Human transforming growth factor-beta activates a receptor serine/threonine kinase from the intravascular parasite Schistosoma mansoni.

The biology of the helminth parasite Schistosoma mansoni is closely integrated with that of its mammalian host. SmRK1, a divergent type I transforming growth factor-beta (TGF-beta) receptor of unknown ligand specificity, was previously identified as a candidate for a receptor that allows schistosomes to respond to host-derived growth factors. The TGF-beta family includes activin, bone morphogenetic proteins (BMPs), and TGF-beta, all of which can play crucial roles in metazoan development. The downstream signaling protein of receptors that respond to TGF-beta and activin is Smad2, whereas the receptors that respond to BMPs signal via Smad1. When a constitutively active mutant of SmRK1 was overexpressed with either schistosome Smad1 (SmSmad1) or SmSmad2, a receptor-dependent modulation of SmSmad phosphorylation and luciferase reporter activity occurred only with SmSmad2. To evaluate potential ligand activators of SmRK1, a chimeric receptor containing the extracellular domain of SmRK1 joined to the intracellular domain of the human type I TGF-beta receptor was used. The chimeric receptor bound radiolabeled TGF-beta and could activate a luciferase reporter gene in response to both TGF-beta 1 and TGF-beta 3 but not BMP7. Confirmatory results were obtained using full-length SmRK1. These experiments implicate TGF-beta as a ligand for SmRK1 and as a potential host-derived regulator of parasite growth and development.     

Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation

Members of the transforming growth factor (TGF)-beta superfamily play pivotal roles in cell migration, differentiation, adhesion, pattern formation, and apoptosis. The family of Smad proteins acts as intracellular signal transducers of TGF-beta and related peptides. Smad4, a common mediator Smad (co-Smad), performs a central role in transmitting signals from TGF-beta, BMP, and activins. Schistosoma mansoni receptor-regulated Smad1 and SmSmad2 were previously identified and shown to act in TGF-beta signaling. Herein, we report the identification and characterization of a Smad4 homologue from S. mansoni and provide details about its role in mediation and down-regulation of TGF-beta signaling in schistosomes. In order to identify the schistosome co-Smad, we designed degenerate primers based on the sequence of the conserved MH1/MH2 domains of Smad4 proteins, which were used in PCR to amplify a 137-bp PCR product. A S. mansoni adult worm pair cDNA library was screened resulting in the isolation of a cDNA clone that encodes a 738 amino acid protein (SmSmad4). SmSmad4 was shown to interact with schistosome R-Smads (SmSmad1 and SmSmad2) in vivo and in vitro. The interaction with SmSmad2 was dependent on the receptor-mediated phosphorylation of SmSmad2. In addition, several potential phosphorylation sites for Erk1/2 kinases were identified in the SmSmad4 linker region and shown to be phosphorylated in vitro by an active mutant of mammalian Erk2. Furthermore, Erk-mediated phosphorylation of SmSmad4 decreased its interaction with the receptor-activated form of SmSmad2, in vitro. SmSmad4 was shown to complement a human Smad4 deficiency through the restoration of TGF-beta-responsiveness in MDA-MB-468 breast cancer cells.     

Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene

Members of transforming growth factor-beta (TGF-beta) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTbetaRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTbetaRII with SmTbetaRI was elucidated and shown to be dependent on TGF-beta ligand binding. Furthermore, in the presence of human TGF-beta1, SmTbetaRII was able to activate SmTbetaRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-beta target gene in schistosomes. Knocking down the expression of SmTbetaRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTbetaRII in mediating TGF-beta-induced activation of the TGF-beta target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-beta signaling pathway in male-induced female reproductive development.     

Semisynthesis of phosphovariants of Smad2 reveals a substrate preference of the activated T beta RI kinase

Transforming growth factor-beta (TGF-beta) signaling regulates a wide range of cellular processes. Aberrant TGF-beta signaling has been implicated in various disease states in humans. A key element in this signaling pathway is phosphorylation of R-Smads such as Smad2 at the last two serine residues of the C-terminal sequence CSSXS (residues 463-467 in Smad2) by the TbetaRI receptor kinase. Phosphorylation results in the release of the R-Smad from the membrane-anchored protein SARA, binding to the co-mediator protein Smad4, translocation into the nucleus, and regulation of target gene expression. Expressed protein ligation was used to probe the contribution of the individual phosphate groups to Smad2 oligomerization and phosphorylation by TbetaRI. Phosphorylation at both positions was required to generate a stable homotrimer; however, the driving force for Smad2 self-association is provided by pSer465. Additionally, SARA was found to modulate the self-association of partially phosphorylated Smad2, which suggests an added role for this protein in preventing premature release of a monophosphorylated substrate from the receptor complex. In related studies, prephosphorylation of Smad2 at Ser465 was found to significantly increase the rate of phosphorylation at Ser467, suggesting that there may be specific recognition determinants within the kinase for the monophosphorylated intermediate. This information was exploited to design an improved peptide substrate for TbetaRI, which may prove valuable in the design of inhibitors of the TGF-beta pathway.     

Divergence and convergence of TGF-beta/BMP signaling

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.     

Novel dominant negative Smad antagonists to TGFbeta signaling

We previously identified a critical serine/threonine residue within the linker domain of Smad2/3, phosphorylated by the kinase GRK2 which plays a critical role in regulating Smad signaling. To define the mechanism by which GRK2-mediated phosphorylation modifies Smad2/3 behavior at the molecular level, we generated mutant Smads where the GRK2 phosphorylation site was replaced with an aspartic acid (D) to mimic the properties of a phospho-residue or an alanine (A) as a control. Interestingly, overexpression of either the D or A mutant inhibits TGFbeta signaling, but through two distinct mechanisms. The D mutant is properly localized and released from the plasma membrane upon ligand stimulation, but fails to interact with the type I receptor kinase. The A mutant properly interacts with and is phosphorylated by the type I receptor, translocates to the nucleus and homodimerizes with wild-type R-Smads, but it fails to form a heterocomplex with Smad4. As a result, both mutants act as antagonists of endogenous TGFbeta signaling through divergent mechanisms. The D mutant acts by blocking endogenous R-Smads phosphorylation and the A mutant acts by preventing endogenous R-Smad/Smad4 heterocomplexes. Thus, mutation of the GRK2 phosphorylation site within the Smad generates dominant negative Smads that efficiently inhibit TGFbeta responses.     

Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.     

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.