Interactions of Smad proteins with TGF-beta receptors

Activins and other members of the transforming growth factor-beta-like superfamily of growth factors transduce their signals by interacting with two types of receptor serine/threonine kinases. The Smad proteins are involved in the signaling pathways of these receptors, but the initial stages of their activation as well as their specific functions remain to be defined. The pathway-specific Smad2 and 3 can form a complex with the activin receptor in a ligand-dependent manner. This complex formation is rapid but also transient. Indeed, soon after their association with the activin receptor, Smad2 and Smad3 are released into the cytoplasm where they interact with the common partner Smad4 (Drosophila homolog: Medea). These Smad complexes then mediate activin-induced transcription. The inhibitory Smad7 can prevent the association of the two pathway-specific Smads with the activin receptor complex, thereby blocking the activin signal (Lebrun, 1999).

MADR2, and not the related protein DPC4 transiently interacts with the TGFß receptor and is directly phosphorylated by the complex on C-terminal serines. Interaction of MADR2 with receptors and phosphorylation requires activation of receptor I by receptor II and is mediated by the receptor I kinase. Mutation of the phosphorylation sites generate a dominant negative MADR2 that blocks TGFß-dependent transcriptional responses. The mutated protein stably associates with receptors, and fails to accumulate in the nucleus in response to TGFß signaling. Thus, transient association and phosphorylation of MADR2 by the TGFß receptor is necessary for nuclear accumulation and initiation of signaling (Macías-Silva, 1996)

TGFbeta signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Smad7 is an inhibitor of TGFbeta signaling. Smad7 prevents TGFbeta-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFbeta type I receptor, thereby blocking association, phosphorylation, and activation of Smad2. Mutations in Smad7 that interfere with receptor binding disrupt Smad7's inhibitory activity. These studies define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFbeta family receptors (Hayashi, 1997).

The Smad proteins function downstream of TGF-beta 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-beta 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, namely, 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-beta, 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).

Smad2 and Smad3 are structurally highly similar and both mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Smad2 and Smad3 interact with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it is phosphorylated by TbetaR-II kinase. TGF-beta1 induces phosphorylation of Smad2 and Smad3 in cultured Mv1Lu mink lung epithelial cells. Smad4 is found to be constitutively phosphorylated in Mv1Lu cells: the phosphorylation level remaining unchanged upon TGF-beta1 stimulation. Similar results are obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interact with Smad4 after TbetaR activation in transfected COS cells. In addition, TbetaR-activation-dependent interaction is observed between Smad2 and Smad3. Smads 2, 3 and 4 accumulate in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and show a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibits the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction (Nakao, 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 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).

TGF-beta mediates phosphorylation of Smad2 at two serine residues in the C terminus (namely Ser465 and Ser467), which are then phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of mutated Smad2 (with the mutation of Ser465 and/or Ser467 to alanine residues) into Mv1Lu cells 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. Mutation of Ser465 and Ser467 in Smad2 abrogate complex formation of this mutant with Smad4 and block 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).

Activin A is a multifunctional protein, which is a member of the transforming growth factor-beta (TGF-beta) superfamily. Smad proteins have recently been shown to transduce signals for the TGF-beta superfamily of proteins, and Smad2 was implicated in activin signaling in Xenopus embryos. The receptors and Smad proteins activated by activin A in a human epidermal keratinocyte cell line, HaCaT, have been identified. The major activin receptors expressed on HaCaT cells are activin type II receptor (ActR-II) and activin type IB receptor (ActR-IB). In HaCaT cells, activin A induces the phosphorylation of Smad3 and, to a lesser extent, of Smad2, while TGF-beta induces an efficient phosphorylation of both Smad2 and Smad3. Activin A preferentially induces the nuclear translocation of Smad3 in HaCaT cells, whereas TGF-beta strongly induces the nuclear translocation of Smad2, as well as other Smads. Moreover, a constitutively active form of ActR-IB efficiently stimulates the formation of a heteromeric complex between Smad3 and Smad4 in COS cells transfected with Smad cDNAs. These results suggest that activin A binds to a receptor complex of ActR-II and ActR-IB, and preferentially activates Smad3 in HaCaT human keratinocytes (Shimizu, 1998).

