Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-β (TGF-β)/Smad Signaling

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.     

Structural basis of heteromeric smad protein assembly in TGF-beta signaling

The formation of protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-beta signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 angstroms, and Smad2/Smad4 to 2.7 angstroms. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad protein assembly in TGF-beta superfamily signaling.     

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.     

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.     

Structural basis for the Smad5 MH1 domain to recognize different DNA sequences

Smad proteins are important intracellular mediators of TGF-β signalling, which transmit signals directly from cell surface receptors to the nucleus. The MH1 domain of Smad plays a key role in DNA recognition. Two types of DNA sequence were identified as Smad binding motifs: the Smad binding element (SBE) and the GC-rich sequence. Here we report the first crystal structure of the Smad5 MH1 domain in complex with the GC-rich sequence. Compared with the Smad5-MH1/SBE complex structure, the Smad5 MH1 domain contacts the GC-rich site with the same β-hairpin, but the detailed interaction modes are different. Conserved β-hairpin residues make base specific contacts with the minimal GC-rich site, 5′-GGC-3′. The assembly of Smad5-MH1 on the GC-rich DNA also results in distinct DNA conformational changes. Moreover, the crystal structure of Smad5-MH1 in complex with a composite DNA sequence demonstrates that the MH1 domain is targeted to each binding site (GC-rich or SBE) with modular binding modes, and the length of the DNA spacer affects the MH1 assembly. In conclusion, our work provides the structural basis for the recognition and binding specificity of the Smad MH1 domain with the DNA targets.     

Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors

TAK1 (transforming growth factor-beta-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-beta/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1/2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development.     

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.     

Identification and expression of amphioxus <Emphasis Type="Italic">AmphiSmad1/5/8</Emphasis> and <Emphasis Type="Italic">AmphiSmad4</Emphasis>

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.