Mothers against dpp


EVOLUTIONARY HOMOLOGS (part 2/3)

Mammalian Mad homologs

A mouse cDNA has been cloned encoding a Mothers-against-dpp (MAD)-related protein, MADR1. Madr1 is ubiquitously expressed in the mouse embryo, indicating a broad function in a variety of tissue during embryogenesis, potentially relaying signals of numerous BMPs. However, its expression in the testis is strictly germ cell-specific and developmentally regulated. Testicular Madr1 expression starts in some seminiferous tubules at 2 weeks of age. After mid-puberty, a stage-specific Madr1 expression is established. During the cycling of the seminiferous epithelium, Madr1 expression initiates in the pachytene spermatocytes of stage V seminiferous tubules, peaks at stage X, then decreases as pachytene spermatocytes differentiate into secondary spermatocytes and then round spermatids. In the testis of adult Bmp8b homozygous mutant males, the Madr1- expressing pachytene spermatocytes are the first cell population to show increased apoptosis. These data suggest that MADR1 serves as a downstream component of the BMP8 signaling pathway during the differentiation of meiotic male germ cells (Zhao, 1997).

Phosphorylation of MADR1, a human homolog of Drosophila MAD, is tightly regulated and rapidly induced by BMP2, but not TGFß or Activin. This phosphorylation is necessary for function, since a point mutant in the human homolog, in the same position that yields a null phenotype in Drosophila (the conserved C-terminal domain), is not phosphorylated. BMP2 treatment results in accumulation of MADR1 in the nucleus (Hoodless, 1996).

Two vertebrate homologs of Drosophila Mothers against dpp (Mad) were isolated from the mouse and the Xenopus embryo. They are are named MusMLP (mad-like protein) and XenMLP, respectively. Overexpression of XenMLP causes ventralization of Xenopus embryos; the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. MLPs are multidomain proteins as shown by a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain. The proline-rich, middle domain maximizes the activity of the C-terminal domain. The MusMLP gene maps to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes (Meersseman, 1997).

The Smad4/DPC4 (Drosophila homolog Medea) tumour suppressor is inactivated in nearly half of pancreatic carcinomas and to a lesser extent in a variety of other cancers. Smad4 is important in that it is the dimerization partner of the other Smads. Smad4/DPC4, and the related tumour suppressor Smad2, belong to the SMAD family of proteins that mediate cell surface to nucleus signaling by the TGF-beta/activin/BMP-2/4 cytokine superfamily; signals from receptor Ser/Thr protein kinases at the cell surface are sent to the nucleus. SMAD proteins, which are phosphorylated by the activated receptor, propagate the signal, in part, through homo- and hetero-oligomeric interactions. Smad4/DPC4 is critical because it is the shared hetero-oligomerization partner for the other SMADs. The conserved carboxy-terminal domains of SMADs are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation. The crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumour suppressor was determined at 2.5 A resolution. The structure reveals that the Smad4/DPC4 CTD forms a crystallographic trimer through a conserved protein-protein interface, to which the majority of the tumour-derived missense mutations map. These mutations disrupt homo-oligomerization in vitro and in vivo, indicating that the trimeric assembly of the Smad4/DPC4 CTD is critical for signaling and is disrupted by tumorigenic mutations. A heterohexamer model is suitable from a structural perspective for Mad protein hetero-oligomerization. The heterohexamer would be formed by a dimerization of Smad4 and Smad2 homotrimers (Shi, 1997).

The conserved C-terminal domains of human Mad proteins mimic the activity of their full-length counterparts in mesoderm induction assays. Furthermore, a MAD fluorescent marker tagged C-terminal domain becomes localized in the nucleus of all cells expressing it. The so called C-domain might be an active domain, whereas the conserved N-domain might act as an inhibitor of nuclear translocation, a direct repressor of the C-domain or both. Thus, an activity of the C-domain appears to be repressed by the N-domain and this repression eliminated by incoming membrane receptor signals (Massague, 1996 and references).

