scholarly journals Transforming Growth Factor-β Induces Nuclear Import of Smad3 in an Importin-β1 and Ran-dependent Manner

2001 ◽  
Vol 12 (4) ◽  
pp. 1079-1091 ◽  
Author(s):  
Akira Kurisaki ◽  
Shingo Kose ◽  
Yoshihiro Yoneda ◽  
Carl-Henrik Heldin ◽  
Aristidis Moustakas
Keyword(s):  
Growth Factor ◽  
Nuclear Import ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Structural Basis ◽  
Type I ◽  
Dependent Manner ◽  
Permeabilized Cells ◽  
Terminal Domain

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-β (TGF-β) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-β type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-β1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-β1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-β1. 4) Importin-β1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-β receptor leads to enhanced interaction with importin-β1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

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.

scholarly journals Transforming Growth Factor-β Receptors Interact with AP2 by Direct Binding to β2 Subunit

2002 ◽  
Vol 13 (11) ◽  
pp. 4001-4012 ◽  
Author(s):  
Diying Yao ◽  
Marcelo Ehrlich ◽  
Yoav I. Henis ◽  
Edward B. Leof
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Coated Pits ◽  
Wide Range ◽  
Cytoplasmic Tails ◽  
Direct Binding

Transforming growth factor-β (TGF-β) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K+ depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the μ2 subunit, AP2/TGF-β receptor binding was mediated by a direct interaction between the β2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the μ2, α, or ς2 subunits of AP2 or to μ1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of β2-adaptin to directly couple TGF-β receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with β2-adaptin.

scholarly journals Functionally diverse heteromeric traps for ligands of the transforming growth factor-β superfamily

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ravindra Kumar ◽  
Asya V. Grinberg ◽  
Huiming Li ◽  
Tzu-Hsing Kuo ◽  
Dianne Sako ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Systematic Analysis ◽  
Activin Receptor ◽  
Naturally Occurring ◽  
Functionally Diverse

AbstractLigands of the transforming growth factor-β (TGF-β) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-β superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-β ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-β superfamily signaling.

scholarly journals The Effects of Platelet-Derived Growth Factor Antagonism in Experimental Glomerulonephritis Are Independent of the Transforming Growth Factor–β System

2002 ◽  
Vol 13 (3) ◽  
pp. 658-667 ◽  
Author(s):  
Tammo Ostendorf ◽  
Uta Kunter ◽  
Claudia van Roeyen ◽  
Steven Dooley ◽  
Nebojsa Janjic ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Matrix Protein ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Platelet Derived Growth Factor ◽  
Renal Diseases ◽  
Protein Accumulation ◽  
Type I

ABSTRACT. Platelet-derived growth factor B-chain (PDGF-B)– and transforming growth factor beta (TGF-β)–mediated accumulation of extracellular matrix proteins contributes to many progressive renal diseases. In vivo, specific antagonism of either PDGF-B or TGF-β in experimental mesangioproliferative glomerulonephritis resulted in an almost complete inhibition of matrix protein accumulation, which suggests an interaction between signaling pathways of these two growth factors. Because nothing is known on the nature of this possible interaction, PDGF-B was antagonized in the rat anti–Thy 1.1 model of glomerulonephritis by use of specific aptamers and its effects on the TGF-β system were investigated. Antagonism of PDGF-B led to a significant reduction of glomerular matrix accumulation compared with scrambled aptamer-treated nephritic controls. PDGF-B antagonism had no effect on the overexpression of glomerular TGF-β mRNA, TGF-β protein, or the expression of TGF-β receptor type I and II mRNA. By immunohistology, it was possible to detect overexpression of the cytoplasmic TGF-β signaling molecules Smad2 (agonistic) and Smad7 (antagonistic) in glomeruli of nephritic control rats which peaked on day 7 after disease induction, i.e., the peak of mesangial cell proliferation in this model. However, immunohistology and Western blot analysis again revealed no difference in the glomerular expression of both Smad proteins between PDGF-B antagonized and nonantagonized nephritic animals. In addition, no difference in the glomerular expression of phosphorylated Smad2 (P-Smad2) was detected between the differently treated nephritic groups. These observations suggest that the effects of PDGF-B antagonism are independent of TGF-β in mesangioproliferative glomerulonephritides.

scholarly journals Transforming Growth Factor β Facilitates β-TrCP-Mediated Degradation of Cdc25A in a Smad3-Dependent Manner

2005 ◽  
Vol 25 (8) ◽  
pp. 3338-3347 ◽  
Author(s):  
Dipankar Ray ◽  
Yasuhisa Terao ◽  
Dipali Nimbalkar ◽  
Li-Hao Chu ◽  
Maddalena Donzelli ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Anaphase Promoting Complex ◽  
Mutant Proteins ◽  
Major Mechanism ◽  
Β Receptor ◽  
Interfering Rna

