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Kohei Miyazono - One of the best experts on this subject based on the ideXlab platform.
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roles for the MH2 Domain of smad7 in the specific inhibition of transforming growth factor β superfamily signaling
Journal of Biological Chemistry, 2004Co-Authors: Toshiaki Mochizuki, Hideyo Miyazaki, Takane Hara, Toshio Furuya, Takeshi Imamura, Tetsuro Watabe, Kohei MiyazonoAbstract:Signals by cytokines of the transforming growth factor-β (TGF-β) superfamily are negatively regulated by inhibitory Smads (I-Smads). Smad7 inhibits signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 inhibits TGF-β signals less effectively. I-Smads have amino-terminal N Domains and carboxyl-terminal Mad homology 2 (MH2) Domains. The N Domains are essential for specific inhibition of TGF-β signaling by Smad7, whereas the MH2 Domains of I-Smads are involved in the inhibition of TGF-β superfamily signals through interaction with type I receptors. Here, we have identified four basic amino acid residues (Lys-312, Lys-316, Lys-401, and Arg-409) in the basic surface of the Smad7 MH2 Domain that play important roles in interaction with type I receptors. Mutations of the four basic amino acid residues to acidic residues (K312E, K316E, K401E, and R409E) abolished the interaction of Smad7 with TGF-β type I receptors, inhibition of Smad2 phosphorylation and transcriptional responses induced by TGF-β, and induction of target genes of endogenous activin/Nodal signals in Xenopus early embryos. The K401E and R409E mutants of Smad7 were also unable to interact with BMP type I receptors (BMPR-I), repress the Smad5 phosphorylation and transcription induced by BMP, and effectively inhibit endogenous BMP signals in Xenopus early embryos. However, the K312E and K316E mutants were able to interact with BMPR-I and retained the ability to inhibit BMP signaling. Thus, the MH2 Domain of Smad7 plays important roles in specific inhibition of TGF-β superfamily signals through differential interaction with type I receptors.
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c ski acts as a transcriptional co repressor in transforming growth factor β signaling through interaction with smads
Journal of Biological Chemistry, 1999Co-Authors: Shingo Akiyoshi, Kohei Miyazono, Hirofumi Inoue, Junichi Hanai, Kiyoshi Kusanagi, Nobuo Nemoto, Masahiro KawabataAbstract:Abstract Smads are intracellular signaling mediators of the transforming growth factor-β (TGF-β) superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-β. We identified c-Ski as a Smad2 interacting protein. c-Ski is the cellular homologue of thev-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 Domain in a TGF-β-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-β. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-β signaling.
Ye-guang Chen - One of the best experts on this subject based on the ideXlab platform.
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Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-β (TGF-β)/Smad Signaling.
The Journal of biological chemistry, 2015Co-Authors: Xiaohua Yan, Xinhua Feng, Xia Lin, Hongwei Liao, Minzhang Cheng, Xiaojing Shi, Ye-guang ChenAbstract:TGF-β is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-β signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-β signaling, Smad7 exerts its inhibitory effects by blocking receptor activity, inducing receptor degradation or interfering with Smad-DNA binding. However, the functions and the molecular mechanisms underlying the actions of Smad7 in TGF-β signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-β signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-β signaling and directly inhibited R-Smad activity, as assessed by Gal4-luciferase reporter assays. Mechanistically, Smad7 competes with Smad4 to associate with R-Smads and recruits the E3 ubiquitin ligase NEDD4L to activated R-Smads, leading to their polyubiquitination and proteasomal degradation. Similar to the R-Smad-Smad4 oligomerization, the interaction between R-Smads and Smad7 is mediated by their mad homology 2 (MH2) Domains. A positive-charged basic region including the L3/β8 loop-strand module and adjacent amino acids in the MH2 Domain of Smad7 is essential for the interaction. These results shed new light on the regulation of TGF-β signaling by Smad7.
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structural basis of smad2 recognition by the smad anchor for receptor activation
Science, 2000Co-Authors: Ye-guang Chen, Joan Massagué, Barish Ozdamar, C A Gyuricza, P A Chong, Jeffrey L Wrana, Yang ShiAbstract:The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. The Smad anchor for receptor activation (SARA) recruits Smad2 to the TGFbeta receptors for phosphorylation. The crystal structure of a Smad2 MH2 Domain in complex with the Smad-binding Domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors.
