Crystal Structure of a Phosphorylated Smad2

Wu, Jiawei; Hu, Min; Chai, Jijie; Seoane, Joan; Huse, Morgan; Li, Carey; Rigotti, Daniel J.; Kyin, Saw; Muir, Tom W.; Fairman, Robert; Massagué, Joan; Shi, Yigong

doi:10.1016/s1097-2765(01)00421-x

Cited by 271 publications

(148 citation statements)

References 47 publications

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“…Mutations of either residue to histidine are found in tumor cells at higher frequency than other missense mutations and completely abolish the interaction with phosphorylated Smad2 and Smad1 (ref. 27 and data not shown). Although these Smad4 mutants lost TGF-␤-dependent function, we tested whether they retained the ability to activate c-myc promoter activity.…”

Section: Smad4 Is Able To Activate the C-myc Promoter In The Absencementioning

confidence: 76%

Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c- myc expression in the absence of TGF-β signaling

Lim

Hoffmann

2006

Proc. Natl. Acad. Sci. U.S.A.

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Section: Smad4 Is Able To Activate the C-myc Promoter In The Absencementioning

confidence: 76%

Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c- myc expression in the absence of TGF-β signaling

Lim

Hoffmann

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Like Smad2, Smad3 loses affinity for SARA as a result of TGF␤-induced phosphorylation of its C terminus (15,23). The crystal structure of phosphorylated Smad2 shows a conformational change in the disposition of the ␤Ј-strand that affects a critical SARA contact site and may explain the observed loss of affinity for SARA (34). This change most likely occurs also in Smad3, as the regions in question are highly conserved (23).…”

Section: Discussionmentioning

confidence: 96%

“…This suggests that Smad4 import machinery is constitutively active and is able to restrict Smad4 in the nucleus, as long as Smad4 can be disengaged from CRM-1 once in the nucleus. We can speculate that TGF␤-promoted association of Smad4 with phosphorylated Smad2/3, other transcription cofactors and DNA all have the potential to interfere with Smad4 interaction with CRM-1, resulting in accumulation of Smad4 in the nucleus (10,34).…”

Section: Discussionmentioning

confidence: 99%

Distinct Domain Utilization by Smad3 and Smad4 for Nucleoporin Interaction and Nuclear Import

Xu¹,

Alarcón

Çöl

et al. 2003

Journal of Biological Chemistry

113

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Smad proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-␤ (TGF␤) 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.Signal from the transforming growth factor-␤ (TGF␤) 1 cytokines originates at the cell surface upon engagement of TGF␤ ligands with their corresponding Type I/Type II receptor complexes and is transduced into the nucleus by the family of Smad proteins (1). Smad proteins are phosphorylated by the corresponding receptor kinases activated upon ligand binding: Smad2 and Smad 3 by TGF␤ and activin receptors; Smads 1, 5, and 8 by bone morphogenetic protein (BMP) receptors. One key event accompanying ligand binding is the rapid movement of Smads, including the unphosphorylated Smad4, into the nucleus. As transcription factors, these nuclear-bound Smads activate or repress gene expression leading to diverse cellular responses to TGF␤ cytokines (2, 3).Translocation of macromolecules across the nuclear envelope occurs via the nuclear pore complex (NPC), which consists of over 20 nucleoporins and many of them contain multiple phenylalanine-glycine (FG) dipeptide repeats (4 -6). In many cases, movement of cargo molecules through the NPC is mediated by the importin ␤ family of transport receptors, which bind cargo molecules and the FG dipeptide repeats simultaneously through separate domains (7). In the case of classical nuclear localization signal (NLS)-containing proteins, importin ␣ bridges the interaction between the NLS motif and importin ␤ (4 -7). Another common element in importin ␤-mediated nuclear import is the small GTPase Ran in its GTP-bound form (RanGTP), whose binding to importin ␤ disrupts all characterized association between importin ␤ and their cargoes (4 -7). Ran is mostly in its GTP-bound form in the nucleus and a GDP-bound form in the cytoplasm. Such Ran-GTP gradient across the nuclear envelope prompts release of cargo only in the nucleoplasm, ensuring that the cargoes will accumulate in the nucleus (4 -7).Since elucidation of the mechanism of importin ␤-mediated nuclear import, there have been efforts to investigate if similar principles apply to Smads. In Smad3, the N-terminal or MH1 domain cont...