Smads are proteins that transduce signals on behalf of members of the TGF beta superfamily of growth factors. Recently, three inhibitory Smads (Smad6, Smad7, and Dad) were isolated from human, mouse, and fly respectively. These anti-Smads were shown to inhibit TGF beta signaling by stably associating to TGF beta type I receptors or, as it was shown for Smad6, by binding to receptor-activated Smad1. Xenopus Smad7 (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. It is suggested that XSmad7 affects distinct TGFbeta pathways at different thresholds: at low doses, it selectively blocks the BMP pathway, whereas at higher concentrations, it is additionally capable of inhibiting the activin/TGFbeta-like pathways. XSmad7 is present maternally and maintains ubiquitous expression at least until the onset of gastrulation when the mesoderm, ectoderm, and endodem are specified (Casellas, 1998).

Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. A cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH: Drosophila homolog CG2224), is a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads (Itoh, 2001).

Interaction of MAD homologs with receptors

Two structural elements, the L45 loop on the kinase domain of the transforming growth factor-beta (TGF-beta) family type I receptors and the L3 loop on the MH2 domain of Smad proteins, determine the specificity of the interactions between these receptors and Smad proteins. The L45 sequence of the TGF-beta type I receptor (TbetaR-I) specifies Smad2 interaction, whereas the related L45 sequence of the bone morphogenetic protein (BMP) type I receptor (BMPR-I) specifies Smad1 interactions. Members of a third receptor group, which includes ALK1 and ALK2 from vertebrates and Saxophone from Drosophila, specifically phosphorylate and activate Smad1 even though the L45 sequence of this group is very divergent from that of BMPR-I. The structural elements that determine the specific recognition of Smad1 by ALK1 and ALK2 have been investigated. In addition to the receptor L45 loop and the Smad1 L3 loop, the specificity of this recognition requires the alpha-helix 1 of Smad1. The alpha-helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the Smad MH2 domain. Thus, Smad1 recognizes two distinct groups of receptors (the BMPR-I group and the ALK1 group) through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the Smad1 MH2 domain (Chen, 1999).

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Reported in this study is the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling (Qin, 2001).

Ligand binding to specific transmembrane receptor kinases induces receptor oligomerization and phosphorylation of the receptor-specific Smad protein (R-Smad) in the cytoplasm. The conserved signaling mechanism is the formation of a heteromeric complex between the phosphorylated R-Smad and the common mediator Smad4. The heteromeric complex enters the nucleus to regulate transcription of target genes. The R-Smads and Smad4 share a common domain configuration consisting of a conserved N-terminal DNA binding domain (MH1 domain) and a C-terminal MH2 domain separated by a variable linker region. The MH2 domain of an R-Smad, but not Smad4, has been shown to homo-oligomerize. Phosphorylation-triggered heteromeric assembly between Smad4 and R-Smad is mediated by the C-terminal MH2 domain. The sites of phosphorylation have been mapped to the last two serine residues within the conserved C-terminal SSXS sequence of the R-Smads. However, the role of phosphorylation in subunit assembly as well as the stoichiometry of the heteromeric complex remains controversial. Phosphorylation has been proposed to contribute directly to subunit assembly by bridging the MH2 domain interaction. Another model, however, suggests that phosphorylation uncouples the intramolecular inhibitory activity of the MH1 domain on the MH2 domain, allowing the MH2 domains to associate constitutively. The work described here suggests that the phosphorylated C-terminal tail of Smad1 functions as a subunit assembly switch by forming specific contacts with the phosphoserine binding pockets of the neighboring molecule. Furthermore, the MH2 domain undergoes concerted conformational changes upon trimerization, which may serve as a signaling switch. The phosphorylation-triggered Smad1-Smad4 complex is a trimer containing two Smad1 and one Smad4 subunits. Conservation of the amino acids involved in subunit assembly demonstrates a unifying structural mechanism of phosphorylation-triggered heteromeric assembly between R-Smads and Smad4 (Qin, 2001).

