Biomedical subjects
Publications and source records attributed to L Attisano.
The discovery of the Mothers against dpp (Mad) gene in Drosophila has opened a window on an entirely unique signalling pathway that functions to mediate responses to the tumour growth factor beta (TGF beta) superfamily. This pathway, which is comprised of a family of proteins related to Mad, acts to convey signals directly from TGF beta receptors to the nucleus and is implicated in the pathogenesis of human diseases.
We identify a mammalian forkhead domain protein, FAST2, that is required for induction of the goosecoid (gsc) promoter by TGF beta or activin signaling. FAST2 binds to a sequence in the gsc promoter, but efficient transcriptional activation and assembly of a DNA-binding complex of FAST2, Smad2, and Smad4 requires an adjacent Smad4 site. Smad3 is closely related to Smad2 but suppresses activation of the gsc promoter. Inhibitory activity is conferred by the MH1 domain, which unlike that of Smad2, binds to the Smad4 site. Through competition for this shared site, Smad3 may prevent transcription by altering the conformation of the DNA-binding complex. Thus, we describe a mechanism whereby Smad2 and Smad3 positively and negatively regulate a TGF beta/activin target gene.
Mothers against dpp (Mad) mediates Decapentaplegic (DPP) signaling throughout Drosophila development. Here we demonstrate that Medea encodes a MAD-related protein that functions in DPP signaling. MEDEA is most similar to mammalian Smad4 and forms heteromeric complexes with MAD. Like dpp, Medea is essential for embryonic dorsal/ventral patterning. However, Mad is essential in the germline for oogenesis whereas Medea is dispensable. In the wing primordium, loss of Medea most severely affects regions receiving low DPP signal. MEDEA is localized in the cytoplasm, is not regulated by phosphorylation, and requires physical association with MAD for nuclear translocation. Furthermore, inactivating MEDEA mutations prevent nuclear translocation either by preventing interaction with MAD or by trapping MAD/MEDEA complexes in the cytosol. Thus MAD-mediated nuclear translocation is essential for MEDEA function. Together these data show that, while MAD is essential for mediating all DPP signals, heteromeric MAD/MEDEA complexes function to modify or enhance DPP responses. We propose that this provides a general model for Smad4/MEDEA function in signaling by the TGF-beta family.
Mothers against Dpp-related or Smad proteins are essential components of serine/threonine kinase receptor signaling pathways that are regulated by phosphorylation. Recently, it was demonstrated that Smad2 interacts transiently with and is a direct substrate of 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. In this report, we show that 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.
The bone morphogenetic proteins, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic protein-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% (P = 0.001). In contrast, 100-fold greater concentrations of OP-1 (28 nM) decreased explant growth by 10% (P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (< 0.5 nM) increased the number of tubular structures formed by 28 +/- 5% (P = 0.01), whereas concentrations > 0.5 nM decreased that number by 22 +/- 8% (P = 0.02). All concentrations of BMP-2 (0.05-10 nM) were inhibitory (maximum inhibition at 10 nM of 88 +/- 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length (P = 0.003) and the number of branch points/structure (3.2-fold increase, P = 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an approximately 80-kDa protein using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 microM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 +/- 1% (P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.
Although mothers against dpp (MAD) and its related proteins (MADR) are believed to be important components of the cell signaling pathway for the transforming growth factor beta (TGFbeta) superfamily, the presence and regulation of these signaling molecules in ovarian cells by TGFbeta is not known. In the present studies, we have examined the presence of MADR2 and MADR1, two members of the MADR family, in hen granulosa cells at different stages of follicular development. The influence of TGFbeta in vitro on their expression was assessed, particularly in the context of TGFbeta-induced down-regulation of cytosolic phospholipase A2 (cPLA2), a key enzyme in the biosynthesis of eicosanoids. We have demonstrated for the first time the presence of MADR2 and MADR1 in hen granulosa cells at different stages of follicular development. The expression of MADR2, but not of MADR1, was up-regulated by TGFbeta in vitro in a concentration- and time-dependent manner. Granulosa cell MADR2 expression was maximal during early stages of follicular development, when the granulosa cell cPLA2 system is most responsive to the growth factor. The changes in MADR2 expression were accompanied by reciprocal alterations in the expression of cPLA2. These findings are consistent with the hypothesis that homologous up-regulation of MADR2 in granulosa cells may be an important determinant in its follicular stage-specific responsiveness to TGFbeta and possibly in the suppression of cPLA2 gene transcription by the growth factor.
