Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Smad proteins are crucial for the intracellular signaling of transforming growth factor-beta (TGF-beta). After receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate transcription of a select set of target genes. Here, we investigated the turnover of Smad3, positively regulating Smad for TGF-beta signaling. In a steady state, the inhibition of proteasome activity leads to stabilization of Smad3 protein. Smad proteins are multi-ubiquitinated and degraded independently of the phosphorylation induced by the TGF-beta receptors. Moreover, the degradation of Smad3 was enhanced by treatment with TGF-beta, and phosphorylated Smad3 was accumulated on proteasome inhibition. Ubiquitination of phosphorylated Smad3 but not Smad3(3SA), a receptor-mediated phosphorylation-incompetent mutant, was observed in the nucleus after treatment with TGF-beta. These findings suggest that, in a steady state, Smad3 is constitutively degraded via the ubiquitin-proteasome pathway in the cytoplasm and that, in response to TGF-beta, it is phosphorylated and translocated into the nucleus, where it is degraded through the ubiquitin-proteasome pathway.
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PMID:Contribution of the constitutive and inducible degradation of Smad3 by the ubiquitin-proteasome pathway to transforming growth factor-beta signaling. 1498 84

Signaling by transforming growth factor-beta (TGF-beta), a regulator of several biological processes, including renal fibrosis, is mediated, in part, by the Smad proteins. Tight control of Smad level and activity is critical for proper TGF-beta biological functions. Here, we have investigated the mechanisms involved in regulating Smad3 expression. In human glomerular mesangial cells, Smad3 protein levels were specifically reduced by 24 h of TGF-beta1 treatment, whereas Smad2 and Smad4 levels were not. TGF-beta1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-beta1-mediated Smad3 down-regulation resulting in accumulation of ubiquitinated Smad3. These data support the concept that Smad3 down-regulation occurs via degradation by the ubiquitin/proteasome machinery. However, changes in Smad3 protein levels were also paralleled by changes in Smad3 mRNA expression. TGF-beta1 did not decrease Smad3 mRNA stability, but it significantly inhibited Smad3 promoter activity. In renal tubular epithelial cells, decreased Smad3 levels were observed only after exposure to TGF-beta1 for longer time periods (5-7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle alpha-actin and decreased expression of E-cadherin. Decline in Smad3 expression also occurred in kidneys after unilateral ureteral obstruction, a model of tubulointerstitial fibrosis associated with TGF-beta up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-beta1 modulates the expression of a receptor-activated Smad at both the protein and transcriptional level. Smad3 down-regulation could represent a feedback loop controlling TGF-beta signaling in a cell phenotype-specific manner.
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PMID:Cell phenotype-specific down-regulation of Smad3 involves decreased gene activation as well as protein degradation. 1740 May 44

We have identified km23-1 as a novel transforming growth factor-beta (TGFbeta) receptor (TbetaR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGFbeta but not in the absence, km23-1 was present in early endosomes with the TbetaRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFbeta treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGFbeta regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGFbeta treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGFbeta/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGFbeta/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGFbeta-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGFbeta signaling pathway.
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PMID:Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway. 1742 Feb 58

Controlling protein function through posttranslational manipulations has emerged as an attractive complementary technology to existing genetic systems. Often these methods involve developing pharmacological agents to probe protein function without the need to generate a unique compound for each protein family. One common strategy uses small molecules that act as chemical inducers of dimerization by mediating the interaction of two proteins. Herein we report the use of a chemical inducer of dimerization for the development of a posttranslational technology for the manipulation of protein function. This system, split ubiquitin for the rescue of function (SURF), places the complementation of genetically split ubiquitin under the control of rapamycin-induced dimerization of FK506-binding protein and FKBP12-rapamycin-binding protein. Before complementation a "degron" dooms a protein of interest for destruction by the proteasome. Addition of rapamycin results in a proteolytic shunt away from degradation by inducing ubiquitin complementation and cleavage of the protein of interest from the degron. Importantly, the native protein is rescued. We characterized this system with firefly luciferase and went on to apply it to members of three important classes of proteins: proteases (caspase-3), kinases (v-Src), and transcription factors (Smad3). This general strategy should allow for inducible rescue of a variety of proteins in such a way that their native structure and function are maintained.
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PMID:Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt. 1760 1