Smads transmit signals from transmembrane ser/thr kinase receptors to the nucleus. SARA (for Smad anchor for receptor activation) is a FYVE domain protein that interacts directly with Smad2 and Smad3. [Cysteine-rich RING domains of the FYVE finger subfamily bind specifically to Ptdlns phosphorylated exclusively by phosphoinositide 3-kinases at the D-3 position of the inositol ring, thereby recruiting and activating downstream effectors of Ptdlns(3)P signaling.] SARA functions to recruit Smad2 to the TGFbeta receptor by controlling the subcellular localization of Smad2 and by interacting with the TGFbeta receptor complex. Phosphorylation of Smad2 induces dissociation from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Furthermore, mutations in SARA that cause mislocalization of Smad2 inhibit TGFbeta-dependent transcriptional responses, indicating that the regulation of Smad localization is important for TGFbeta signaling. These results thus define SARA as a component of the TGFbeta pathway that brings the Smad substrate to the receptor (Tsukazaki, 1998).

Activins, members of the transforming growth factor-ß family, are pleiotropic growth and differentiation factors. Activin A induces B-cell apoptosis. To identify the genes responsible for activin-induced apoptosis, retrovirus-mediated gene trap screening was performed in a mouse B-cell line. The rasGAP-binding protein Dok-1 (p62) was identified as an essential molecule that links activin receptors with Smad proteins. In B cells overexpressing Dok-1, activin A-induced apoptotic responses are augmented. The expression of bcl-XL is down-regulated by inhibition of the ras/Erk pathway. Activin stimulation triggers association of Dok-1 with Smad3, as well as association of Smad3 with Smad4. Dok-1 also associates with both the type I and type II activin receptors. Dok-1 has been characterized previously as a tyrosine-phosphorylated protein acting downstream of the protein tyrosine kinase pathway: intriguingly, activin signaling does not induce tyrosine phosphorylation of Dok-1. These findings indicate that Dok-1 acts as an adaptor protein that links the activin receptors with the Smads, suggesting a novel function for Dok-1 in activin signaling leading to B-cell apoptosis (Yamakawa, 2000).

Function of Type I Activin receptors and Smads in Xenopus

During early embryogenesis of Xenopus, dorsoventral polarity of the mesoderm is established by dorsalizing and ventralizing agents, which are presumably mediated 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 Smad2 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).

Transforming growth factor (TGF)-beta family members play a central role in mesoderm induction during early embryogenesis in Xenopus. Although a number of target genes induced as an immediate-early response to activin-like members of the family have been described, little is known about the molecular mechanisms involved. Systematic analysis of the activin induction of the target gene XFKH1 reveals two regions that mediate activin-responsive transcription: one, in the first intron, is targeted directly by the activin-signalling pathway; the other, in the 5' flanking sequences, responds to activin indirectly, possibly being required for the maintenance of gene expression. A 107 bp region of the XFKH1 first intron acts as an enhancer and confers activin inducibility onto a minimal uninducible promoter in the absence of new protein synthesis. It bears little sequence similarity to other activin responsive sequences. Overexpression of a constitutively active derivative of Xenopus Smad2 (XSmad2), which has been implicated as a component of the activin signaling pathway, is sufficient for direct activation of transcription via this enhancer. XSmad2 acts indirectly on the proximal promoter element induced by activin via an indirect mechanism. These results establish the XFKH1 intron enhancer as a direct nuclear target of the activin signaling pathway in Xenopus embryos, and provide strong new evidence that XSmad2 is a transducer of activin signals (Howell, 1997).

The normal expression pattern of the Wnt responsive homeobox gene Siamois is restricted to the dorso-vegetal region of the Xenopus embryo. Since the Wnt signaling pathway (via beta-catenin) is active on the entire dorsal side of the early embryo, it seemed curious that Siamois expression is not seen in the dorsal ectoderm. It turns out that only Wnt signaling, via activation of beta-catenin, can directly induce Siamois; induction is not induced by signaling via the SMAD1 (BMP2/4) or SMAD2 (activin/Vg-1) pathways. In normal embryos, the SMAD2 pathway can cooperate with the Wnt pathway to induce expression of Siamois much more strongly than does the Wnt pathway alone. The significance of this cooperation is demonstrated in normal embryos by blocking the SMAD2 signaling pathway with a dominant negative activin receptor. The activin dominant negative receptor blocks this cooperative effect and reduces the expression of Siamois by threefold in early embryos. This cooperative relationship between the SMAD2 and Wnt pathways is reciprocal. Thus, in normal embryos, the Wnt pathway can enhance induction, by the SMAD 2 pathway, of the organizer genes Goosecoid and Chordin but not the pan-mesodermal marker genes Xbra and Eomes. The SMAD 1 pathway, which functions to transduce zygotic BMP2/4 signals, fails to induce Siamois. It is concluded that the Wnt and SMAD2 signaling pathways cooperate to induce the expression of Spemann-organizer specific genes and so help to localize their spatial expression (Crease, 1998).

