Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling

The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.     

Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser/Thr kinase-mediated TGF-beta 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.     

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.     

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.     

Degradation of the tumor suppressor Smad4 by WW and HECT domain ubiquitin ligases

Smad4 mediates signaling by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-beta receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-beta signaling may be critical for proper physiological response to this pathway.     

SnoN co-repressor binds and represses smad7 gene promoter

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.     

Repression of endogenous Smad7 by Ski

The Ski protein has been proposed to serve as a corepressor for Smad4 to maintain a transforming growth factor-beta (TGF-beta)-responsive promoter at a repressed, basal level. However, there have been no reports so far that it indeed acts on a natural promoter. We have previously cloned the human Smad7 promoter and shown that it contains the 8-base pair palindromic Smad-binding element (SBE) necessary for TGF-beta induction. In this report, we have characterized the negative regulation of Smad7 promoter basal activity by Ski. We show that Ski inhibits the Smad7 promoter basal activity in a SBE-dependent manner. Mutation of the SBE abrogates the inhibitory effect of Ski on the Smad7 promoter. Moreover, mutation of the SBE increases the Smad7 promoter basal activity. Using the chromatin immunoprecipitation assay, we further show that Ski together with Smad4 binds to the endogenous Smad7 promoter. Finally, we show that RNAi knockdown of Ski increases Smad7 reporter gene activity in transient transfection assays as well as elevating the endogenous level of Smad7 mRNA. Taken together, our results provide the first evidence that Ski is indeed a corepressor for Smad4, which can inhibit a natural TGF-beta responsive gene at the basal state.     

Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers

Smad proteins form multimeric complexes consisting of the 'common partner' Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-β and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein-protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.