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R Derynck

Publications and source records attributed to R Derynck.

At least 55 records · Page 3Linked to original sources
PubMed1997

A kinase subdomain of transforming growth factor-beta (TGF-beta) type I receptor determines the TGF-beta intracellular signaling specificity.

Transforming growth factor-beta (TGF-beta) signals through a heteromeric complex of related type I and type II serine/threonine kinase receptors. In Mv1Lu cells the type I receptor TbetaRI mediates TGF-beta-induced gene expression and growth inhibition, while the closely related type I receptors Tsk7L and TSR1 are inactive in these responses. Using chimeras between TbetaRI and Tsk7L or TSR1, we have defined the structural requirements for TGF-beta signaling by TbetaRI. The extracellular/transmembrane or cytoplasmic domains of TbetaRI and Tsk7L were functionally not equivalent. The juxtamembrane domain, including the GS motif, and most regions in the kinase domain can functionally substitute for each other, but the alphaC-beta4-beta5 region from kinase subdomains III to V conferred a distinct signaling ability. Replacement of this sequence in TbetaRI by the corresponding domain of Tsk7L inactivated TGF-beta signaling, whereas its introduction into Tsk7L conferred TGF-beta signaling. The differential signaling associated with this region was narrowed down to a sequence of eight amino acids, the L45 loop, which is exposed in the three-dimensional kinase structure and diverges highly between TbetaRI and Tsk7L or TSR1. Replacement of the L45 sequence in Tsk7L with that of TbetaRI conferred TGF-beta responsiveness to the Tsk7L cytoplasmic domain in Mv1Lu cells. Thus, the L45 sequence between kinase subdomains IV and V specifies TGF-beta responsiveness of the type I receptor.

Activin Receptors, Type I↗
PubMed1997

Impaired lung branching morphogenesis in the absence of functional EGF receptor.

The mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome.

Animals↗
PubMed1997

The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues.

The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.

Adenosine Triphosphate↗
PubMed1997

The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function.

BACKGROUND: The invertebrate and vertebrate Smad proteins have recently been identified as important mediators of the responses to transforming growth factor beta (TGF-beta) and related factors. We have previously shown that Smad3 and Smad4 (the product of the tumor suppressor gene DPC 4) strongly synergize as mediators of TGF-beta signaling, and that inactive carboxy-terminally truncated mutants of either Smad act as dominant-negative inhibitors of the natural TGF-beta response. The finding that Smad4, unlike Smad3, does not interact with the TGF-beta receptor, coupled with the distinct structural features of Smad4, raises the possibility that Smad4 cooperates not only with Smad3, but also with Smad1 and Smad2 to mediate signaling by TGF-beta family members. RESULTS: Overexpression of Smad4 in 'animal caps' taken from Xenopus embryos induced both ventral and dorsal mesoderm, thereby mimicking the effects of TGF-beta family members - bone morphogenetic protein-2 (BMP-2) or BMP-4 and activin, respectively. Low levels of Smad4 mRNA coinjected with Smad1 or Smad2 mRNA also synergized to induce ventral or dorsal mesoderm, respectively. In addition, Smad4 synergized Smad2, as it does with Smad3, to induce gene expression from the promoter for plasminogen activator inhibitor-1. The carboxy-terminal domains of both Smad3 and Smad4 were required for this synergy. Finally, a short carboxy-terminal truncation of Smad4, previously identified as a mutation of DPC 4 in tumors, blocked nuclear translocation of wild-type Smads 1, 2, 3 and 4, consistent with our observation of a physical interaction between truncated Smad4 and the other Smads. CONCLUSIONS: Our observations indicate that Smad4 cooperates with Smad1, Smad2 and Smad3 to act as a common mediator of signaling by TGF-beta-related factors, and provide a mechanism that explains the dominant-negative interference with receptor signaling that results from expression of the naturally occurring Smad4/DPC 4 truncation mutant.

Animals↗
PubMed1997

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition.

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.

Amino Acid Sequence↗
PubMed1997

Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4.

Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor beta (TGF-beta)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-beta activities and are essential mediators of the natural TGF-beta response. We now show that Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses. Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4. In Smad3, the homomeric interaction is mediated by the same domain. In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize. Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homo- and heteromeric associations. Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homo- and heteromeric interactions. Mutations that interfere with these interactions result in decreased signaling activity. Finally, we evaluated the ability of Smad3 or Smad4 to induce transcriptional activation in yeast. These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells.

Animals↗
PubMed1997

Cellular interpretation of multiple TGF-beta signals: intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads.

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 (BMP), respectively. 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. In this study, we have exploited the well characterized model of Xenopus mesoderm induction 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, we demonstrate that signals provided by activin/BVg1 and BMP modulate each other's activity and that this crosstalk occurs through intracellular mechanisms. In assays using BMP-2/4 and activin/BVg1-specific reporters, we 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 signaling possibly 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, and a dominant negative variant of Smad4 can inhibit both activin/BVg1 and BMP-2/4 mediated signaling Finally, we demonstrate that 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.

