Domain structure of Medea homologs

An assay based on the restoration of ligand-dependent transcriptional responses in a Smad4 null cell line was used to characterize functional domain structures within Smad4. Restoration of TGF-beta-induced transcriptional responses by Smad4 is inhibited by co-transfection with a kinase dead (mutationally inactivated) TGF-beta type II receptor; constitutive activation is blocked with TGF-beta neutralizing antibodies, confirming the essential role of Smad4 in TGF-beta signaling. A 47-amino acid deletion within the middle-linker region of Smad4 has been identified that is essential for the mediation of signaling responses. The NH2-terminal domain of Smad4 augments ligand-dependent activation associated with the middle-linker region, indicating that there is a distinct ligand-response domain within the N terminus of this molecule (de Caestecker, 1997).

Interaction of Medea related proteins with other Smads and with DNA

The Smad proteins function downstream of TGF-ß receptor serine/threonine kinases and undergo serine phosphorylation in response to receptor activation. Smad1 is regulated in this fashion by BMP receptors, and Smad2 and Smad3 by TGF-ß and activin receptors. BMP receptors phosphorylate and activate Smad1 directly. Phosphorylation of Smad1 in vivo involves serines in the carboxy-terminal motif SSXS. These residues are phosphorylated directly by a BMP type I receptor in vitro. Mutation of these carboxy-terminal serines prevents several Smad1 activation events: Smad1 association with the related protein DPC4, accumulation in the nucleus, and gain of transcriptional activity. Similar carboxy-terminal serines in Smad2 are required for its phosphorylation and association with DPC4 in response to TGF-ß, indicating the general nature of the Smad activation process. As a direct physiological substrate of BMP receptors, Smad1 provides a link between receptor serine/threonine kinases and the nucleus (Kretzschmar, 1997).

TGF-beta mediates phosphorylation of Smad2 at two serine residues in the C terminus (Ser465 and Ser467), which are then phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 first be phosphorylated. Transfection of Smad2 into Mv1Lu cells with the mutation of Ser465 and/or Ser467 into alanine residues results in dominant-negative inhibition of TGF-beta signaling. These Smad2 mutants stably interact with an activated TGF-beta receptor complex, in contrast to wild-type Smad2, which interacts only transiently. The mutation of Ser465 and Ser467 within Smad2 abrogates complex formation of this mutant with Smad4 and blocks the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides are less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation (Souchelnytskyi, 1997).

Smad2 interacts transiently with and is a direct substrate for the transforming growth factor-beta (TGF-beta) type I receptor, TbetaRI. Phosphorylation sites on Smad2 were localized to a carboxyl-terminal fragment containing three serine residues at positions 464, 465, and 467. TbetaRI specifically phosphorylates Smad2 on serines 465 and 467. Serine 464 is not a site of phosphorylation, but is important for efficient phosphorylation of Smad2. Phosphorylation at both sites is required to mediate association of Smad2 with Smad4 in mammalian cells, while in yeast, Smad2 interacts directly with Smad4 and does not require phosphorylation. Mutation of either serine residue 465 or 467 prevents the dissociation of Smad2 from activated TbetaRI and blocks TGF-beta-dependent signaling and Smad2 transcriptional activity. These results indicate that receptor-dependent phosphorylation of Smad2 on serines 465 and 467 is required in mammalian cells to permit association with Smad4 and to propagate TGF-beta signals (Abdollah, 1997).

Signal transduction by the TGF-beta family involves sets of receptor serine/threonine kinases, Smad proteins that act as receptor substrates, and Smad-associated transcription factors that target specific genes. Discrete structural elements have been identified that dictate the selective interactions between receptors and Smads and between Smads and transcription factors in the TGF-beta and BMP pathways. Of particular interest is a nine-amino-acid segment in the receptor kinase domain, known as the L45 loop. Replacement of all but the L45 loop in the kinase domain of TbetaR-I with the corresponding regions from ALK2 yields a construct that still mediates TGF-beta responses. As predicted from the conserved structure of protein kinases, the L45 loop links beta-strands 4 and 5, and is not part of the catalytic center. The L45 loop differs between type I receptors of different signaling specificity, such as the TGF-beta receptors and the BMP receptors, but is highly conserved between receptors of similar signaling specificity such as TbetaR-I and the activin receptor ActR-IB, or the BMP receptors from human (BMPR-IA and BMPR-IB) and Drosophila (Thick veins). A cluster of four residues in the L45 loop of the type I receptor kinase domain, and a matching set of two residues in the L3 loop of the Smad carboxy-terminal domain establish the specificity of receptor-Smad interactions. The L3 loop of Smads has drawn attention as a target of inactivating mutations in Drosophila and Caenorhabditis elegans Smad family members. As inferred from the effect of similar mutations in vertebrate Smads, the L3 loop participates in different interactions that are essential for signaling. In Smad4 the L3 loop is required for interaction with activated receptor regulated Smads (R-Smads), whereas in R-Smads the L3 loop is required for interaction with the receptors and, furthermore, it specifies these interactions. The present results show that matching combinations of L45 loops and L3 loops determine the specificity of the receptor-Smad interaction. Exchanging the subtype-specific residues in either the L45 loop or the L3 loop causes a switch in the specificity of this interaction, with an attendant switch in the signaling specificity of the pathway. As evidence of a functional match between a receptor L45 loop and an R-Smad L3 loop, the switch in the signaling specificity of a TGF-beta receptor construct containing the BMP receptor L45 loop can be reversed by a Smad2 construct containing the matching L3 loop sequence from Smad1. A cluster of residues in the highly exposed alpha-helix 2 of the Smad carboxy-terminal domain specifies the interaction with the DNA-binding factor Fast1 and, as a result, the gene responses mediated by the pathway. By establishing specific interactions, these determinants keep the TGF-beta and BMP pathways segregated from each other (Chen, 1998).

Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor beta superfamily. A member of the Smad family, Smad8, has been isolated from a rat brain cDNA library; its ability to transduce signals from serine kinase receptors has been characterized. In the Xenopus embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not ALK-4, the related receptor serine kinase. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4. This association leads to a synergistic effect on gene transcription. Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a downstream signaling mediator of ALK-2 (Chen, 1997).

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. 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; 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 suggests that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrate that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevents Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4; these trimers 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 has 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 (Kawabata, 1998).

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps, although the mechanism by which this modulation occurs remains unknown. Notch signaling also delays the onset of LMC in whole embryos, as it does in animal caps. To better understand this effect and the mechanism of LMC itself, an investiation was carried out to discover at which step of activin signal transduction pathway the Notch signaling acts to affect the timing of the LMC. In this system, ALK4 (activin type I receptor) maintains the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 can bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. It is concluded that LMC is induced because C-terminal-phosphorylated smad2 loses its ability to bind to smad4, and consequently can not accumulate in the nucleus. Notch signal activation restores the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC (Abe, 2005).

Transcriptional regulation by TGFbeta signaling is mediated by the Smad family of transcription factors. It is generally accepted that Smads must interact with other transcription factors in order to bind to their targets. However, recently it has been shown that a complex of the Drosophila Smad proteins, Mad and Medea, binds with high affinity to silencer elements that repress brinker and bag of marbles in response to Dpp signaling. These silencers are bound by a heterotrimer containing two Mad subunits and one Medea subunit. The MH1 domains of all three subunits contribute directly to sequence-specific DNA contact, thus accounting for the exceptionally high stability of the Smad-silencer complex. The Medea MH1 domain binds to a canonical Smad box (GTCT), while the Mad MH1 domains bind to a GC-rich sequence resembling Mad binding sites previously identified in Dpp-responsive enhancer elements. The consensus for this sequence, GRCGNC, differs from that of the canonical Smad box, but it was found that Mad binding nonetheless required the same beta-hairpin amino acids that mediate base-specific contact with GTCT. Binding is also affected by alanine substitutions in Mad and Med at a subset of basic residues within and flanking helix 2, indicating a contribution to binding of the GRCGNC and GTCT sites. Slight alteration of the Dpp silencers causes them to activate transcription in response to Dpp signaling, indicating that the potential for Smad complexes to recognize specific targets need not be limited to repression (Gao, 2005).

This work demonstrates that Mad binds to GCrich sites -- originally defined as GCCGNCG and more recently in the context of silencers as GRCGNC -- by sequence-specific contact with two subunits. Sequence specific interaction with GC-rich sites has been demonstrated previously for Smad1 and for Mad, but the stoichiometry of these interactions was not documented. Finding that the 6 bp site is contacted by two Mad subunits raises new questions, since it is not clear how the two MH1 domains are arranged on the DNA, nor is it known why GNCG or GNCN (depending on orientation) is recognized rather than the GTCT site preferred by Smad3 and Smad4. Previous work involving Smad1- Smad3 chimeras identified Lys36 and Ser37 of Smad3 helix 2 vs. Asp35 and Ala36 in Smad1 as a key difference necessary for activation of a (CAGA)9-luc reporter. However, mutation of the corresponding residue in Mad (D49A) has little or no effect on binding to BrkS, and thus the structural determinants for the GRCGNC binding site preference of Mad (and by inference, Smad1) remain to be identified (Gao, 2005).

These findings contradict a previous report that Mad and Med bind to BrkS as a dimer. Although no documentation was provided for how this was determined experimentally, attempts to determine stoichiometry by means of antibody supershift experiments suggest a possible explanation for the discrepancy. It was found that epitope tags at the Mad amino terminus are only readily accessible to antibody on one of the two Mad subunits, possibly due to crowding by the adjacent DNA-bound MH1 domain of Med. This problem led to the use instead of protein fusions as a strategy for assessing stoichiometry (Gao, 2005).

Mad and Med appear to be the only factors that directly contact the bam and brk silencers. This conclusion is based on mutational analysis of the BrkS element which showed that mutations that disrupt silencing also disrupt binding of the Mad-Med complex. The only exceptions were mutation of GTCT to GTCG or to GGCG, and a 1-bp deletion between the Mad and Med sites, each of which allowed binding of the Mad- Med complex but disrupted recruitment of Shn (Gao, 2005).

Over-expression of activated Mad-Med complexes is sufficient to generate these gel-shift complexes and therefore it is unlikely that an unknown cofactor is required since it would presumably need to be expressed at high levels endogenously in both Drosophila and human cells. The demonstration that binding of the Mad-Med heterotrimer requires all three MH1 domains also weighs against cofactor involvement since it becomes difficult to explain how GRCGNC could be contacted by a cofactor in addition to two Mad MH1 domains (the Med MH1 contacting GTCT). Nonetheless, it is possible that a cofactor present in Drosophila and human extracts has gone undetected, although the evidence suggests it could not play a direct role in sequence-specific DNA contact (Gao, 2005).

