP), and qRT-PCR. Results The expression of SNHG16 was significantly up-regulated in bleomycin-(BLM) induced lung fibrosis and transforming growth factor-β (TGF-β)-induced fibroblast. Knockdown of SNHG16 could attenuate fibrogenesis. Mechanistically, SNHG16 was able to bind and regulate the expression of miR-455-3p. Moreover, SNHG16 also regulated the expression of Notch2 by targeting miR-455-3p. Finally, SNHG16 could promote fibrogenesis by regulating the expression of Notch2. Conclusion Taken together, our study demonstrated that SNHG16 promoted pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway. These findings might provide a novel insight into pathologic process of lung fibrosis and may provide prevention strategies in the future.

scholarly journals Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Allison B. Powell ◽  
Sridevi Yadavilli ◽  
Devin Saunders ◽  
Stacey Van Pelt ◽  
Elizabeth Chorvinsky ◽  
...  
Keyword(s):  
Nk Cells ◽  
Transforming Growth Factor ◽  
Nk Cell ◽  
Transforming Growth Factor Β ◽  
In Vitro Models ◽  
Dominant Negative ◽  
Conditioned Media ◽  
Retroviral Transduction ◽  
Effector Cells

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

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