scholarly journals Degradation of the Tumor Suppressor Smad4 by WW and HECT Domain Ubiquitin Ligases

2005 ◽  
Vol 280 (23) ◽  
pp. 22115-22123 ◽  
Author(s):  
Anita Morén ◽  
Takeshi Imamura ◽  
Kohei Miyazono ◽  
Carl-Henrik Heldin ◽  
Aristidis Moustakas
Keyword(s):  
Transforming Growth Factor ◽  
Direct Interaction ◽  
Ubiquitin Ligases ◽  
Transforming Growth Factor Β ◽  
Down Regulation ◽  
Peripheral Cell ◽  
Hect Domain ◽  
E3 Ligases ◽  
Mechanism Of Degradation ◽  
Signaling Receptors

Smad4 mediates signaling by the transforming growth factor-β (TGF-β) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-β receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4–2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-β signaling may be critical for proper physiological response to this pathway.

scholarly journals Kelch-like protein 42 is a profibrotic ubiquitin E3 ligase involved in systemic sclerosis

2020 ◽  
Vol 295 (13) ◽  
pp. 4171-4180 ◽  
Author(s):  
Travis B. Lear ◽  
Karina C. Lockwood ◽  
Mads Larsen ◽  
Ferhan Tuncer ◽  
Jason R. Kennerdell ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Transforming Growth Factor ◽  
Nuclear Translocation ◽  
E3 Ligase ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Regulatory Subunit ◽  
Connective Tissues ◽  
E3 Ligases ◽  
Ubiquitin E3 Ligase

Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor β (TGF-β) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-β signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc–TGF-β signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-β signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-β–dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-β–mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-β–mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42–PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc.

scholarly journals NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) negatively regulates TGF-β (transforming growth factor-β) signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-β type I receptor

2005 ◽  
Vol 386 (3) ◽  
pp. 461-470 ◽  
Author(s):  
Go KURATOMI ◽  
Akiyoshi KOMURO ◽  
Kouichiro GOTO ◽  
Masahiko SHINOZAKI ◽  
Keiji MIYAZAWA ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Ubiquitin Ligases ◽  
Transforming Growth Factor Β ◽  
Precursor Cell ◽  
Neural Precursor ◽  
Type I ◽  
Dependent Manner ◽  
E3 Ubiquitin Ligases ◽  
Neural Precursor Cell

Inhibitory Smad, Smad7, is a potent inhibitor of TGF-β (transforming growth factor-β) superfamily signalling. By binding to activated type I receptors, it prevents the activation of R-Smads (receptor-regulated Smads). To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified NEDD4-2 (neural precursor cell expressed, developmentally down-regulated 4-2) as a direct binding partner of Smad7. NEDD4-2 is structurally similar to Smurfs (Smad ubiquitin regulatory factors) 1 and 2, which were identified previously as E3 ubiquitin ligases for R-Smads and TGF-β superfamily receptors. NEDD4-2 functions like Smurfs 1 and 2 in that it associates with TGF-β type I receptor via Smad7, and induces its ubiquitin-dependent degradation. Moreover, NEDD4-2 bound to TGF-β-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3. However, in contrast with Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN (Ski-related novel protein N), although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2. We showed further that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-β and BMP, whereas silencing of the NEDD4-2 gene by siRNA (small interfering RNA) resulted in enhancement of the responsiveness to TGF-β superfamily cytokines. These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT (WW is Trp-Trp and HECT is homologous to the E6-accessory protein) type E3 ubiquitin ligases, which negatively regulate TGF-β superfamily signalling through similar, but not identical, mechanisms to those used by Smurfs.

Antagonistic Effects of Insulin and TGF-β3 during Chondrogenic Differentiation of Human BMSCs under a Minimal Amount of Factors

2016 ◽  
Vol 201 (2) ◽  
pp. 88-96 ◽  
Author(s):  
Emilio Satoshi Hara ◽  
Mitsuaki Ono ◽  
Yuya Yoshioka ◽  
Junji Ueda ◽  
Yuri Hazehara ◽  
...  
Keyword(s):  
Chondrogenic Differentiation ◽  
Transforming Growth Factor ◽  
Direct Interaction ◽  
Transforming Growth Factor Β ◽  
Treated Group ◽  
Minimal Amount ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Antagonistic Effects

Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-β (TGF-β) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-β-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-β3 or in combination, and found that TGF-β3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-β3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-β3 significantly decreased the TGF-β3-induced increase in mRNA levels of the direct downstream factor of TGF-β/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-β3 alone. On the other hand, insulin/TGF-β3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-β3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-β3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.

scholarly journals Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

2013 ◽  
Vol 288 (12) ◽  
pp. 8737-8749 ◽  
Author(s):  
Huibin Yang ◽  
Gangyong Li ◽  
Jing-Jiang Wu ◽  
Lidong Wang ◽  
Michael Uhler ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transforming Growth Factor ◽  
Direct Interaction ◽  
Transforming Growth Factor Β ◽  
Smad4 Protein ◽  
Kinase A

scholarly journals A Transforming Growth Factor β-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A