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“…The ''AAVEL'' sequence of Smad3 and its corresponding part of Smad1 (STIEN) are different from each other in its hydrophobic properties. To identify locations of these two parts in the three-dimensional structure of monomer and trimer forms of Smads, we explored the crystal structure of the Smad1 MH2 domain (15) and homology-modeled Smad3 structure based on the crystal structure of the Smad2 MH2 domain (16). Interestingly, SE and AAVEL are distantly located, facing the N-terminal upper and bottom sides of the toroidal structure of MH2 domain trimer, respectively (Fig.…”

Section: Se Is Responsible For Differential Binding Of Smad1 and Smadmentioning

confidence: 99%

Two Short Segments of Smad3 Are Important for Specific Interaction of Smad3 with c-Ski and SnoN

Mizuide

Hara²,

Furuya³

et al. 2003

Journal of Biological Chemistry

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Cytokines of the transforming growth factor-␤ (TGF-␤)1 superfamily bind to two different serine/threonine kinase receptors, type I and type II, and transmit intracellular signals through Smad proteins (1). Receptor-regulated Smads (RSmads) are directly phosphorylated by type I receptors, and form complexes with common-partner Smad (Co-Smad). The R-Smad⅐Co-Smad complexes translocate into the nucleus, where they regulate transcription of target genes through interacting with various transcription factors and transcriptional co-activators or co-repressors. Of the eight different mammalian Smad proteins, Smad1, Smad5, and Smad8 are R-Smads activated by bone morphogenetic proteins (BMPs) and anti-Mü llerian hormone, whereas Smad2 and Smad3 are R-Smads activated by TGF-␤s, activins, and nodal. Smad4 is the only Co-Smad in mammals.Transcription of target genes by TGF-␤ is up-regulated by binding of Smads to transcriptional co-activators, including p300 and CBP, which induce acetylation of histones (2-4). In contrast, transcriptional co-repressors, including c-Ski and its related protein SnoN, physically interact with Smads and repress TGF-␤ superfamily signaling through recruitment of histone deacetylases (5-9). Ski was originally identified as the oncogene present in the avian Sloan-Kettering retroviruses (10). Although c-Ski regulates transcriptional activities of some other proteins (11,12), Smads may be one of the most important partners of c-Ski in transcriptional regulation.R-Smads and Co-Smads are composed of the N-terminal MH1 domains, linker regions, and the C-terminal MH2 domains. Among the various regions of Smads, the MH2 domains play important roles in binding to type I receptors, formation of Smad oligomers, and interaction with DNA-binding proteins (13). Upon ligand stimulation, R-Smads and Co-Smads may form heterotrimers or heterodimers through their MH2 domains, although their exact oligomeric structures have not been fully determined (14 -16). Smads interact with various transcription factors and transcriptional co-activators/co-repressors through the MH2 domains. Among them, Mixer and FoxH3 (originally termed FAST1) have been shown to bind to ␣-helix 2 (H2) of the MH2 domains of Smad2/3 (17-19). However, sites of binding to other proteins in Smads have not been determined in detail. Interestingly, SnoN was shown to be degraded by a HECT type E3 ubiquitin ligase Smurf2, which binds to the linker region of Smad2 (20). SnoN does not directly bind to Smurf2, but it may be located in the vicinity of Smurf2 when it binds to Smad2. It is thus important to determine through which regions c-Ski/SnoN bind to Smad2 and Smad3.Among mammalian Smad proteins, Smad2, Smad3, and Smad4 interact with c-Ski, but Smad1 or Smad5 does so only weakly (6). In the present study, we have shown that the SE and QPSMT parts in the MH2 domain of Smad3 interact with c-Ski and SnoN. SE and QPSMT are located on the N-terminal upper side of the toroidal structure of the MH2 oligomer, toward which the MH1 domain and the linker region f...

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Crystal Structure of a Phosphorylated Smad2

Cited by 271 publications

References 47 publications

Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c- myc expression in the absence of TGF-β signaling

Smad4 cooperates with lymphoid enhancer-binding factor 1/T cell-specific factor to increase c- myc expression in the absence of TGF-β signaling

Distinct Domain Utilization by Smad3 and Smad4 for Nucleoporin Interaction and Nuclear Import

Two Short Segments of Smad3 Are Important for Specific Interaction of Smad3 with c-Ski and SnoN

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