Activins↗
PubMed1996

Cysteines 153 and 154 of transmembrane transforming growth factor-alpha are palmitoylated and mediate cytoplasmic protein association.

Transforming growth factor-alpha (TGF-alpha) is synthesized as a transmembrane protein with a highly conserved, short cytoplasmic domain that is rich in cysteines. TGF-alpha is a prototype of a large family of growth factors involved in cell-cell communication. We have shown previously that transmembrane TGF-alpha associates with a kinase activity and two proteins of 106 and 86 kDa. In this study, we have used site-directed mutagenesis of the cytoplasmic domain of TGF-alpha to define the structural requirements for these protein interactions. Whereas the cytoplasmic domain of TGF-alpha was not essential for association with transmembrane p106, deletion of the C-terminal 8 amino acids, including a cysteine pair, abolished the interaction with p86 and greatly reduced the kinase activity associated with transmembrane TGF-alpha. Replacement of these 2 cysteines by serines similarly reduced the association of p86 with transmembrane TGF-alpha. Using a combination of mutational analysis and direct microsequencing, we have determined that this cysteine pair was palmitoylated. We therefore conclude that these cysteines play a critical role in the interaction of TGF-alpha with associated proteins and in the function of this protein complex. The palmitoylation of these cysteines suggests a possibly dynamic role of fatty acid modification in the integrity and function of the transmembrane TGF-alpha complex.

Animals↗
PubMed1996

Intracellular signalling: the mad way to do it.

A family of proteins has been identified that transduce a signal inside cells following binding of a TGF-beta family ligand to its cognate receptor. Like the prototype Drosophila protein Mad, many members of the family play key roles in developmental signalling.

Animals↗
PubMed1996

Induction of myogenesis in mesenchymal cells by MyoD depends on their degree of differentiation.

Expression of a transfected MyoD gene induces myogenic differentiation of most cell types. In this study, we evaluated the ability of an exogenous MyoD gene to induce myogenic conversion in two pairs of matched cell lines with different degrees of differentiation within either the osteoblastic or chondrocytic lineage. We show that osteoblasts and chondrocytes are resistant to the myogenic effects of MyoD alone. However, in their less-differentiated cell line counterparts, MyoD induces expression of muscle-cell-specific markers. Less-differentiated osteoblasts can be made resistant to MyoD-induced myogenic conversion by induction of adipogenic differentiation using dexamethasone. Finally, a dominant positive form of MyoD, one which is tethered to a partner, E47, activates muscle-specific gene expression in osteoblasts. Our results suggest that the response of a cell to MyoD depends on its lineage and its degree of differentiation. Furthermore, commitment of cells to the osteoblastic or chondrocytic lineage may involve inhibition of alternative pathways, such as those leading to myoblastic differentiation. Finally, osteoblasts may express a protein(s) which interferes with the activity of MyoD by inhibiting its association with E proteins. This interference can be overcome by expression of the MyoD-E47 hybrid, suggesting that osteoblasts are otherwise competent to undergo myogenic conversion.

Animals↗
PubMed1996

Receptor-associated Mad homologues synergize as effectors of the TGF-beta response.

Transforming growth factor-beta TGF-beta is the prototype for a family of extracellular proteins that affect cell proliferation and tissue differentiation. TGF-beta-related factors, including BMP-2/4, Dpp and activin, act through two types of serine/threonine kinase receptors which can form a heteromeric complex. However, the mechanism of signal transduction by these receptors is largely unknown. In Drosophila, Mad is required for signalling by Dpp. We have isolated complementary DNAs for four human Mad homologues, one of which, hMAD-4, is identical to DPC-4, a candidate tumour suppressor. hMAD-3 and -4 synergized to induce strong ligand-independent TGF-beta-like responses. When truncated at their carboxy termini, hMAD-3 and -4 act as dominant-negative inhibitors of the normal TGF-beta response. The activity of hMAD-3 and -4 was regulated by the TGF-beta receptors, and hMAD-3 but not hMAD-4 was phosphorylated and associated with the ligand-bound receptor complex. These results define hMAD-3 and -4 as effectors of the TGF-beta response and demonstrate a function for DPCA-4/hMAD-4 as a tumour suppressor.

Activin Receptors, Type I↗
PubMed1996

Ligand-independent activation of transforming growth factor (TGF) beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors.