Mothers against dpp (Mad) is the prototype of a family of genes required for signaling by TGF-beta related ligands. In Drosophila, Mad is specifically required in cells responding to Decapentaplegic (DPP) signals. We further specify the role of Mad in DPP-mediated signaling by utilizing tkvQ199D, an activated form of the DPP type I receptor serine-threonine kinase thick veins (tkv). In the embryonic midgut, tkvQ199D mimics DPP-mediated inductive interactions. Homozygous Mad mutations block signaling by tkvQ199D. Appropriate responses to signaling by tkvQ199D are restored by expression of MAD protein in DPP-target cells. Endogenous MAD is phosphorylated in a ligand-dependent manner in Drosophila cell culture. DPP overexpression in the embryonic midgut induces MAD nuclear accumulation; after withdrawal of the overexpressed DPP signal, MAD is detected only in the cytoplasm. However, in three different tissues and developmental stages actively responding to endogenous DPP, MAD protein is detected in the cytoplasm but not in the nucleus. From these observations, we discuss possible roles for MAD in a DPP-dependent serine-threonine kinase signal transduction cascade integral to the proper interpretation of DPP signals.
MAD-related (MADR) proteins are essential intracellular components of TGFbeta signaling pathways and are regulated by phosphorylation. Here, we demonstrate that MADR2 and not the related protein DPC4 transiently interacts with the TGFbeta 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 generates a dominant negative MADR2 that blocks TGFbeta-dependent transcriptional responses, stably associates with receptors, and fails to accumulate in the nucleus in response to TGFbeta signaling. Thus, transient association and phosphorylation of MADR2 by the TGFbeta receptor is necessary for nuclear accumulation and initiation of signaling.
The MAD-related (MADR) family of proteins are essential components in the signaling pathways of serine/threonine kinase receptors for the transforming growth factor beta (TGFbeta) superfamily. We demonstrate that MADR2 is specifically regulated by TGFbeta and not bone morphogenetic proteins. The gene for MADR2 was found to reside on chromosome 18q21, near DPC4, another MADR protein implicated in pancreatic cancer. Mutational analysis of MADR2 in sporadic tumors identified four missense mutations in colorectal carcinomas, two of which display a loss of heterozygosity. Biochemical and functional analysis of three of these demonstrates that the mutations are inactivating. These findings suggest that MADR2 is a tumor suppressor and that mutations acquired in colorectal carcinomas may function to disrupt TGFbeta signaling.
noggin is expressed in the Spemann organizer region of the Xenopus embryo and can promote dorsal cell fates within the mesoderm and neural development within the overlying ectoderm. Here, we show that noggin promotes ventral development in Drosophila, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. We utilize constitutively active forms of the dpp receptors to demonstrate that noggin blocks dpp signaling upstream of dpp receptor activation. These results suggest a mechanistic basis for the action of Spemann's organizer during Xenopus development and provide further support for the conservation of dorsal-ventral patterning mechanisms between arthropods and chordates.
Components of the signaling pathways that lie downstream of Ser/Thr kinase receptors and are required for signaling by the TGF beta superfamily have been poorly defined. The Drosophila gene Mothers against dpp (MAD) and the C. elegans sma genes are implicated in these signaling pathways. We show that MAD functions downstream of DPP receptors and is required for receptor signaling. Phosphorylation of MADR1, a human homolog of MAD, is tightly regulated and rapidly induced by BMP2, but not TGF beta or activin. This phosphorylation is necessary for function, since a point mutant that yields a null phenotype in Drosophila is not phosphorylated. BMP2 treatment results in accumulation of MADR1 in the nucleus. MAD proteins may thus define a novel class of signaling molecules with nuclear function in Ser/Thr kinase receptor signaling pathways.
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Transforming growth factor-beta (TGF beta) superfamily members exert their diverse biological effects through their interaction with heteromeric receptor complexes of transmembrane serine/threonine kinases. Both components of the receptor complex, known as receptor I and receptor II are essential for signal transduction. The composition of these complexes can vary significantly due to the promiscuous nature of the ligands and the receptors, and this diversity of interactions can yield a variety of biological responses. Several receptor interacting proteins and potential mediators of signal transduction have now been identified. Recent advances, particularly in our understanding of the function of Mothers against dpp-related (MADR) proteins, are providing new insights into how the TGF beta superfamily signals its diverse biological activities.