The apparent ability of a Mad-Med complex to bind silencers without cofactors contrasts with the general reliance of Smads on DNA-binding cofactors for target specificity. brk could be considered a special case since it is negatively regulated by Dpp globally, while other Dpp targets require tissue-specific regulation. However, bam expression is specific to germline cells, and thus it is unexpected that DNA contact by Mad and Med would be sufficient for the regulatory specificity of the bam silencer. The existence of a similar Dpp-responsive silencer regulating gooseberry provides further evidence that these novel arrangements of Smad binding sites provide sufficient specificity for regulation in response to signaling. Nonetheless, tissue specificity might be augmented by cooperative interaction of DNA-bound corepressors with Smads bound to BrkS/BamSE-like sites (Gao, 2005).

The high affinity of Mad and Med for these silencers is explained by the trimeric stoichiometry and involvement of all MH1 domains in directly contacting DNA. The ability of a single Smad complex to engage all three MH1 domains in DNA contact has several implications. The most obvious is that Smad complexes may in some cases make a greater contribution to target recognition than was previously apparent. As in the case of brk, this provides a mechanism by which Dpp, BMP or even TGFbeta signaling might trigger a general response without the need for a tissue-specific cofactor. Such a response need not be limited to silencing since slight alterations in the silencers transform them into Dpp-responsive activating elements, possibly by allowing CBP to interact with the Mad-Med complex in the absence of Shn. Conserved sites exhibiting the BrkS/BamSE motif have been identified within BMP-response elements for the Id genes, which as a class are responsive to BMP signaling. Conversely, tissue-specificity might be conferred upon such tripartite Smad response elements by adjacent binding sites for other transcription factors (Gao, 2005).

A second implication is that Smad complexes may have greater flexibility in their ability to recognize binding sites than was previously apparent, particularly for moderate affinity sites to which only two MH1 domains make sequence-specific contact. The observed flexibility in spacing between the Mad and Med sites in BrkS and BamSE suggests that DNA binding by Med plus just one Mad MH1 domain might be able to occur in a variety of permutations. However, loss of binding when the Med site was reversed shows that the topology of Smad sites has strict limits. A moderate affinity site might also consist of just the GRCGNC Mad binding site without an adjacent Med binding site, as appears to be the case for many Dpp responsive enhancer elements. Such moderate affinity sites would activate transcription in response to Dpp signaling, but only when the necessary tissue-specific cofactor is present. Some Mad sites serve also as binding sites for the Brk protein, which opposes Dpp signaling by direct competition for Mad binding and by functioning as an active repressor. The third implication points to complexity in the response to BMP signaling. In vertebrates that possess three BMP responsive rSmads -- Smad1, Smad5 and Smad8 -- BMP signaling might trigger the formation of a variety of trimeric rSmad complexes with Smad4 (e.g., a Smad1-Smad5- Smad4 complex). There is the potential for six such combinations. If such mixed complexes do form, as has been shown for Smad2, Smad3 and Smad4 in activation of p15Ink4B, it will be important to determine whether differences exist among them in the range of binding sites that can be bound by means of two or three MH1 domains. The likelihood of differential cofactor interactions by Smad1, Smad5 and Smad8 adds an additional layer of complexity (Gao, 2005).

Smad4 interacts with YY1 which acts to repress Smad transcriptional activities

Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. YY1, a transcription factor that positively or negatively regulates transcription of many genes, has been identified as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies result in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways (Kurisaki, 2003).

A mutant-p53/Smad complex opposes p63 to empower TGFβ-induced metastasis

TGFβ ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. This study shows that TGFβ-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFβ acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, two candidate metastasis suppressor genes associated with metastasis risk were identified in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFβ-dependent inhibition of p63 function (Adorno, 2009)

C. elegans Medea related proteins

Three C. elegans genes (sma-2, sma-3, and sma-4) have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a family of proteins, the dwarfins, that includes the MAD gene product, a participant in the decapentaplegic pathway in Drosophila. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. Highly conserved dwarfins from vertebrates have been isolated, indicating that these components are not idiosyncratic to invertebrates. All the described null mutations in Mad, sma2 and sma3 are missense or nonsense mutations that fall within a highly conserved, short portion of the C-terminal domain (Savage, 1996)

Signals from TGF-beta superfamily receptors are transduced to the nucleus by Smad proteins, which transcriptionally activate target genes. In C. elegans, defects in a TGF-beta-related pathway cause a reversible developmental arrest and metabolic shift at the dauer larval stage. Null mutations in daf-3 (now termed Smad4) suppress mutations in genes encoding this TGF-beta signal, its receptors, and associated Smad signal transduction proteins. daf-3 encodes a Smad protein that is most closely related to mammalian DPC4, and is expressed throughout development in many of the tissues that are remodeled during dauer development. DAF-4, the type II TGF-beta receptor in this pathway, is also expressed in remodeled tissues. These data suggest that the DAF-7 signal from sensory neurons acts as a neuroendocrine signal throughout the body to directly regulate developmental and metabolic shifts in tissues that are remodeled during dauer formation. A full-length functional DAF-3/GFP fusion protein is predominantly cytoplasmic; this localization is independent of activity in the upstream TGF-beta-related pathway. However, this fusion protein is associated with chromosomes in mitotic cells, suggesting that DAF-3 binds DNA either directly or indirectly. DAF-3 transgenes also interfere with dauer formation, perhaps attributable to a dosage effect. A truncated DAF-3/GFP fusion protein that is predominantly nuclear interferes with dauer formation, implying a role for DAF-3 in the nucleus. These data suggest that DAF-7 signal transduction antagonizes or modifies DAF-3 Smad activity in the nucleus to induce reproductive development; when DAF-7 signals are disabled, unmodified DAF-3 Smad activity mediates dauer arrest and its associated metabolic shift. Therefore, daf-3 is unique in that it is antagonized, rather than activated, by a TGF-beta pathway (Patterson, 1997).

Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the phyrangeal muscle specific myosin gene, myo-2, target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, a yeast one-hybrid screen was performed for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. DAF-3 was shown to bind subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interferes with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, expression of a gfp reporter controlled by a concatenated C subelement oligonucleotide was examined in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. Expression of this gfp reporter was examined in dauer larvae and no daf-3-dependent repression of C activity was observed. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development. How does DAF-3 repress enhancer activity? Three possibilities are suggested: DAF-3 may compete with a transcritional activator binding to the enhancer; DAF-3 may inhibit an activator bound to another site in the enhancer, or DAF-3 may directly interact with the transcriptional machinery, preventing it from responding to a bound activator. What ever the mechanism, TGFbeta signaling could antagonize the DAF-3 repressor by phosphorylating a factor that competes or interacts with DAF-3, or by phosphorylating an unidentified DAF-3 site (Thatcher, 1999).

A TGFbeta-like signal is required for spicule development in Caenorhabditis elegans males. This signal appears to originate in the male-specific musculature and is required for the migrations of cells within the proctodeum. The migrations of these cells form cellular molds, the spicule traces, in which the cuticle of the spicules is secreted, thus determining spicule morphology. Mutations in daf-4, sma-2, sma-3, and sma-4, which disrupt TGFbeta-like signaling, result in aberrant migrations and morphologically abnormal spicules. daf-4 codes for a type II TGFbeta-like receptor and the smas code for smad family proteins. daf-4, and hence the TGFbeta-like signal, is required prior to or during cell migrations. Therefore, the TGFbeta-like signal may act to prime the migrating cells or as a guidance cue. Mutations in lin-31 result in identical cell migration and spicule morphology defects. Thus, lin-31, which encodes a "winged helix" protein, may be a component of this TGFbeta-like signaling pathway. The TGFbeta-like signal required for spicule formation likely is coded for by the dbl-1 gene. Mutations in dbl-1 result in adult spicule, ray pattern, and body size defects. Spicule development in dbl-1 mutant males has not been analyzed. A possible source of the TGFbeta-like signal required for spicule development is the male-specific musculature. These muscles are derived from a single postembryonic myoblast, M. Expression of the TGFbeta-like signal in M cell descendants may require the egl-5 gene product, an Abdominal B homolog (Baird, 1999).

An unconventional TGFß superfamily pathway plays a crucial role in the decision between dauer diapause and reproductive growth. The daf-5 gene, along with the daf-3 Smad gene (homolog of Drosophila Medea), is antagonized by upstream receptors and receptor-regulated Smads. DAF-5 is a novel member of the Sno/Ski superfamily (see Drosophila snoN) that binds to DAF-3 Smad, suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a TGFß superfamily signaling pathway. However, evidence is presented that DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor, rather than an antagonist, of a Smad protein. Expressing DAF-5 in the nervous system rescues a daf-5 mutant, whereas muscle or hypodermal expression does not. Previous work suggested that DAF-5 and DAF-3 function in pharyngeal muscle to regulate gene expression, but analysis of regulation of a pharynx specific promoter suggests otherwise. A model is presented in which DAF-5 and DAF-3 control the production or release of a hormone from the nervous system by either regulating the expression of biosynthetic genes or by altering the connectivity or the differentiated state of neurons (da Graca, 2004).

Sno and Ski are found in humans and all major groups of vertebrates, but in insects have only one ortholog of these two proteins. Sno and Ski are more similar to each other than either is to the single Drosophila or mosquito ortholog; therefore Sno and Ski are probably paralogs that were duplicated after the divergence of the protostome and deuterostome lineages. The insect genes have been named Snowski (Snk) to reflect the orthology to both Sno and Ski (da Graca, 2004).

A new family of proteins closely related to the Snowski group is described. Humans have two genes in this group. These genes have been named Skate (for Ski-related gene) and Icy (for Ski sequence family). Drosophila and mosquito each have a gene that is much more similar to human Icy and Skate than to Drosophila Snk. The single Drosophila and mosquito genes have been named iceskate (isk) to reflect their orthology to both Icy and Skate (da Graca, 2004).

It is suggested that DAF-5 is an ortholog of Snowski or Iceskate. (1)DAF-5 clearly has an SDS box, which is not found in any other protein in C. elegans or C. briggsae. This SDS box is more similar to the Snowski group than to the Iceskate group, including amino acids that are important for the ability of Ski to bind Smad proteins. (2) DAF-5 binds the DAF-3 Smad. This binding is mediated by the SDS box in Ski, and may thus be a conserved function of the SDS box. (3) The rate of divergence in the Snowski/Iceskate family is so high that relatively modest sequence conservation is not surprising. This rapid change can be seen when examining the SDS box of B. malayi and potato cyst nematode Snowski. These two nematode proteins are more different from each other than insect Snowski is from Human Sno and Ski. The DAF-5 gene is even more rapidly diverging in the Caenorhabditis genus. C. briggsae and C. elegans proteins average more than 70% amino acid identity. The DAF-5 sequence is only 40% identical overall between C. briggsae and C. elegans. In fact, in the Dachbox and SDS box, the difference between C. elegans and C. briggsae DAF-5 is greater than the difference between insect and human Snowski (da Graca, 2004).