Smads are proteins that transduce signals on behalf of members of the TGF beta superfamily of growth factors. Recently, inhibitory Smads (Smad6, Smad7, and Dad) were isolated from human, mouse, and fly. These anti-Smads were shown to inhibit TGF beta signaling by stably associating to TGF beta type I receptors or, as has been shown for Smad6, by binding to receptor-activated Smad1. The cloning, distribution, and embryological activity of the Xenopus Smad7 (XSmad7) is reported. XSmad7 inhibits signaling from the activin and BMP pathways in animal explants although at different thresholds. When expressed in the embryo, low concentrations of XSmad7 dorsalize the ventral mesoderm, thus inducing a secondary axis. At higher concentrations however, XSmad7 inhibits both mesoderm induction and primary axis specification. In addition, XSmad7 acts as a direct neural inducer both in the context of ectodermal explants and in vivo (Casellas, 1998).

Smads are central mediators of signal transduction for the TGFbeta superfamily. However, the precise functions of Smad-mediated signaling pathways in early development are unclear. A requirement for Smad2 signaling is demonstrated in dorsoanterior axis formation during Xenopus development. Using two point mutations of Smad2 previously identified in colorectal carcinomas, it has been shown that Smad2 ushers Smad4 to the nucleus to form a transcriptional activation complex with the nuclear DNA-binding protein FAST-1 and that the mutant proteins interact normally with FAST-1 but fail to recruit Smad4 into the nucleus. This mechanism of inhibition specifically restricts the dominant-negative activity of these mutants to the activin/Vg1 signaling pathway without inhibiting BMPs. Furthermore, expression of these mutants in Xenopus animal caps inhibits but does not abolish activin and Vg1 induction of mesoderm and in the embryo results in a truncated dorsoanterior axis. These studies define a mechanism through which mutations in Smad2 may block TGFbeta-dependent signaling and suggests a critical role for inductive signaling mediated by the Smad2 pathway in Xenopus organizer function (Hoodless, 1999).

Function of Type I Activin receptors and Smads in mammals

Mammalian primordial germ cells (PGCs) proliferate as they migrate from their initial location in the extraembryonic mesoderm to the genital ridge, the gonadal anlage. Once in the genital ridge, PGCs cease dividing and differentiate according to their gender. To identify ligands that might limit PGC proliferation, growth factor receptors encoded in RNA obtained from purified germ cells shortly after their arrival in the genital ridge were examined. Receptors for two members of the TGFbeta superfamily were found, TGFbeta1 and activin. Partial sequence determinations of 30 PCR-generated clones indicate the presence of TGFbeta receptors TbetaR-I and TbetaR-II, as well as the activin receptors ActR-IB and ActR-IIB. Since the signal-transducing domains of both receptor systems are highly conserved, the effects of both TGFbeta1 and activin on PGCs would be expected to be similar. Both ligands limit the accumulation of germ cells in primary PGC cultures. BrdU incorporation assays demonstrate that either ligand inhibits PGC proliferation. These results suggest that these signal transduction pathways are important elements of the mechanism that determines germ cell endowment (Richards, 1999).

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI/ALK2 in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos are rescued from the gastrulation defect of Acvr1 null embryos but exhibit abnormal heart looping and embryonic turning. High mutant contribution chimeras express left-side markers, such as nodal, bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 is absent in the midline of chimeric embryos, but shh, a midline marker, is expressed normally, suggesting that, despite formation of midline, its barrier function is abolished. High-contribution chimeras also lack asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse (Kishigami, 2004).

Function and targets Smad2, and SMad3 homologs in mammals

Continued: baboon Evolutionary homologs part 3/3 | back to part 1/3 |

baboon: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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