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...
c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor- (TGF-) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF--induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-/activin signaling. INTRODUCTIONCytokines of the transforming growth factor- (TGF-) superfamily are multifunctional proteins that regulate growth, differentiation, apoptosis, and morphogenesis of a wide variety of cell types. TGF- and related factors bind to two different types of serine/threonine kinase receptors, termed type I and type II (Derynck et al., 1998;Attisano and Wrana, 2000;Shi and Massagué, 2003). Type I receptor is activated by type II receptor upon ligand binding and transduces signals into cytoplasm through phosphorylation of receptorregulated Smads (R-Smads). Phosphorylated R-Smads interact with Co-Smad (Smad4) and translocate into the nucleus. Nuclear Smad complexes regulate transcription of target genes in cooperation with transcriptional activators/repressors as well as with coactivators/corepressors . Of the eight different mammalian Smad proteins, Smad1, Smad5, and Smad8 are R-Smads activated by BMP family members, whereas Smad2 and Smad3 are R-Smads activated by TGF- and activin.Transcription of target genes by TGF- is upregulated by binding of Smads to transcriptional coactivators, including p300 and CBP, which induce acetylation of histones and loosen chromatin structure (Massagué and Chen, 2000;Miyazono et al., 2001). In contrast, transcriptional corepressors, including TGIF, c-Ski, and SnoN, physically interact with Smads, and repress TGF- signaling through recruitment of histone deacetylases (HDACs) (Massagué and Chen, 2000;Liu et al., 2001).c-Ski was originally identified as a cellular counterpart of a retroviral oncogene product, v-Ski (Li et al., 1986). c-Ski physically interacts with Smad2, Smad3, and Smad4, thus antagonizing signal transduction in the TGF- pathway (Akiyoshi et al., 1999;Luo et al., 1999;Sun et al., 1999;Xu et al., 2000). Overexpression of c-Ski abolished TGF--induced growth inhibition (Luo et al., 1999;Sun et al., 1999;Xu et al., 200...
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