Tissue-specific expression of daf-5 was used to identify cells in which it functions. daf-5cDNA::GFP was expressed with various tissue-specific promoters; these constructs had GFP inserted at the same site as a genomic construct that rescued a daf-5 mutant. Rescue was assayed in daf-7; daf-5 double mutants. Rescued animals would be expected to have the Daf-c phenotype of a daf-7 mutant. pF25B3.3 strongly expressed daf-5::GFP exclusively in nervous system and rescued daf-5 mutants as well as two positive controls. Similarly, punc-14, which expresses daf-5::GFP in nervous system at high level in addition to some non-neuronal expression, also shows strong rescue. Weak ubiquitous expression of daf-5::GFP from the pdpy-30 promoter gives partial rescue. unc-119::daf-5::GFP is weakly expressed in the nervous system but does not rescue, perhaps owing to the low level of expression. Strong expression of daf-5::GFP from the muscle promoter pmyo-3 do not rescue at all. Initial tests of two strains of pdpy-7::daf-5::GFP gave very weak expression and no rescue. Therefore, additional lines were isolated that had strong hypodermal expression of daf-5::GFP, and these did not rescue either. Overall, the results show neuronal expression of daf-5 is sufficient to rescue daf-5 dauer formation defect, while muscle or hypodermal expression is not (da Graca, 2004).

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-β (TGF-β) signal mediators. However, the involvement of RUNX in TGF-β signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-β signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed a downstream target of rnt-1. These data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans (Ji, 2004).

TGF-ß signaling in the nematode Caenorhabditis elegans plays multiple roles in the development of the animal. The Sma/Mab pathway controls body size, male tail sensory ray identity and spicule formation. Three Smad genes, sma-2, sma-3 and sma-4, are all required for signal transduction, suggesting that the functional complex could be a heterotrimer. Because the C termini of Smads play important roles in receptor-mediated activation and heteromeric complex formation, C-terminal mutations were generated in the C. elegans Smad genes and their activities were tested in vivo in each of their distinct developmental roles. Pseudophosphorylated SMA-3 is dominant negative in body size, but functional in sensory ray and spicule development. Somewhat differently, pseudophosphorylated SMA-2 is active in any tissue. The C-terminal mutants of SMA-4 function like wild type, suggesting that the SMA-4 C terminus is dispensable. Using a combination of different C-terminal mutations in SMA-2 and SMA-3, a complex set of requirements was found for Smad-phosphorylation state that are specific to each outcome. Finally, a physical interaction of SMA-3 was detected with the forkhead transcription factor LIN-31, that is enhanced by SMA-3 pseudophosphorylation and reduced in an unphosphorylatable mutant. It is concluded that the tissue-specific requirements for Smad phosphorylation may result, in part, from the need to interact with tissue-specific transcription co-factors that have different affinities for phosphorylated and unphosphorylated Smad protein (Wang, 2005).

In the heterotrimer, the evidence suggests that two R-Smads will interact with one Co-Smad. The Smad2-Smad4 trimer contains one Smad4 and two Smad2 molecules. The complex of Smad4 and ppSmad3 shows a similar ratio. The pSmad2:Smad4 and pSmad3:Smad4 crystal structures contain a 2:1 R-Smad:Co-Smad ratio. Finally, a trimer formed by Smad2, Smad3 and Smad4 has also been reported. The simplest model of heterotrimer formation would suggest that both R-Smad subunits are phosphorylated. It was found, however, that in C. elegans male tail sensory rays, phosphorylatable SMA-3 is not required for function. Thus, it is possible that a Smad heterotrimer may contain one pR-Smad and one unphosphorylated R-Smad. In the case of male sensory rays, the functional complex must contain both SMA-2 and SMA-3, but only one of them needs to be phosphorylated. This finding has implications for the interpretation of morphogen gradients in other contexts. At high levels of ligand, cells may contain a large proportion of Smad heterotrimers containing two pR-Smads, whereas at lower levels of ligand more of the trimers may contain a single pR-Smad and a single unphosphorylated R-Smad. Thus, the Smad heterotrimer composition may provide a direct measure of ligand concentration that can then be translated into differential gene expression (Wang, 2005).

BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

Planarians can be cut into irregularly shaped fragments capable of regenerating new and complete organisms. Such regenerative capacities involve a robust ability to restore bilateral symmetry. Three genes needed for bilaterally asymmetric fragments to regenerate missing body parts have been identified. These genes are candidate components of a signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene (smedolloid-1), a SMAD4-like gene (smedsmad4-1), and a BMP2/4/DPP-like gene (smedbmp4-1). BMP signaling is involved in the formation of new tissues at the midline of regeneration, the dorsal-ventral patterning of new tissues, and the maintenance of the dorsal-ventral pattern of existing adult tissue in homeostasis. smedbmp4-1 is normally expressed at the dorsal midline. Asymmetric fragments lacking a midline display new smedbmp4-1 expression prior to formation of a regenerative outgrowth (blastema). Asymmetric fragments containing the midline display expanded smedbmp4-1 expression towards the wound. It is suggested injured animals that lack left-right symmetry reset their midline through modulation of BMP activity as an early and necessary event in regeneration (Reddien, 2007; full text of article).