Joan Massagué - One of the best experts on this subject based on the ideXlab platform.
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Distinct Domain Utilization by Smad3 and Smad4 for Nucleoporin Interaction and Nuclear Import
Journal of Biological Chemistry, 2003Co-Authors: Lan Xu, Claudio Alarcón, Joan MassaguéAbstract:Abstract Smad proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-β (TGFβ) and in so doing transduce the signal into the nucleus. In this report we unraveled nuclear import mechanisms of Smad3 and Smad4 that are dependent on their interaction with FG-repeat-containing nucleoporins such as CAN/Nup214, without the involvement of importin molecules that are responsible for most of the known nuclear import events. A surface hydrophobic corridor within the MH2 Domain of Smad3 is critical for association with CAN/Nup214 and nuclear import, whereas Smad4 interaction with CAN/Nup214, and nuclear import requires structural elements present only in the full-length Smad4. As exemplified by the different susceptibility to inhibition of import by cytoplasmic retention factor SARA (Smad anchor for receptor activation), such utilization of distinct Domains for nuclear import of Smad3 and Smad4 suggests that nuclear transport of Smad3 and Smad4 is subject to control by different retention factors.
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crystal structure of a phosphorylated smad2 recognition of phosphoserine by the MH2 Domain and insights on smad function in tgf β signaling
Molecular Cell, 2001Co-Authors: Jijie Chai, Joan Massagué, Joan Seoane, Morgan Huse, Daniel J Rigotti, Saw Kyin, Tom W Muir, Robert Fairman, Yigong ShiAbstract:Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-β signaling. The crystal structure of a phosphorylated Smad2, at 1.8 A resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 Domain, frequently targeted for inactivation in cancers, is highly conserved among the Co- and R-Smads. This finding, together with mutagenesis data, pinpoints a functional interface between Smad2 and Smad4. In addition, the pSer binding surface on the MH2 Domain coincides with the surface on R-Smads that is required for docking interactions with the serine-phosphorylated receptor kinases. These observations define a bifunctional role for the MH2 Domain as a pSer-X-pSer binding module in receptor Ser/Thr kinase signaling pathways.
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structural basis of smad2 recognition by the smad anchor for receptor activation
Science, 2000Co-Authors: Ye-guang Chen, Joan Massagué, Barish Ozdamar, C A Gyuricza, P A Chong, Jeffrey L Wrana, Yang ShiAbstract:The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. The Smad anchor for receptor activation (SARA) recruits Smad2 to the TGFbeta receptors for phosphorylation. The crystal structure of a Smad2 MH2 Domain in complex with the Smad-binding Domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors.
David Danielpour - One of the best experts on this subject based on the ideXlab platform.
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Hic-5 Controls BMP4 Responses in Prostate Cancer Cells through Interacting with Smads 1, 5 and 8
2016Co-Authors: Dorjee T. N. Shola, Hui Wang, Reema Wahdan-alaswad, David DanielpourAbstract:Hydrogen peroxide-inducible clone-5 (Hic-5, or androgen receptor-associated protein 55) is a transforming growth factor-β (TGF-β)-inducible LIM protein whose deregulation is implicated in the progression of prostate cancer. Here we report that Hic-5 binds to Smads 1, 5 and 8, and represses bone morphogenetic protein (BMP) signaling responses. Myc-Hic-5 but not Myc-paxillin was specifically immunoprecipitated with anti-FLAG IgG1 from lysates of HEK293 co-transfected with either Myc-Hic-5 or Myc-paxillin and FLAG-tagged Smads 1, 5 or 8. We showed that such interactions require the LIM3 Domain of Hic-5 and the MH2 Domain of those Smads. Anti-Hic-5 antibody specifically pulled down endogenous Smad1 in both the PC3 human prostate cell line and primary cultures of rat prostate fibroblasts, supporting that Hic-5 binds to Smad1 at the endogenous level. Bacterially expressed GST-Smads 1, 5 or 8, but not GST alone, pulled down in vitro transcribed and translated Hic-5, implicating that Hic-5 binds directly to Smads 1, 5 and 8. Significantly, using Hic-5 shRNA silencing and overexpression systems, we show that Hic-5 (at both the endogenous and exogenous levels) represses the ability of BMP4 to induce expression of the inhibitor of differentiation-1 (Id1) (a downstream target gene of BMP), activate the Id1 gen