2004 ◽  
Vol 24 (5) ◽  
pp. 2169-2180 ◽  
Author(s):  
Lizhi Zhang ◽  
Chao Jun Duan ◽  
Charles Binkley ◽  
Gangyong Li ◽  
Michael D. Uhler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transforming Growth Factor ◽  
Direct Interaction ◽  
Transforming Growth Factor Β ◽  
Regulatory Subunit ◽  
Smad Proteins ◽  
Kinase A

ABSTRACT Transforming growth factor β (TGFβ) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFβ signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFβ activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFβ activation of CREB, induction of p21Cip1, and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFβ and PKA signaling pathways.

scholarly journals Dab2 inhibits the cholesterol-dependent activation of JNK by TGF-β

2014 ◽  
Vol 25 (10) ◽  
pp. 1620-1628 ◽  
Author(s):  
Keren E. Shapira ◽  
Tal Hirschhorn ◽  
Lior Barzilay ◽  
Nechama I. Smorodinsky ◽  
Yoav I. Henis ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Lateral Diffusion ◽  
Adaptor Protein ◽  
Transforming Growth Factor Β ◽  
Cholesterol Depletion ◽  
Type I ◽  
Dependent Manner ◽  
A Cell ◽  
Β Receptor ◽  
Signaling Receptors

Transforming growth factor-β (TGF-β) ligands activate Smad-mediated and noncanonical signaling pathways in a cell context–dependent manner. Localization of signaling receptors to distinct membrane domains is a potential source of signaling output diversity. The tumor suppressor/endocytic adaptor protein disabled-2 (Dab2) was proposed as a modulator of TGF-β signaling. However, the molecular mechanism(s) involved in the regulation of TGF-β signaling by Dab2 were not known. Here we investigate these issues by combining biophysical studies of the lateral mobility and endocytosis of the type I TGF-β receptor (TβRI) with TGF-β phosphoprotein signaling assays. Our findings demonstrate that Dab2 interacts with TβRI to restrict its lateral diffusion at the plasma membrane and enhance its clathrin-mediated endocytosis. Small interfering RNA–mediated knockdown of Dab2 or Dab2 overexpression shows that Dab2 negatively regulates TGF-β–induced c-Jun N-terminal kinase (JNK) activation, whereas activation of the Smad pathway is unaffected. Moreover, activation of JNK by TGF-β in the absence of Dab2 is disrupted by cholesterol depletion. These data support a model in which Dab2 regulates the domain localization of TβRI in the membrane, balancing TGF-β signaling via the Smad and JNK pathways.

Free fucose is a danger signal to human intestinal epithelial cells

2008 ◽  
Vol 99 (3) ◽  
pp. 449-454 ◽  
Author(s):  
Wai Ling Chow ◽  
Yuan Kun Lee
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Intestinal Epithelial Cells ◽  
Direct Interaction ◽  
Transforming Growth Factor Β ◽  
Intestinal Lumen ◽  
Mucosal Barrier ◽  
Intestinal Epithelial ◽  
Regulated Gene Expression

Fucose is present in foods, and it is a major component of human mucin glycoproteins and glycolipids.l-Fucose can also be found at the terminal position of many cell-surface oligosaccharide ligands that mediate cell-recognition and adhesion-signalling pathways. Mucin fucose can be released through the hydrolytic activity of pathogens and indigenous bacteria, leading to the release of free fucose into the intestinal lumen. The immunomodulating effects of free fucose on intestinal epithelial cells (enterocyte-like Caco-2) were investigated. It was found that the presence ofl-fucose up regulated genes and secretion of their encoded proteins that are involved in both the innate and adaptive immune responses, possibly via the toll-like receptor-2 signalling pathway. These include TNFSF5, TNFSF7, TNF-α, IL12, IL17 and IL18.Besides modulating immune reactions in differentiated Caco-2 cells, fucose induced a set of cytokine genes that are involved in the development and proliferation of immune cells. These include the bone morphogenetic proteins (BMP) BMP2, BMP4, IL5, thrombopoietin and erythropoietin. In addition, the up regulated gene expression of fibroblast growth factor-2 may help to promote epithelial cell restitution in conjunction with the enhanced expression of transforming growth factor-β mRNA. Since the exogenous fucose was not metabolised by the differentiated Caco-2 cells as a carbon source, the reactions elicited were suggested to be a result of the direct interaction of fucose and differentiated Caco-2 cells. The presence of free fucose may signal the invasion of mucin-hydrolysing microbial cells and breakage of the mucosal barrier. The intestinal epithelial cells respond by up regulation and secretion of cytokines, pre-empting the actual invasion of pathogens.

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