Xenopus Smad4, a Medea related protein

A Xenopus homolog of human DPC4 is XSmad4. Smad4/DPC4 is the shared hetero-oligomerization partner for the other SMADs. XSmad4 has 89% identity to human DPC4 and only 40-46% similarity to human or Xenopus XSMad1 and XSMad2 (having undergone a name change acceptable to both C. elegans and Drosophila biologists, and formerly known as XMad1 and XMad2). XSmad4 transcripts are present in the maternal RNA pool and are ubiquitously expressed at least until the neural-groove stage. Smad4 proteins are important because they are the dimerization partners of the other Smads. Human DPC4 transcripts were injected into the animal pole of two-cell Xenopus embryos. Human DPC4 alone can act as a ventral mesoderm inducer in the context of Xenopus animal cap explants, thus mimicking the effect of low concentrations of activin. A mutant form of DPC4, with a small C-terminal deletion, acts as a dominant negative form, failing to induce mesoderm in animal cap assays. Injection of the dominant negative DPC4 prevents brachyury expression in embryos, indicating the potential requirement for endogenous DPC4 in mesoderm induction. Mesoderm induction by XSmad2, an activin mediatior, is completely inhibited by coinjection of dominant negative DPC4. The action of XSmad1, a BMP mediator, is likewise inhibited by coinjection of dominant negative DPC4. DPC4 can physically interact with both XSmad1 and XSmad2. These complexes are formed when cells are stimulated with BMP4 for XSmad1 and activin or TGF-ß for XSmad2. The XSmads become phosphorylated upon stimulation. Thus each member of the TGF-ß family signals through its own SMad, requiring partnership with DPC4/SMad4 (Lagna, 1996).

Misexpression of Smad5 in the Xenopus embryo causes ventralization and induces ventral mesoderm. Moreover, Smad5 induces epidermis in dissociated ectoderm cells that would otherwise form neural tissue. Both of these activities require Smad4 (DPC4) activity, the promiscuous partner of the other Smads. It is proposed that Smad5 acts downstream of the BMP4 signaling pathway in Xenopus embryos and directs the formation of ventral mesoderm and epidermis (Suzuki, 1997).

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated (respectively) by the activity of an activin/BVg1-like protein and bone morphogenetic proteins (BMPs). Interestingly, these two TGF-beta subfamilies are found in overlapping regions during mesoderm patterning. This raises the question of how the presumptive mesodermal cells recognize the multiple TGF-beta signals and differentially interpret this information to assign a particular cell fate. The well characterized model of Xenopus mesoderm induction was exploited to determine the intracellular interactions between BMP-2/4 and activin/BVg1 signaling cascades. Using a constitutively active BMP-2/4 receptor that transduces BMP-2/4 signals in a ligand-independent fashion, it has been demonstrated that signals provided by activin/BVg1 and BMP modulate each other's activity; this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, it has been determined that the specificity of BMP-2/4 and activin/BVg1 signaling is mediated by Smad1 and Smad2, respectively. These Smads should be considered as the mediators of the intracellular antagonism between BMP-2/4 and activin/BVg1, possibly signaling through sequestration of a limited pool of Smad4. Consistent with such a mechanism, Smad4 interacts functionally with both Smad1 and -2 to potentiate their signaling activities; a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling. An activin/BVg1-dependent transcriptional complex contains both Smad2 and Smad4 and thereby provides a physical basis for the functional involvement of both Smads in TGF-beta-dependent transcriptional regulation. Thus, Smad4 plays a central role in synergistically activating activin/BVg1 and BMP-dependent transcription, and functions as an intracellular sensor for TGF-beta-related signals (Candia, 1997).

Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. A natural inhibitor of this process, Smad6, is a longer version of the previously reported JV15-1. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1-Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy. In Xenopus, Smad6 can induce cement gland and neural tissues in ectodermal explants in a cell-autonomous and dose-dependent manner, without inducing mesoderm. Smad6 also inhibits induction of a ventral mesoderm marker by a BMP receptor. Similar effects are observed with Dad, a Drosophila homolog of Smad6. Smad6 does interfere with formation of the primary axis, a process that requires signaling via the activin receptor. When directly challenged by Smad6 in a Xenopus animal cap assay, Smad1, but not Smad2, action is inhibited by Smad6. Smad6 inhibits BMP/Smad1 signaling selectively, without inhibiting either Smad2 signaling in Xenopus embryos or TGFbeta and activin effects in mammalian cells. Smad6 may be an intracellular complement to the BMP inhibitory functions of Noggin, Chordin, and/or Follistatin and may play a key role in cell-autonomous determination of cell fate (Hata, 1998).

The Spemann organizer induces neural tissue, dorsalizes mesoderm and generates a second dorsal axis. Smad10, is shown to have all three of these Spemann activities. The primary structure of Smad 10 is most closely related to the common-partner Smad, Smad4. Smad 10 also contains carboxyl-terminal serines that are sites of phosphorylation in the ligand-activated Smads. therefore, Smad10 may be a hybrid of these two classes of Smads and function in a new manner. Smad10 is expressed at the appropriate time to transduce Spemann signals endogenously. Like the organizer, Smad10 generates anterior and posterior neural tissues. Smad10 appears to function downstream of the Spemann organizer, consistent with a role in mediating organizer-derived signals. Interestingly, Smad10, unlike previously characterized mediators of Spemann activity, does not appear to block BMP signals. This finding, coupled with the functional activity and expression profile, suggests that Smad10 mediates Spemann action in a novel manner. Absence of BMP signaling is not the only condition for dorsal mesodermal induction. A TGFbeta signal transduced via a Smad is thought to be essential for induction of dorsal mesoderm. Although BMP4 signaling counteracts neural induction by BMP inhibitors, it does not reverse Smad10-mediated neural induction. It is plausible that Smad10 is the transducer of the neural induction signal and that the pattern of the induced neural tissue is then modified by factors such as the BMP inhibitors, FGF signals or Wnt signals. Discovery of a ligand that activates Smad10 will provide insights into this novel signaling cascade (LeSueur, 1999)