Transforming growth factor beta (TGF-beta) transduces signals through two related serine/threonine kinase receptors, the type I and type II receptors, which have the ability to interact with each other. In the heteromeric complex, the type II receptor is the primary determinant of ligand binding and phosphorylates the cytoplasmic domain of the type I receptor. Using a chimeric receptor strategy, we and others have shown previously that a functional TGF-beta receptor complex requires heteromerization of both extracellular and intracellular domains of type I and type II receptors. In the current study, we show that overexpression of two receptors carrying a heteromeric combination of cytoplasmic domains resulted in ligand-independent responses, further supporting the functional requirement of the two heterologous cytoplasmic domains in TGF-beta signaling. Furthermore, coexpression of only the cytoplasmic domains of both the type I and II receptors or tethering the type II to the type I cytoplasmic domain activated TGF-beta responses in a ligand-independent manner. In cotransfected COS-1 cells, both cytoplasmic domains are associated with each other. Our results indicate that the cytoplasmic domains of the type I and type II TGF-beta receptors physically and functionally interact with each other in the heteromeric complex.

Animals↗
PubMed1996

Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype.

The development of the skeleton requires the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The activities of these two cell types are likely to be regulated by TGF-beta, which is abundant in bone matrix. We have used transgenic mice to evaluate the role of TGF-beta 2 in bone development and turnover. Osteoblast-specific overexpression of TGF-beta 2 from the osteocalcin promoter resulted in progressive bone loss associated with increases in osteoblastic matrix deposition and osteoclastic bone resorption. This phenotype closely resembles the bone abnormalities seen in human hyperparathyroidism and osteoporosis. Furthermore, a high level of TGF-beta 2 overexpression resulted in defective bone mineralization and severe hypoplasia of the clavicles, a hallmark of the developmental disease cleidocranial dysplasia. Our results suggest that TGF-beta 2 functions as a local positive regulator of bone remodeling and that alterations in TGF-beta 2 synthesis by bone cells, or in their responsiveness to TGF-beta 2, may contribute to the pathogenesis of metabolic bone disease.

Age Factors↗
PubMed1995

Transforming growth factor-beta (TGF-beta)-induced down-regulation of cyclin A expression requires a functional TGF-beta receptor complex. Characterization of chimeric and truncated type I and type II receptors.

Transforming growth factor-beta (TGF-beta) inhibits the proliferation of epithelial cells by altering the expression or function of various components of the cell cycle machinery. Expression of one of these components, cyclin A, is inhibited by TGF-beta treatment. We have identified a 760-base pair fragment of the human cyclin A gene promoter that is sufficient to confer TGF-beta responsiveness. Using this promoter fragment, we have developed a cyclin A-based luciferase reporter assay that quantitates the growth inhibitory effect of TGF-beta in transient transfection assays. This assay was used to determine which domains of the type I (RI) and type II (RII) receptors were required for the antiproliferative effect of TGF-beta. In parallel, the functionality of chimeric receptors, between RI and RII (RI-RII or RII-RI), was tested for TGF-beta effect on gene expression using a reporter assay based on the plasminogen activator inhibitor type 1 (PAI-1) promoter. We found that TGF-beta-induced inhibition of cyclin A expression was absent in RI or RII-deficient Mv1Lu cells and that this response was restored by expression of wild-type type I or type II receptors in these cells. Furthermore, expression of a single chimeric receptor, either RI-RII or RII-RI, did not confer cyclin A regulation by TGF-beta. However, expression of two reciprocal chimeras (RI-RII and RII-RI) resulted in growth inhibition, similarly to wild-type receptors. In addition, chimeric receptors as well as mutant receptors with a deleted cytoplasmic domain and kinase-negative receptors inhibited TGF-beta responsiveness in the cyclin A reporter assay in a dominant negative fashion. Finally, in both receptor types, the juxtamembrane domain preceding the kinase domain was essential for receptor function but the cytoplasmic tail was dispensable. Our results suggest that a functional TGF-beta receptor complex is required for TGF-beta-dependent down-regulation of cyclin A gene expression and illustrate the identical receptor requirements for TGF-beta-induced growth inhibition and gene expression.

Animals↗
PubMed1995

A WD-domain protein that is associated with and phosphorylated by the type II TGF-beta receptor.

Transforming growth factor-beta (TGF-beta) is the prototype for a family of extracellular polypeptides that affect cell proliferation and differentiation, and tissue morphogenesis. TGF-beta signalling is mediated by two types of serine/threonine kinase receptors, the type I and II receptors, which are able to form a heteromeric complex. No cytoplasmic proteins that associate with these receptors in vivo, or are their kinase targets, have yet been described. We have now identified a WD-domain-containing protein, TRIP-1, which specifically associates with the type II TGF-beta receptor in a kinase-dependent way. TRIP-1 does not interact with the type II activin or type I receptors, but associates with the heteromeric TGF-beta receptor complex. TRIP-1 is phosphorylated on serine and threonine by the receptor kinase, strongly suggesting that it has a role in TGF-beta signalling. This is supported by coexpression of TRIP-1 and type II receptor during development. The existence of TRIP-1 homologues in plant and yeast suggests a conserved function in all eukaryotes.

Amino Acid Sequence↗