Hereditary haemorrhagic telangiectasia, or Osler-Rendu-Weber (ORW) syndrome, is an autosomal dominant vascular dysplasia. So far, two loci have been demonstrated for ORW. Linkage studies established an ORW locus at chromosome 9q3; endoglin was subsequently identified as the ORW1 gene. A second locus, designated ORW2, was mapped to chromosome 12. Here we report a new 4 cM interval for ORW2 that does not overlap with any previously defined. A 1.38-Mb YAC contig spans the entire interval. It includes the activin receptor like kinase 1 gene (ACVRLK1 or ALK1), a member of the serine-threonine kinase receptor family expressed in endothelium. We report three mutations in the coding sequence of the ALK1 gene in those families which show linkage of the ORW phenotype to chromosome 12. Our data suggest a critical role for ALK1 in the control of blood vessel development or repair.
Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals.
Transforming growth factor-beta (TGF-beta) signaling in Mv1Lu lung epithelial cells requires coexpression of TGF-beta receptors I (T beta R-I) and II (T beta R-II), two distantly related transmembrane serine/threonine kinases that form a heteromeric complex upon ligand binding. Here, we examine the formation of TGF-beta receptor homo-oligomers and their possible contribution to signaling. T beta R-I can contact ligand bound to T beta R-II, but not ligand free in the medium, and thus cannot form ligand-induced homo-oligomers. T beta R-II, which binds ligand on its own, formed oligomeric complexes when overexpressed in transfected COS cells. However, these complexes were largely ligand-independent and involved immature receptor protein. Since ligand-induced homo-oligomers could not be obtained with the wild-type TGF-beta receptors, we studied receptor cytoplasmic domain homo-oligomerization by using receptor chimeras. The extracellular domain of T beta R-II was fused to the transmembrane and cytoplasmic domains of T beta R-I, yielding T beta R-II/I, and the extracellular domain of T beta R-I was fused to the transmembrane and cytoplasmic domains of T beta R-II, yielding T beta R-I/II. When contransfected with wild-type receptors and exposed to ligand, T beta R-II/I formed a complex with T beta R-I, and T beta R-I/II formed a complex with T beta R-II, thus yielding complexes with homologous cytoplasmic domains. T beta R-II/I transfected alone or with T beta R-I did not restore TGF-beta responsiveness in T beta R-II-defective cell mutants. Furthermore, T beta R-II/I acted in a dominant negative fashion, inhibiting restoration of TGF-beta responsiveness by a cotransfected T beta R-II in T beta R-II-defective cells and by a cotransfected T beta R-I in T beta R-I-defective cells. Similarly, T beta R-I/II transfected alone or with T beta R-II did not restore TGF-beta responsiveness and acted in a dominant negative fashion against T beta R-I. Together with previous genetic and biochemical evidence, these results suggest that TGF-beta mediates transcriptional and antiproliferative responses through the heteromeric T beta R-I.T beta R-II complex and not through homo-oligomeric T beta R-I or T beta R-II complexes.
Activin is a potent inducer of mesoderm in frog embryos. We showed previously that in the mouse, activin beta A is expressed in the uterine decidua near the embryo before and during the first appearance of mesoderm (E4.5-E6.5). Here, using Northern blotting and in situ hybridization, we show that mouse oocytes, E6.5 and E7.5 embryos, and E6.5 and E7.5 decidua contain mRNAs for both activin receptors type II and IIB. The expression of activin receptor type IIB is particularly strong in embryonic ectoderm apparent at E5.5 and continuing through E8.5. These results support the hypothesis that activin derived from the decidua promotes development of mesoderm in the period E5.5-E6.5.
Transforming growth factor-beta (TGF-beta) signals by contacting two distantly related transmembrane serine/threonine kinases called receptors I and II. The role of these molecules in signalling has now been determined. TGF-beta binds directly to receptor II, which is a constitutively active kinase. Bound TGF-beta is then recognized by receptor I which is recruited into the complex and becomes phosphorylated by receptor II. Phosphorylation allows receptor I to propagate the signal to downstream substrates. This provides a mechanism by which a cytokine can generate the first step of a signalling cascade.