Smad4 is defined as the common-mediator Smad (co-Smad) required for transducing signals for all TGF-beta superfamily members. This paper describes two Smad4s in Xenopus: XSmad4alpha, which is probably the Xenopus ortholog of human Smad4, and a distinct family member, XSmad4beta, which differs primarily at the extreme N-terminus and in the linker region. Both XSmad4s act as co-Smads, forming ligand-dependent complexes with receptor-regulated Smads 1 and 2 and synergizing with them to activate transcription of mesodermal genes in Xenopus embryos. The two XSmad4 genes have reciprocal temporal expression patterns in Xenopus embryos and are expressed in varying ratios in adult tissues, suggesting distinct functional roles in vivo. XSmad4beta is the predominant maternal co-Smad and it plays a role in the transcriptional regulation of early mesodermal genes. Two distinct nuclear complexes have been identified that bind the activin-responsive element of the Xenopus Mix.2 promoter: one formed in response to high levels of activin signaling and the other activated by endogenous signaling pathways. Using specific antisera, the presence of endogenous XSmad4beta and also XSmad2 in both of these complexes has been demonstrated, and the DNA-binding components of the complexes are different from one another. Furthermore, the presence of these complexes in the nucleus perfectly correlates with the transcriptional activity of the target gene, Mix.2, and one of the XSmad4beta-containing transcription factor complexes undergoes a developmentally regulated nuclear translocation (Howell, 1999).

The role of the maternally encoded transcription factor FAST-1 in the establishment of the mesodermal transcriptional program was examined in Xenopus embryos. FAST-1 has been shown to associate with Smad2 and Smad4, transducers of TGFbeta superfamily signals, in response to stimulation by several TGFbeta superfamily ligands. The FAST-1/Smad2/Smad4 complex binds and activates a 50 bp activin responsive element identified in the promoter of the meso-endodermal marker Mix.2. Three complementary approaches have been used to demonstrate that FAST-1 is a central regulator of mesoderm induction by ectopic TGFbeta superfamily ligands and is a central regulator during endogenous patterning: ectopic expression of mutationally activated FAST-1; ectopic expression of dominant inhibitory FAST-1, and injection of a blocking antibody specific for FAST-1. Expression of constitutively transcriptionally active FAST-1 fusion protein in prospective ectoderm can directly induce the same set of general and dorsal mesodermal genes, as well as some endodermal genes, as are induced by activin or Vg1. In intact embryos, this construct can induce secondary axes similar to those induced by activin or Vg1. Conversely, expression of a FAST-1-repressor fusion (FAST-1 carrying the Engrailed repressor domain in prospective ectoderm blocks induction of mesodermal genes by activin, while expression of this FAST-1 repressor domain construct in intact embryos prevents general/dorsal mesodermal gene expression and axial development). Injection of a blocking antibody specific for FAST-1 prevents induction of mesodermal response genes by activin or Vg1, but not by FGF. In intact embryos, this antibody can prevent the expression of early mesodermal markers and inhibit axis formation, demonstrating that FAST-1 is a necessary component of the first steps in the specification of mesoderm (Watanabe, 1999).

In addition to the activin response element (ARE) from the Mix.2 gene, activin-responsive promoter elements have been identified in genomic sequence from several other mesoderm early response genes: Gsc, XFKH-1, Xbra, HNF1 alpha, Xlim-1 and Xombi. These elements do not share extensive sequence similarity to the Mix.2 ARE or to one another. It was therefore initially surprising that all of the activin response genes that were examined, including Gsc, Xbra, XFKH-1, Xlim-1 and Xombi, can be activated by FAST-1 in a cycloheximide-insensitive manner. This observation indicates that FAST-1 can directly bind to regulatory sites controlling expression of these genes. However, the recent definition of a FAST-1 consensus binding site, does permit the identification by inspection of potential FAST-1 target sites in these promoters, consistent with their direct regulation by FAST-1. FAST-1-binding sites and/or FAST-1-dependent regulation of enhancer sequences from the Xombi, Xbra, Gsc and Xlim-1 promoters have been confirmed. FAST-1/FAST2 binding and regulation of the mouse goosecoid promoter do occur, supporting the direct regulation of these genes by FAST-1 in vivo (Watanabe, 1999).

Since the original identification of FAST-1 as a transcription factor that targets activated Smad2 to an activin responsive promoter element, both Drosophila and vertebrate Smads have been shown to have intrinsic DNA-binding activities. These observations raise the issue of the extent to which Smads have intrinsic affinity for regulatory element in mesoderm early response genes, and to what extent they require additional factors, such as FAST-1, to regulate these responses. Cell types that lack FAST-1 but express Smad2 and Smad4 cannot activate an ARE-luciferase reporter construct unless FAST-1 is ectopically expressed, indicating that Smad activation in the absence of FAST-1 is not sufficient for regulation of the ARE. Both FAST-1 and Smad4 (but not Smad2)-binding sites have been identified in the ARE; elimination of the FAST-1-binding site eliminates activin responsiveness, while elimination of the Smad4-binding site reduces, but does not eliminate, activin responsiveness. This result also indicates that FAST-1 is the major determinant of targeting of the ARE by the FAST-1/Smad2/Smad4 complex (Watanabe, 1999).