The broad range of biological responses elicited by transforming growth factor-beta (TGF-beta) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. Here we show that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-beta (GSK3-beta). Smad3 physically interacts with Axin and GSK3-beta only in the absence of TGF-beta. Reduction in the expression or activity of Axin/GSK3-beta leads to increased Smad3 stability and transcriptional activity without affecting TGF-beta receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-beta. Mechanistically, Axin facilitates GSK3-beta-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-beta, and suggest a new function of the Axin/GSK3-beta complex in modulating critical TGF-beta/Smad3-regulated processes during development and tumor progression.
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PMID:Axin and GSK3- control Smad3 protein stability and modulate TGF- signaling. 1817 67

Smurf2 is an E3 ubiquitin ligase that targets TGF-beta receptor activated Smad2 and Smad3 for the proteasome in primary articular chondrocytes, thus stimulating their hypertrophic differentiation. Comparatively, how Smurf2 functions in growth plate chondrocytes in a developing long bone is an open question. In this study, we measured the mRNA levels of endogenous Smurf2 and type X collagen in chick growth plate at different embryonic stages to monitor the correlation between the level of Smurf2 expression and chondrocyte maturational stage. We found that high levels of Smurf2 were associated with the differentiative and proliferative stages, while Smurf2 levels were thereafter decreased as the chondrocytes matured toward hypertrophy. In addition, we injected Smurf2-RCAS into chick wing buds at HH stage 20-23 and examined how the ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme affects the subsequent process of chondrocyte maturation and ossification during embryonic development. Histological analysis showed that overexpression of Smurf2 in a developing wing bud accelerated chondrocyte maturation and endochondral ossification, which may result from a decrease in TGF-beta signaling in the infected chondrocytes with Smurf2-RCAS.
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PMID:Regulation of embryonic endochondral ossification by Smurf2. 1817 45

Several models have been suggested above, describing possible modes of spindle checkpoint action: 1. Cdc20 sequestration (by Mad2-Cdc20 and/or MCC). 2. Stable MCC-APC/C association. 3. Cdc20 turnover (in budding yeast). 4. Cdc20-APC/C modification (by Mps1, Bub1, MAPK, Aurora B or BubR1 kinases). Several of these mechanisms could affect APC/C activity by modifying, competing for, and/or blocking the binding site(s) for its substrates. Alternatively, they could reduce the processivity of ubiquitination of substrates, or prevent the release of substrates and thereby reduce substrate turnover. Indeed, the processivity of ubiquitination can determine the order of destruction of APC/C substrates (Rape et al., 2006). Most substrates require multiple APC/C binding events in order to build polyubiquitin chains, and only polyubiquitinated substrates are recognised by the 26S proteasome for destruction. Thus, if the processivity of ubiquitination or the turnover of APC/C substrates were impaired in mitosis, the degradation of securin and cyclin would no longer take place, which would result in mitotic arrest. Our results have highlighted the importance of Mad3 as an anaphase inhibitor, and suggest that it usually acts in concert with Mad2 to efficiently inhibit Cdc20-APC/C. Further experiments are necessary to fully understand their mechanism of action, and this will require a wide range of approaches including dynamic studies of the 'flux' of Mad2 and BubR1 through signalling scaffolds, further structural insights, the identification of important phosphorylation sites on both the checkpoint proteins and Cdc20-APC/C, and an in vitro reconstitution of MCC inhibition of the APC/C. We look forward to seeing the complex regulation of mitotic progression being described over the coming years.
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PMID:The spindle checkpoint: how do cells delay anaphase onset? 1836 27

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.
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PMID:Transforming growth factor-beta suppresses the ability of Ski to inhibit tumor metastasis by inducing its degradation. 1845 Nov 54

Transforming growth factor-beta (TGF-beta) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-beta signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-beta and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-beta-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-beta signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.
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PMID:Pin1 down-regulates transforming growth factor-beta (TGF-beta) signaling by inducing degradation of Smad proteins. 1912 40

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.
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PMID:Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling. 1991 53


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