The current model suggests two mechanisms controlling specific phosphorylation of the R-Smads by the receptor kinase complexes. (1) A loop structure in the receptor kinase domain, referred to as the L45 loop, specifically interacts with the L3 loop of an R-Smad protein. (2) The receptor-associated recruiting molecule, such as SARA in the TGF-ß/activin pathway, can recruit specific R-Smad protein to the receptor kinase for phosphorylation. Although these mechanisms warrant specific phosphorylation of the R-Smads, how the recruited R-Smads release after phosphorylation is unknown. The current work provides insight into how the R-Smad disengages from the receptor kinase and the recruiting molecule after phosphorylation. The structure of the trimeric Smad1 reveals that the L3 loop and the three-helix bundle structure undergo concerted conformational changes upon activation, which may signal Smad1 dissociation from the receptor kinase complex. At the basal state, in which Smad1 is monomeric, the L3 loop of Smad1 interacts with the L45 loop of the receptor kinase. Upon activation and formation of a trimer, the L3 loop of Smad1 undergoes conformation change and interacts with the phosphorylated C-terminal tail of another Smad1 molecule. Thus, by employing distinct conformations to interact with mutually exclusive signaling partners, the L3 loop can serve as a switch for R-Smad dissociation from the receptor. Consistent with this model, the interaction between the receptor kinase complex and R-Smad is stronger when either the C-terminal phosphorylation sites of the R-Smad are mutated or when the kinase activity is rendered inactive by the catalytic site mutation. In addition, the trimerization-induced tilting of the three-helix bundle structure toward the subunit interface could also serve as a conformational switch to direct Smad1 dissociation from the receptor complex. In the analogous TGF-ß/activin pathway, SARA recruits and stabilizes the monomeric form of Smad2/3. Structural comparison between the trimeric Smad1 structure and the Smad2/SARA 1:1 complex structure reveals that SARA inhibits Smad2 trimerization by restricting the three-helix bundle movement, which is an essential mechanism of Smad protein trimerization. It is suggested that phosphorylation energetically favors trimerization, and that formation of trimers is sterically incompatible with SARA association. Similar mechanisms may exist for Smad1 through other receptor-associated molecules (Qin, 2001).

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 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 was 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).

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, 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-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).

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 (Drosophila homolog: Thick veins) by receptor II (Drosophila homolog: Punt) 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)

BMP7 and activin are members of the transforming growth factor beta superfamily. Endogenous activin and BMP7 signaling pathways are characterized in P19 embryonic carcinoma cells. BMP7 and activin bind to the same type II receptors, ActRII and IIB, but recruit distinct type I receptors into heteromeric receptor complexes. The major BMP7 type I receptor observed is ALK2, while activin binds exclusively to ALK4 (ActRIB). BMP7 and activin elicit distinct biological responses and activate different Smad pathways. BMP7 stimulates phosphorylation of endogenous Smad1 and 5 and the formation of complexes with Smad4, and induces the promoter for the homeobox gene, Tlx2. In contrast, activin induces phosphorylation of Smad2, association with Smad4, and induction of the activin response element from the Xenopus Mix.2 gene. Biochemical analysis reveals that constitutively active ALK2 associates with and phosphorylates Smad1 on the COOH-terminal SSXS motif, and also regulates Smad5 and Smad8 phosphorylation. Activated ALK2 also induces the Tlx2 promoter in the absence of BMP7. ALK1 (TSRI), an orphan receptor that is closely related to ALK2 also mediates Smad1 signaling. Thus, ALK1 and ALK2 induce Smad1-dependent pathways. ALK2 functions to mediate BMP7 but not activin signaling (Macias-Silva, 1998).

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).

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) have opposite effects on diverse cellular functions, but the basis for this antagonism is not known. TGF-beta signals through a receptor serine kinase that phosphorylates and activates the transcription factors Smads 2 and 3, whereas the IFN-gamma receptor and its associated protein tyrosine kinase Jak1 mediate phosphorylation and activation of the transcription factor Stat1. A basis for the integration of TGF-beta and IFN-gamma signals is presented. IFN-gamma inhibits the TGF beta-induced phosphorylation of Smad3 and its attendant events, namely, the association of Smad3 with Smad4, the accumulation of Smad3 in the nucleus, and the activation of TGFbeta-responsive genes. Acting through Jak1 and Stat1, IFN-gamma induces the expression of Smad7, an antagonistic SMAD, which prevents the interaction of Smad3 with the TGF-beta receptor. The results indicate a mechanism of transmodulation between the STAT and SMAD signal-transduction pathways (Ulloa, 1999).