In early Xenopus embryos, the prototypical XFast-1/Smad2/Smad4 complex ARF1 is induced at the Mix.2 activin responsive element (ARE) by activin overexpression. ARF2, a related, but much more abundant, complex formed during gastrulation in response to endogenous TGFß family members has been characterized and a novel Fast family member, XFast-3, has been identified as its transcription factor component. Endogenous ARF2 efficiently competes out ARF1 at early gastrulation, due to the ability of XFast-3 to interact with activated Smads (Smad2 and Smad4) with much higher affinity than XFast-1. ARF1 and ARF2 are activated by distinct TGFß family members (activin and Xnr1, and weakly by Xnr2). Using morpholino antisense oligonucleotides to deplete levels of the constituent transcription factors XFast-1 and XFast-3 specifically, an important role has been identified for ARF1 and ARF2 in early Xenopus embryos in controlling the convergent extension movements of gastrulation (Howell, 2002).

Xenopus Smad10 and nervous system development

Before the nervous system establishes its complex array of cell types and connections, multipotent cells are instructed to adopt a neural fate and an anterior-posterior pattern is established. Smad10, a Medea related member of the Smad family of intracellular transducers of TGFß signaling, is required for formation of the nervous system. In addition, two types of molecules proposed as key to neural induction and patterning, bone morphogenetic protein (BMP) antagonists and fibroblast growth factor (FGF), require Smad10 for these activities. These data suggest that Smad10 may be a central mediator of the development of the frog nervous system (LeSueur, 2002).

Two separate classes of organizer-derived secreted molecules -- FGFs and BMP antagonists -- are thought to be key to neural induction and anterior-posterior patterning. In Xenopus, FGFs induce posterior neural tissue, and FGFs are required for neural induction in the chick and spinal cord formation in the frog. Since Smad10 is necessary for formation of neural structures, including the spinal cord, it follows that FGF might require Smad10 for its neuralizing properties. To test this notion, embryos were injected with control morpholino-modified oligonucleotide (morpholinos or MO), Smad10 MO, DNS10 mRNA, or ß-galactosidase mRNA and explanted animal caps. During gastrulation, the caps were cultured in the presence of FGF under conditions that induce formation of neural tissue and then the caps were analyzed for expression of neural markers. Smad10 does not block all FGF activities, but rather is required specifically for neural induction by FGF (LeSueur, 2002).

If Smad10 transduces an RTK signal, what ligand might activate the cascade? One plausible candidate is FGF. FGFs signal via an RTK pathway that involves phosphorylation and activation of Erks. This FGF pathway has similar biological functions to Smad10; both induce posterior neural fates. Furthermore, FGF requires Smad10 for this activity. These data, coupled with the in vitro phosphorylation results and the inability of the Smad10-PXAPx3 mutant to induce spinal cord formation, are consistent with the idea that FGF initiates an RTK pathway that leads to activation of Erk, subsequent phosphorylation of Smad10, and induction of posterior neural fates. Additional biochemical experiments will be required to test this hypothesis (LeSueur, 2002).

The data suggest that a RTK pathway may regulate the function of Smad10 and may do so in a direct biochemical sense. Smad10 contains Erk consensus phosphorylation sites, Erk2 directly phosphorylates Smad10 in vitro, and the PX(S/T)P consensus phosphorylation sites on Smad10 are required for this Erk-dependent phosphorylation. Of note, when the Erk consensus sites are mutated to alanine, Smad10 remains functional and generates anterior neural fates; however, the mutant no longer produces posterior neural fates. This suggests that the nonphosphorylated and phosphorylated forms of Smad10 might interact with different subsets of transcription factors to generate distinct cell fates. Erks often phosphorylate and activate transcription factors that regulate gene expression. Smad10 may be another example of such a transcription factor. Taken together, these data suggest that an RTK signal, rather than a TGFß signal, might control Smad10's biological function in anterior versus posterior neural development (LeSueur, 2002).

The tumor suppressor Smad4/DPC4 is regulated by phosphorylations that integrate FGF, Wnt, and TGF-beta signaling

Smad4 is a major tumor suppressor currently thought to function constitutively in the transforming growth factor beta (TGF-beta)-signaling pathway. This study reports that Smad4 activity is directly regulated by the Wnt and fibroblast growth factor (FGF) pathways through GSK3 (see Drosophila Shaggy) and mitogen-activated protein kinase (MAPK; see Drosophila Rolled) phosphorylation sites. FGF activates MAPK, which primes three sequential GSK3 phosphorylations that generate a Wnt-regulated phosphodegron bound by the ubiquitin E3 ligase beta-TrCP (see Drosophila Slmb). In the presence of FGF, Wnt potentiates TGF-beta signaling by preventing Smad4 GSK3 phosphorylations that inhibit a transcriptional activation domain located in the linker region. When MAPK is not activated, the Wnt and TGF-beta signaling pathways remain insulated from each other. In Xenopus embryos, these Smad4 phosphorylations regulate germ-layer specification and Spemann organizer formation. The results show that three major signaling pathways critical in development and cancer are integrated at the level of Smad4 (Demagny, 2014: PubMed).

Mammalian Medea related proteins

continued: Medea Evolutionary homologs part 2/3 | part 3/3 |

Medea: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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