ABSTRACT Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-β-TrCP (SCFβ-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor β (TGF-β)-Smad3 pathway promotes SCFβ-TrCP-mediated Cdc25A ubiquitination. Cells treated with TGF-β, as well as cells transfected with Smad3 or a constitutively active type I TGF-β receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-β-induced ubiquitination is associated with Cdc25A phosphorylation at the β-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and β-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-β-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits β-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-β. β-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFβ-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-β-Smad3 pathway.

scholarly journals Smad7 Antagonizes Transforming Growth Factor β Signaling in the Nucleus by Interfering with Functional Smad-DNA Complex Formation

2007 ◽  
Vol 27 (12) ◽  
pp. 4488-4499 ◽  
Author(s):  
Suping Zhang ◽  
Teng Fei ◽  
Lixia Zhang ◽  
Ran Zhang ◽  
Feng Chen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Feedback Regulation ◽  
Transforming Growth Factor Β ◽  
Binding Activity ◽  
Type I ◽  
Negative Feedback Regulation ◽  
Dna Complex

ABSTRACT Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor β (TGF-β) signaling by inhibiting TGF-β signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-β signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-β target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-β-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-β signaling in the nucleus by a novel mechanism.

scholarly journals STRAP and Smad7 Synergize in the Inhibition of Transforming Growth Factor β Signaling

2000 ◽  
Vol 20 (9) ◽  
pp. 3157-3167 ◽  
Author(s):  
Pran K. Datta ◽  
Harold L. Moses
Keyword(s):  
Growth Factor ◽  
Transcriptional Activation ◽  
Intracellular Signaling ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Transcriptional Responses ◽  
C Terminus ◽  
Smad Proteins

ABSTRACT Smad proteins play a key role in the intracellular signaling of the transforming growth factor β (TGF-β) superfamily of extracellular polypeptides that initiate signaling from the cell surface through serine/threonine kinase receptors. A subclass of Smad proteins, including Smad6 and Smad7, has been shown to function as intracellular antagonists of TGF-β family signaling. We have previously reported the identification of a WD40 repeat protein, STRAP, that associates with both type I and type II TGF-β receptors and that is involved in TGF-β signaling. Here we demonstrate that STRAP synergizes specifically with Smad7, but not with Smad6, in the inhibition of TGF-β-induced transcriptional responses. STRAP does not show cooperation with a C-terminal deletion mutant of Smad7 that does not bind with the receptor and consequently has no inhibitory activity. STRAP associates stably with Smad7, but not with the Smad7 mutant. STRAP recruits Smad7 to the activated type I receptor and forms a complex. Moreover, STRAP stabilizes the association between Smad7 and the activated receptor, thus assisting Smad7 in preventing Smad2 and Smad3 access to the receptor. STRAP interacts with Smad2 and Smad3 but does not cooperate functionally with these Smads to transactivate TGF-β-dependent transcription. The C terminus of STRAP is required for its phosphorylation in vivo, which is dependent on the TGF-β receptor kinases. Thus, we describe a mechanism to explain how STRAP and Smad7 function synergistically to block TGF-β-induced transcriptional activation.

scholarly journals FKBP12 functions as an adaptor of the Smad7–Smurf1 complex on activin type I receptor

2006 ◽  
Vol 36 (3) ◽  
pp. 569-579 ◽  
Author(s):  
T Yamaguchi ◽  
A Kurisaki ◽  
N Yamakawa ◽  
K Minakuchi ◽  
H Sugino
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Regulatory Factor ◽  
Fk506 Binding Protein ◽  
Adaptor Molecule ◽  
Inhibitory Function ◽  
Inhibitory Molecule

The cytoplasmic immunophilin FKBP12, a 12 kDa FK506-binding protein, has been shown to act as an inhibitor for transforming growth factor-β (TGF-β) signaling. FKBP12 binds to the glycine- and serine-rich motif (GS motif) of the TGF-β type I receptor, and functions as a secure switch to prevent the leaky signal. Upon stimulation with ligand, FKBP12 is released from the receptor to fully propagate the signal. We found that activin, a member of TGF-β superfamily, also induced the dissociation of FKBP12 from the activin type I receptor (ALK4). However, we observed that the released FKBP12 associates again with the receptor a few hours later. FKBP12 also interacted with another inhibitory molecule of activin signal, Smad7, in an activin-dependent manner, and formed a complex with Smad7 on the type I receptor. FK506, a chemical ligand for FKBP12, which dissociates FKBP12 from the receptor, decreased the interaction between Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1). FK506 also inhibited the ubiquitination of the type I receptor by Smurf1. These findings indicate a new inhibitory function of FKBP12 as an adaptor molecule for the Smad7–Smurf1 complex to regulate the duration of the activin signal.

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