The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. To initiate a particular TGFbeta response, a specific TGFbeta ligand binds to a specific pair of transmembrane Ser-Thr receptor kinases, the type I and type II receptors, and this activates the Ser-Thr kinase in the cytoplasmic domain of the type I receptor. The signal is then propagated by type I receptor-mediated phosphorylation of specific R-Smads. The R-Smads, Smad2 and 3, are recruited to the TGFbeta receptors by SARA (Smad anchor for receptor activation). This process appears to involve direct interactions between SARA and Smad2, SARA and TGF receptors, and Smad2 and TGF receptors. SARA does not interact with either Smad1 or Smad5, which share ~80% sequence identity with Smad2. These interactions are important in regulating specific Smad phosphorylation. The phosphorylated R-Smad hetero-oligomerizes with the co-Smad, Smad4, translocates into the nucleus and associates with sequence-specific DNA binding proteins, resulting in the positive or negative regulation of agonist-responsive genes. The crystal structure of a Smad2 MH2 domain in complex with the Smad-binding domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors. Despite having generally similar structures, the MH2 domains of Smad2 and Smad4 have different surface features. A direct comparison of the electrostatic potential reveals the presence of a highly positively charged groove next to the L3 loop on Smad2, but not on Smad4. This basic surface contains residues that are conserved in R-Smads but not in co-Smads, suggesting that this region might be important for receptor binding. Analysis of the type I TGFbeta receptor cytoplasmic domain reveals a complementary pattern on its surface. Specifically, the L45 loop, which specifies interactions with Smad2, is located immediately adjacent to the flexible GS region, which is phosphorylated upon ligand binding and becomes very acidic. Thus, the phosphorylated GS region on the type I receptors might interact with a highly basic surface groove on R-Smads to provide binding affinity, whereas the L45 loop on the type I receptors recognizes the L3 loop of specific R-Smads to provide specificity (Wu, 2000).

Degradation of Smads and Smad role in TGFbeta receptor degradation

The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signaling pathway. On the inside of the cell membrane, members of a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Smurf1, a new member of the Hect family of E3 ubiquitin ligases is described. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals (Zhu, 1999).

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Smurf2, a C2-WW-HECT domain ubiquitin ligase has been identified and Smurf2 has been shown to associate constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGFß receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFNgamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGFß receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGFß receptor for degradation. Although these studies have focused on the role of Smad7 as the receptor component of a ubiquitin ligase complex, Smad6 and the R-Smads all contain PY motifs in their linker regions and have the potential to stably assemble with other ubiquitin ligases. Smads could thus fulfill a more general function in regulating protein degradation in response to TGFbeta signaling. Consistent with this, the transcriptional corepressor SnoN is degraded in response to TGFbeta signaling through interaction with Smad2 and Smad3. Thus, in addition to their role as transcriptional comodulators, Smads may function as receptor components of E3 ubiquitin ligases that target specific proteins for degradation in response to TGFbeta signaling. It will be interesting to determine what role this activity may fulfill in mediating TGF biology (Kavsak, 2000).

To understand the role of Smad1 in mouse development, a Smad1 loss-of-function allele was generated using homologous recombination in ES cells. Smad1-/- embryos die by 10.5 dpc because they fail to connect to the placenta. Mutant embryos are first recognizable by 7.0 dpc, owing to a characteristic localized outpocketing of the visceral endoderm at the posterior embryonic/extra-embryonic junction, accompanied by a dramatic twisting of the epiblast and nascent mesoderm. Chimera analysis reveals that these two defects are attributable to a requirement for Smad1 in the extra-embryonic tissues. By 7.5 dpc, Smad1-deficient embryos show a marked impairment in allantois formation. By contrast, the chorion overproliferates, is erratically folded within the extra-embryonic space and is impeded in proximal migration. BMP signals are known to be essential for the specification and proliferation of primordial germ cells. A drastic reduction of primordial germ cells is found in Smad1-deficient embryos, suggesting an essential role for Smad1-dependent signals in primordial germ cell specification. Surprisingly, despite the key involvement of BMP signaling in tissues of the embryo proper, Smad1-deficient embryos develop remarkably normally. An examination of the expression domains of Smad1, Smad5 and Smad8 in early mouse embryos shows that, while Smad1 is uniquely expressed in the visceral endoderm at 6.5 dpc, in other tissues Smad1 is co-expressed with Smad5 and/or Smad8. Collectively, these data have uncovered a unique function for Smad1 signaling in coordinating the growth of extra-embryonic structures necessary to support development within the uterine environment (Tremblay, 2001).

