Conservation and evolutionary divergence in the activity of receptor-regulated smads

Abstract

BackgroundActivity of the Transforming growth factor-β (TGFβ) pathway is essential to the establishment of body axes and tissue differentiation in bilaterians. Orthologs for core pathway members have been found in all metazoans, but uncertain homology of the body axes and tissues patterned by these signals raises questions about the activities of these molecules across the metazoan tree. We focus on the principal canonical transduction proteins (R-Smads) of the TGFβ pathway, which instruct both axial patterning and tissue differentiation in the developing embryo. We compare the activity of R-Smads from a cnidarian (Nematostella vectensis), an arthropod (Drosophila melanogaster), and a vertebrate (Xenopus laevis) in Xenopus embryonic assays.ResultsOverexpressing NvSmad1/5 ventralized Xenopus embryos when expressed in dorsal blastomeres, similar to the effects of Xenopus Smad1. However, NvSmad1/5 was less potent than XSmad1 in its ability to activate downstream target genes in Xenopus animal cap assays. NvSmad2/3 strongly induced general mesendodermal marker genes, but weakly induced ones involved in specifying the Spemann organizer. NvSmad2/3 was unable to induce a secondary trunk axis in Xenopus embryos, whereas the orthologs from Xenopus (XSmad2 and XSmad3) and Drosophila (dSmad2) were capable of doing so. Replacement of the NvSmad2/3 MH2 domain with the Xenopus XSmad2 MH2 slightly increased its inductive capability, but did not confer an ability to generate a secondary body axis.ConclusionsVertebrate and cnidarian Smad1/5 have similar axial patterning and induction activities, although NvSmad1/5 is less efficient than the vertebrate gene. We conclude that the activities of Smad1/5 orthologs have been largely conserved across Metazoa. NvSmad2/3 efficiently activates general mesendoderm markers, but is unable to induce vertebrate organizer-specific genes or to produce a secondary body axis in Xenopus. Orthologs dSmad2 and XSmad3 generate a secondary body axis, but activate only low expression of organizer-specific genes that are strongly induced by XSmad2. We suggest that in the vertebrate lineage, Smad2 has evolved a specialized role in the induction of the embryonic organizer. Given the high level of sequence identity between Smad orthologs, this work underscores the functional importance of the emergence and fixation of a few divergent amino acids among orthologs during evolution.

Phylogenetic evidence for the modular evolution of metazoan signalling pathways

“…The BMP arm of the TGF-b signalling is asymmetrically expressed along the directive axis (orthogonal to the anterior/posterior axis) of the sea anemone Nematostella vectensis [38,53,54] and has been implicated in defining the bilateral axis of symmetry in cnidarians [39,43]. Furthermore, ectopic expression of NvSMAD (a SMAD homologue isolated from Nematostella vectensis) in Xenopus during development has demonstrated that the Nv homologue of this gene is sufficient to recapitulate the ventralized phenotype generated by vertebrate homologues in similar studies [40]. Similarly, using in situ hybridization, Adamska et al [30] identified asymmetrical expression of TGF-b along the larval swimming axis in sponge embryos and Pang et al [31] revealed that TGF-b …”

Conformational landscape of multidomain SMAD proteins

“…SMAD linkers are inter-domain sequences of approximately 100–120 residues, characteristic of each type of SMAD protein [5] , [31] . There is no structural information available, except for short fragments of SMAD1, SMAD2 and SMAD7, which were studied as complexes bound to transcription activators and ubiquitin ligases [14] .…”

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation

“…This rapid change indicates that the effects are at least partly mediated by the non‐genomic function of RARγ rather than through genomic action that would require changes in gene expression and production of new proteins, both likely requiring more than 1 h. In vitro studies showed that RARγ physically interacts with the MH2 domain of Smad3 and down‐regulates Smad3/4‐dependent transcription in the presence of RARγ agonists, while Smad3/4‐dependent transcription activity was markedly increased by RAR pan‐antagonist treatment . The MH2 domain is used for protein–protein interactions, including hetero‐trimerization of Smads, and is highly conserved especially among the R‐Smad proteins . Thus, RARγ may directly interact with Smad1/5/8 proteins and modulate their function.…”

“…29 The MH2 domain is used for protein-protein interactions, including heterotrimerization of Smads, and is highly conserved especially among the R-Smad proteins. 30 Thus, RARg may directly interact with Smad1/5/8 proteins and modulate their function. On the other hand, RARg is also known to interact with Src, a non-receptor tyrosine kinase, and ligand-bound RARg enhances Src kinase activity.…”