The N domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling

Hanyu, Aki; Ishidou, Yasuhiro; Ebisawa, Takanori; Shimanuki, Tomomasa; Imamura, Takeshi; Miyazono, Kohei

doi:10.1083/jcb.200106023

Cited by 210 publications

(196 citation statements)

References 37 publications

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“…These results also indicate that the de novo induction of Smad7 is not a mechanism involved in the inhibitory action of Ac-SDKP. Previous reports indicate that Smad7 is localized in the nucleus and that TGF-␤-mediated nuclear export of Smad7 is associated with its inhibitory effects (38,45); we therefore clarified the influence of Ac-SDKP on Smad7 localization. Although we could not clearly detect endogenous Smad7 in human mesangial cells by the antibody we used (data not shown), we confirmed the localization of Smad7 by the transfection study.…”

Section: Discussionmentioning

confidence: 84%

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Inhibits TGF-β–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells

Kanasaki

Koya

Sugimoto

et al. 2003

Journal of the American Society of Nephrology

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Abstract. Recent large clinical trials indicate that angiotensinconverting enzyme inhibitors (ACE-I) attenuate the detrimental outcome of progressive renal disease. The hemoregulatory tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP, AcSDKP) is hydrolyzed by ACE, and plasma Ac-SDKP level is increased by fivefold after treatment with ACE-I. Ac-SDKP was found to ameliorate cardiac and renal fibrosis in hypertensive animal models. However, the molecular mechanisms by which Ac-SDKP mediates anti-fibrotic effects remain unclear. This study is an examination of the interaction between Ac-SDKP and transforming growth factor-␤ (TGF-␤), one of the key cytokines in the progression of renal disease, in human mesangial cells. Ac-SDKP inhibited TGF-␤1-induced plasminogen activator inhibitor-1 (PAI-1) and alpha2 (I) collagen mRNA. Ac-SDKP suppressed not only TGF-␤1-induced Smad2 phosphorylation at Ser-465/467 in a dose-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. As expected, Ac-SDKP inhibited TGF-␤-responsive Smad-dependent luciferase reporters, 3TP-luc and 4xSBE-luc. Immunofluorescence analysis revealed that the inhibitory Smad, Smad7, was exported to the cytoplasm from the nucleus by the treatment with Ac-SDKP. These findings provide novel evidence that Ac-SDKP inhibits TGF-␤ signal transduction through the suppression of R-Smad activation via nuclear export of Smad7, highlighting an alternative mechanism involved in the reno-protective efficacy of ACE-I.

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Section: Discussionmentioning

confidence: 84%

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Inhibits TGF-β–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells

Kanasaki

Koya

Sugimoto

et al. 2003

Journal of the American Society of Nephrology

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“…At 24 h after transfection, cell lysates were prepared, and luciferase activity was measured by the Dual-Luciferase Reporter System (Promega) as described previously [7]. Values were normalized to Renilla luciferase activity.…”

Section: Luciferase Assaymentioning

confidence: 99%

“…Among the R-Smads, Smad2 and Smad3 act in TGF-β, activin and nodal pathways, whereas Smad1, Smad5 and Smad8 act in BMP and anti-Müllerian hormone pathways. I-Smads (inhibitory Smads), including Smad6 and Smad7, bind to type I receptors and prevent phosphorylation of R-Smads, resulting in the inhibition of TGF-β superfamily signalling [6,7].…”

Section: Introductionmentioning

confidence: 99%

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

Kuratomi

Komuro

Goto

et al. 2005

Biochemical Journal

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197

143

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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.

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“…We have demonstrated that the identified NLS mediates not only LMB-induced nuclear import of intact Smad4 as well as its isolated MH1 domain, but also the nuclear Since the isolated MH1 domains of R-Smads (such as Smad1 and 3) and I-Smads (such as Smad7) also mostly accumulate inside the nucleus (Xiao et al, 2000a;Hanyu et al, 2001;Kurisaki et al, 2001), we were interested to know whether the Smad4 NLS region is conserved among the R-and I-Smads. We aligned the NLS regions of representative R-Smads (Smad1, 2 and 3), Co-Smads (Smad4) and I-Smads (Smad7) in Figure 8a.…”

Section: Smad4 Nls Resembles An Extended Bipartite Nlsmentioning

confidence: 99%

An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity

2003

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Smad proteins are a class of tumor suppressors that play critical roles in inhibiting the proliferation of a variety of cell types by modulating the transcriptions of target genes. Despite recent advances, the mechanism of their nuclear import is not completely understood. Smad proteins contain a conserved basic motif in their N-terminal MH1 domains that resembles a nuclear localization signal (NLS). Previous studies indicate that in receptor-regulated Smads such as Smad1 and Smad3 this motif determines their interactions with nuclear import receptors and mediates their ligand-induced nuclear translocation. Common-Smads such as Smad4 display constant nucleocytoplasmic shuttling and are capable of autonomous nuclear import and export. Mutations of the basic motif in Smad4 disrupted its nuclear accumulation. However, this motif is not sufficient to confer nuclear translocation to a fused heterologous protein, suggesting that it is only part of the bona fide Smad4 NLS. We mapped the Smad4 NLS by fusing various segments of Smad4 sequence covering the basic motif to GFP and tested the localization of the fusion proteins. We identified an extended NLS, starting from the basic motif and extending into the DNA-binding region , that is sufficient to confer nuclear localization to GFP. Among the 14 basic residues in the NLS, only four (K45, K46, K48 and R81) are critical for import. This NLS is critical not only for autonomous nuclear import of Smad4, but also for its nuclear translocation in the presence of activated R-Smads, further confirming the functional relevance of the Smad4 NLS in TGF-b signal transduction. Structural modeling demonstrated that the four critical basic residues are all solvent exposed and map to a single localized segment on one surface of the Smad4 MH1 domain. Their distribution and spacing resemble a classical bipartite NLS. Smad4 displays specific binding to importin a through its MH1 domain, which was abrogated by loss-of-function mutations in Smad4 NLS. Finally, the Smad4 NLS is essential for its transcriptional activity since loss-of-function NLS mutants are also transcriptionally inactive.

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The N domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling

Cited by 210 publications

References 37 publications

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Inhibits TGF-β–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Inhibits TGF-β–Mediated Plasminogen Activator Inhibitor-1 Expression via Inhibition of Smad Pathway in Human Mesangial Cells

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

An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity

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