Members of the Smad family of intracellular signaling intermediates transduce signals downstream from the TGF-beta family of receptor serine threonine kinases. The original member of this family, Smad1, has been shown to mediate signals from receptors for the bone morphogenetic proteins (BMPs), a large group of ligands in the TGF-beta superfamily that mediate important developmental events. The Smad1 gene has been targeted in mice and mutants null at this locus have been created. Smad1 mutant mice die at approximately 9.5 days postcoitum due to defects in allantois formation. In Smad1 mutant mice, the allantois fails to fuse to the chorion, resulting in a lack of placenta and failure to establish a definitive embryonic circulation. Although vasculogenesis is initiated in the mutant allantois, the vessels formed are disorganized, and VCAM-1 protein, a marker for distal allantois development, is not expressed. Smad1 null fibroblasts are still able to respond to BMP2, however, suggesting that the defect observed in the developing extraembryonic tissue is caused by a very specific loss of transcriptional activity regulated by Smad1. The data further demonstrate that although highly similar structurally, Smad proteins are not functionally homologous (Lechleider, 2001).

The bone morphogenetic proteins (BMPs) regulate early embryogenesis and morphogenesis of multiple organs, such as bone, kidney, limbs, and muscle. Smad1 is one of the key signal transducers of BMPs and is responsible for transducing receptor activation signals from the cytoplasm to the nucleus, where Smad1 serves as a transcriptional regulator of various BMP-responsive genes. Based upon the ability of Smad1 to bind multiple proteins involved in proteasome-mediated degradation pathway, whether Smad1 could be a substrate for proteasome was investigated. Smad1 is targeted to proteasome for degradation in response to BMP type I receptor activation. The targeting of Smad1 to proteasome involves not only the receptor activation-induced Smad1 ubiquitination but also the targeting functions of the ornithine decarboxylase antizyme (See Drosophila Ornithine decarboxylase antizyme) and the proteasome beta subunit HsN3. These studies provide the first evidence for BMP-induced proteasomal targeting and degradation of Smad1 and also reveal new players and novel mechanisms involved in this important aspect of Smad1 regulation and function (Gruendler, 2001).

Smad1 binds to multiple proteins involved in proteasome-mediated degradation pathways such as HsN3, antizyme (Az), and ubiquitin. HsN3 is one of the seven subunits of the 20 S proteasome, the catalytic core of the 26 S proteasome. HsN3 was also shown to be involved in the targeting of p105 NF-kappaB subunit to proteasome for processing. Ubiquitin is well known for its role in covalently modifying proteasomal substrates for ubiquitin-dependent degradation. Az is a protein previously known to bind ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis. Interestingly, its physical interaction with ODC is necessary and sufficient for targeting ODC to the 26 S proteasome for ubiquitin-independent degradation. Thus, ubiquitin and Az are two types of proteasome targeting proteins that mark proteins for both ubiquitin-dependent and ubiquitin-independent degradation by the 26 S proteasome. Currently, it is not clear how proteasome recognizes ubiquitinated proteins or Az-bound ODC. The ability of Smad1 to bind to ubiquitin and Az as well as HsN3, which is a proteasome component, suggests an interesting link between Smad1 and the proteasome targeting events involving ubiquitin, Az and HsN3. Studies were carried out to test whether the physical interaction between Smad1 and proteins involved in proteasomal degradation pathways (HsN3, Az, and Ub) may lead to: (1) proteasomal degradation of Smad1 or (2) proteasomal degradation of Smad1 interacting proteins. Concomitant with these studies, recent studies by others in the field have demonstrated several important roles of proteasomal degradation in regulating the protein levels of Smads and Smad-interacting proteins (28-35). In the signaling pathways of BMPs, it has been shown that Smad1 interacts with an ubiquitin E3 ligase, Smurf1, which regulates proteasomal degradation of Smad1 independent of BMP type I receptor activation. This study provides the first evidence that proteasomal degradation of Smad1 is also induced upon the activation by the BMP type I receptor. Furthermore, the data reveal novel roles of two Smad1 interactors, Az and HsN3, in proteasomal targeting and degradation of Smad1, in addition to Smad1 ubiquitination (Gruendler, 2001 and references therein).

Convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis

Integration of diverse signaling pathways is essential in development and homeostasis for cells to interpret context-dependent cues. BMP and MAPK signaling converge on Smads, resulting in differential phosphorylation. To understand the physiological significance of this observation, Smad1 mutant mice were generated carrying mutations that prevent phosphorylation of either the C-terminal motif required for BMP downstream transcriptional activation (Smad1C mutation) or of the MAPK motifs in the linker region (Smad1L mutation). Smad1C/C mutants recapitulate many Smad1-/- phenotypes, including defective allantois formation and the lack of primordial germ cells (PGC), but also show phenotypes that are both more severe (head and branchial arches) and less severe (allantois growth) than the null. Smad1L/L mutants survive embryogenesis but exhibit defects in gastric epithelial homeostasis correlated with changes in cell contacts, actin cytoskeleton remodeling, and nuclear ß-catenin accumulation. In addition, formation of PGCs is impaired in Smad1L/L mutants, but restored by allelic complementation in Smad1C/L compound mutants. These results underscore the need to tightly balance BMP and MAPK signaling pathways through Smad1 (Aubin, 2004).

Histological analysis reveals that the cytology of the Smad1L/L gastric mucosa was perturbed. The stomach of rodents consists of a proximal keratinized epithelium and a distal glandular mucosa. The glandular stomach is subdivided in three zones: a proximal zymogenic, a middle mucoparietal, and a distal pure mucus zone. In the zymogenic zone, the four main cell types show a stereotyped distribution: mucus-producing and zymogenic cells are found in the upper third and at the base of the unit, respectively, whereas parietal and enteroendocrine cells are distributed along the entire length. A common stem cell progenitor located in the isthmus repopulates each unit. In the Smad1L/L stomach, all the expected cell types were represented albeit with variations in their relative proportion. The zymogenic cells recognizable by their strong affinity for hematoxylin were severely depleted in linker mutants compared with wild-type samples. In addition, the parietal cells were more numerous in mutant compared with wild-type stomachs, as revealed by immunostaining with the H+K+ATPase proton pump antibody specific for this cell type. Mucous-producing and enteroendocrine cells were not substantially affected. Stomach morphogenesis and primordial gastric unit formation was similar in wild-type and Smad1L/L mutants (tested at E13.5, E18.5, and postnatal day 0). The altered cellularity of Smad1L/L gastric epithelium was not associated with altered specification of the stomach epithelium into an intestinal identity, as revealed at P0 and in adults by the absence of alkaline phosphatase staining, a hallmark of intestinal transformation. Thus, preventing MAPK phosphorylation of Smad1 alters stomach homeostasis (Aubin, 2004).

The analysis of the Smad1 linker phenotype provides the first evidence for a role of integrating BMP and MAPK signaling in epithelial homeostasis. Rendering the Smad1 protein resistant to MAPK-mediated phosphorylation alters homeostasis of the gastric epithelium as reflected by the increased parietal cells and decreased zymogenic cell populations. This characterization provides some hints on how MAPK signals affect homeostasis via Smad1. Smad1 is expressed at high levels in parietal cells, a cell population that is expanded in Smad1L/L mutants. Parietal cell depletion experiments in transgenic mice have demonstrated that this cell type influences decision-making among gastric epithelial cell precursors and modulates the migration-associated terminal differentiation programs of the pit (mucous-producing) and zymogenic lineages. Changes in the parietal cell population in Smad1L/L mutants are therefore most likely to impede this migration-associated differentiation and underlie the perturbed homeostasis. However, a cell-autonomous role in the differentiation of zymogenic cells cannot be excluded because Smad1 is also expressed at low levels in these cells (Aubin, 2004).