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novel function of androgen receptor associated protein 55 hic 5 as a negative regulator of smad3 signaling
Journal of Biological Chemistry, 2005Co-Authors: Hui Wang, Kyung Song, Tracy L Sponseller, David DanielpourAbstract:Androgen receptor-associated protein 55 (ARA55/Hic-5) belongs to the LIM protein superfamily and is featured by three or four N-terminal LD motifs and four C-terminal zinc finger-like LIM Domains. Both LD motifs and LIM Domains can serve as protein-protein interaction interfaces. Recently, we found that enforced expression of ARA55 inhibits transforming growth factor-beta-mediated up-regulation of Smad binding element-luciferase reporter activity in NRP-154 and NRP-152 rat prostate and LNCaP human prostate cell lines. Moreover, ARA55 also inhibits the induction of Smad-binding element 4-luciferase and 3TP-luciferase (a plasminogen activator inhibitor-1 (PAI-1) promoter construct) reporters by constitutively active (CA)-Smad3 in these cell lines. Co-immunoprecipitation studies suggest an interaction between ARA55 and either CA-Smad3 or wild-type Smad3 in HEK293 cells that occurs through the MH2 Domain of Smad3 and the C terminus of ARA55 with wild-type Smad3 having stronger affinity than CA-Smad3 to ARA55. Glutathione S-transferase pull-down assays demonstrate that this interaction can occur in a cell-free system. These results are consistent with the luciferase data showing that the C terminus of ARA55 is critical for suppression of Smad3 activity. Furthermore, using a mammalian two-hybrid system, we confirmed that ARA55 interacts with the MH2 Domain of Smad3 and suppresses CA-Smad3-induced transcriptional responses. In conclusion, these results support that ARA55 selectively intercepts transforming growth factor-beta signaling through an interaction of the LIM Domain of ARA55 with the MH2 Domain of Smad3.
Toshiaki Mochizuki - One of the best experts on this subject based on the ideXlab platform.
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roles for the MH2 Domain of smad7 in the specific inhibition of transforming growth factor β superfamily signaling
Journal of Biological Chemistry, 2004Co-Authors: Toshiaki Mochizuki, Hideyo Miyazaki, Takane Hara, Toshio Furuya, Takeshi Imamura, Tetsuro Watabe, Kohei MiyazonoAbstract:Signals by cytokines of the transforming growth factor-β (TGF-β) superfamily are negatively regulated by inhibitory Smads (I-Smads). Smad7 inhibits signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 inhibits TGF-β signals less effectively. I-Smads have amino-terminal N Domains and carboxyl-terminal Mad homology 2 (MH2) Domains. The N Domains are essential for specific inhibition of TGF-β signaling by Smad7, whereas the MH2 Domains of I-Smads are involved in the inhibition of TGF-β superfamily signals through interaction with type I receptors. Here, we have identified four basic amino acid residues (Lys-312, Lys-316, Lys-401, and Arg-409) in the basic surface of the Smad7 MH2 Domain that play important roles in interaction with type I receptors. Mutations of the four basic amino acid residues to acidic residues (K312E, K316E, K401E, and R409E) abolished the interaction of Smad7 with TGF-β type I receptors, inhibition of Smad2 phosphorylation and transcriptional responses induced by TGF-β, and induction of target genes of endogenous activin/Nodal signals in Xenopus early embryos. The K401E and R409E mutants of Smad7 were also unable to interact with BMP type I receptors (BMPR-I), repress the Smad5 phosphorylation and transcription induced by BMP, and effectively inhibit endogenous BMP signals in Xenopus early embryos. However, the K312E and K316E mutants were able to interact with BMPR-I and retained the ability to inhibit BMP signaling. Thus, the MH2 Domain of Smad7 plays important roles in specific inhibition of TGF-β superfamily signals through differential interaction with type I receptors.