The severity of the stomach alteration in some of the Smad1L/- mutants unveils the importance of fine-tuning BMP signaling during organogenesis. Stomach morphogenesis is known to rely on several signaling cascades, and requires integration of TGFß and FGF signals. The source of MAPK signaling that is involved in this process remains to be identified, but FGF10 and its cognate receptor FGFR2b constitute potential candidates. The glandular stomach is particularly sensitive to the action of BMP and FGF signaling; they are both essential for its formation . This potential scenario is reminiscent of the role of FGF/IGF signaling in antagonizing BMP action in neural development in Xenopus. The extent of the defect on both the epithelium and the muscular layer in Smad1L/- stomach, however, may indicate that cell autonomous as well as non-cell-autonomous factors are involved in this phenotype (Aubin, 2004).

Taken together, this study underscores the importance of fine-tuning the balance of BMP and MAPK signaling through Smad1 in a physiological context. The unforeseen germ-cell and gastric epithelial phenotype, as well as the cellular consequences of the linker mutation, raise interesting questions about the underlying mechanisms of BMP-MAPK cross-talk. They also support the notion that MAPK-dependent Smad1 phosphorylation may not only serve to inhibit BMP signaling but may serve other important cellular functions as well. An outstanding issue remains to identify the source of MAPK signaling in affected tissues. Another interesting aspect is how Smad1 linker phosphorylation impinges on Wnt signaling through ß-catenin. The Smad1C and Smad1L mutant lines provide useful tools to tackle these questions and to further dissect the functional significance of integrating diverse signaling pathways (Aubin, 2004).

Cytoplasmic-Nuclear Shuttling of Smads

Upon transforming growth factor beta (TGF-beta) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, the kinetics of Smad nucleocytoplasmic shuttling was studied in a quantitative manner in vivo. Rate constants were obtained for import and export of Smad2; it was shown that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. TGF-beta-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-beta-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-beta-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties (Schmierer, 2005; full text of article).

Smad transcription factors are key signal transducers for the TGF-beta/BMP family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-beta and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. This study provides functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes (Knockaert, 2006; full text of article).

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. This study found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, this study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation (Duan, 2006; full text of article).

Non-canonical Wnt signalling involving TAK1 (TGF-beta-activated kinase) and NLK (Nemo-like kinase) and MAPK

The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and C. elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. This study shows that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway (Ishitani, 1999).

Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor [beta(2)AR-Rfz-2] containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor [beta(2)AR] and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling (Ishitani, 2003a; full text of article).

The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. A specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. This study investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. It was found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity (Ishitani, 2003b; full text of article).

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. This study reports that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps (Kanei-Ishii, 2004; full text of article).

Genetic studies on endoderm-mesoderm specification in C. elegans have demonstrated a role for several Wnt cascade components as well as for a MAPK-like pathway in this process. The latter pathway includes the MAPK kinase kinase-like MOM-4/Tak1, its adaptor TAP-1/Tab1, and the MAPK-like LIT-1/Nemo-like kinase. A model has been proposed in which the Tak1 kinase cascade counteracts the Wnt cascade at the level of beta-catenin/TCF phosphorylation. In this model, the signal that activates the Tak1 kinase cascade is unknown. As an alternative explanation of these genetic data, whether Tak1 is directly activated by Wnt was explored. It was found that Wnt1 stimulation results in autophosphorylation and activation of MOM-4/Tak1 in a TAP-1/Tab1-dependent fashion. Wnt1-induced Tak1 stimulation activates Nemo-like kinase, resulting in the phosphorylation of TCF. These results combined with the genetic data from C. elegans imply a mechanism whereby Wnt directly activates the MOM-4/Tak1 kinase signaling pathway. Thus, Wnt signal transduction through the canonical pathway activates beta-catenin/TCF, whereas Wnt signal transduction through the Tak1 pathway phosphorylates and inhibits TCF, which might function as a feedback mechanism (Smit, 2004; full text of article).

Smads and integration of signaling pathways in the nucleus

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Mothers against dpp: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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