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G Weinmaster

Publications and source records attributed to G Weinmaster.

At least 55 records · Page 3Linked to original sources
PubMed1996

Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3 genes identify ligand-receptor pairs that may function in neural development.

Notch genes encode receptors for a signaling pathway that regulates neurogenesis. The DSL (Delta/Serrate/lag-2) genes encode ligands that bind and activate Notch. In situ hybridization was used to determine the spatiotemporal expression of Notch1, Notch2, and Notch3, and the DSL ligands, Jagged and Delta 1, in an effort to identify potential ligand-receptor pairs that function during development of the rat nervous system. Here we describe both distinct and overlapping expression patterns for these genes in neural progenitors that form both the central and the peripheral nervous systems. The punctate expression patterns we detected for Jagged and Delta 1 are consistent with their role in mediating lateral inhibition, a process proposed to regulate neural determination. Furthermore, within the ventricular zone of the neural tube and retina, Jagged and Delta 1 were expressed in complementary regions, suggesting that different DSL-Notch combinations may direct the development of distinct neural subtypes.

Animals↗
PubMed1996

Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway.

Notch controls cell fate by inhibiting cellular differentiation, presumably through activation of the transcriptional regulator human C promoter Binding Factor (CBF1), which transactivates the hairy and Enhancer of split (HES-1) gene. However, we describe constitutively active forms of Notch1, which inhibit muscle cell differentiation but do not interact with CBF1 or upregulate endogenous HES-1 expression. In addition, Jagged-Notch interactions that prevent the expression of muscle cell specific genes do not involve the upregulation of endogenous HES-1. In fact, exogenous expression of HES-1 in C2C12 myoblasts does not block myogenesis. Our data demonstrate the existence of a CBF1-independent pathway by which Notch inhibits differentiation. We therefore propose that Notch signaling activates at least two different pathways: one which involves CBF1 as an intermediate and one which does not.

Animals↗
PubMed1995

Jagged: a mammalian ligand that activates Notch1.

Here we report the isolation of a rat cDNA clone, Jagged, which we show encodes a ligand for vertebrate Notch. Our conclusion is based on three observations. First, sequence analysis reveals substantial homology between Jagged and invertebrate ligands for the LIN-12/Notch proteins. Second, in situ hybridization of rat embryos identifies both distinct and overlapping patterns of gene expression for Jagged with those for Notch1, Notch2, and Notch3. Finally, the biological activity of Jagged was tested using a cell culture assay in which Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Our data support the hypothesis that Notch-ligand interactions function in maintaining mammalian cells in an undifferentiated state.

Amino Acid Sequence↗
PubMed1994

Notch1 is essential for postimplantation development in mice.

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell fate determination during embryonic and larval development. This gene is evolutionarily conserved, and Notch homologs have been cloned from several vertebrate species. To examine the in vivo role of the Notch1 gene, a mouse homolog of Notch, a mutation was introduced by targeted disruption in embryonic stem cells, and these cells were used to generate mutant mice. Intercrosses of animals heterozygous for the Notch1 mutation yielded no live-born homozygous mutant offspring. Homozygous mutant embryos died before 11.5 days of gestation. Morphological and histological analysis of the homozygous mutant embryos indicated that pattern formation through the first nine days of gestation appeared largely normal. However, histological analysis of mutant embryos subsequent to this stage revealed widespread cell death. Death of mutant embryos did not appear to be attributable to defects in placentation or vascularization. Examination of the RNA expression pattern of the Notch2 gene, another Notch gene family member, indicated that it partially overlapped the Notch1 expression pattern. Genetic analysis of the Notch1 mutation also demonstrated that it was not allelic to a mouse mutation described previously, Danforth's short tail (Sd). These results demonstrate that the Notch1 gene plays a vital role during early postimplantation development in mice.

Animals↗
PubMed1992

Notch2: a second mammalian Notch gene.

Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.

Amino Acid Sequence↗
PubMed1991

A homolog of Drosophila Notch expressed during mammalian development.

Drosophila Notch and the related Caenorhabditis elegans proteins lin-12 and glp-1 function as mediators of local cell-cell interactions required for cell-fate decisions during invertebrate development. To investigate the possibility that similar proteins play determinative roles during mammalian development, we isolated cDNA clones encoding rat Notch. The deduced amino acid sequence of this protein contains 36 epidermal growth factor (EGF)-like repeats, and is remarkably similar in both its extracellular and cytoplasmic domains to the sequence of Xenopus Xotch and Drosophila Notch. In the developing central nervous system, in situ hybridisation analyses revealed that Notch transcripts were dramatically restricted to the ventricular proliferative zones of embryonic neuroepithelia. Notch was also strongly expressed during development of non-neural tissues, such as hair follicles and tooth buds, whose correct differentiation requires epithelial-mesenchymal interactions. These data support the hypothesis that Notch plays an essential role in mammalian development and pattern formation that closely parallels its role in the development of invertebrates.

Amino Acid Sequence↗
PubMed1990

Expression and activity of the POU transcription factor SCIP.

POU proteins have been shown to transcriptionally active cell-specific genes and to participate in the determination of cell fate. It is therefore thought that these proteins function in development through the stable activation of genes that define specific developmental pathways. Evidence is provided here for an alternative mode of action. The primary structure of SCIP, a POU protein expressed by developing Schwann cells of the peripheral nervous system, was deduced and SCIP activity was studied. Both in normal development and in response to nerve transection, SCIP expression was transiently activated only during the period of rapid cell division that separates the premyelinating and myelinating phases of Schwann cell differentiation. In cotransfection assays, SCIP acted as a transcriptional repressor of myelin-specific genes.

Amino Acid Sequence↗
PubMed1990

Cell-specific cyclic AMP-mediated induction of the PDGF receptor.

Cyclic AMP (cAMP) cooperates with a wide variety of polypeptide growth factors to synergistically stimulate the proliferation of many vertebrate cell types. However, the cellular mechanisms underlying these cooperative interactions are for the most part unknown. We have identified one such mechanism by observing that (i) cultured rat Schwann cells proliferate in response to platelet-derived growth factor (PDGF) only if simultaneously cultured in the presence of agents that elevate intracellular cAMP and (ii) this unmasked PDGF response is accounted for by a dramatic cAMP-mediated induction of PDGF receptor mRNA and protein. cAMP-mediated induction of the PDGF receptor results in enhanced, ligand dependent receptor autophosphorylation, and in enhanced PDGF activation of c-fos gene expression. In addition, this induction is unique to those cells, such as Schwann cells, for which cAMP is itself mitogenic. These results indicate that the synergistic proliferative effect obtained from the combination of cAMP and polypeptide growth factors may in large result from the cAMP-mediated induction of growth factor receptors.

Animals↗
PubMed1989

SCIP: a glial POU domain gene regulated by cyclic AMP.

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.

Amino Acid Sequence↗
PubMed1987

Modifications of tumor histology by point mutations in the v-fps oncogene: possible role of extracellular matrix.

Fujinami sarcoma virus (FSV) encodes a protein-tyrosine kinase, p130gag-fps, whose enzymatic activity and ability to transform cultured cells to a neoplastic phenotype are reduced by substitution of the major autophosphorylation site tyrosine-1073 with other amino acids. We compared the histopathology of tumors formed in syngeneic immunocompetent rats by Rat-2 cells and by Rat-2 cells transformed in culture with (a) wild type (wt) FSV, (b) mutant FSV where the codon for tyrosine-1073 of p130gag-fps had been changed to codons for phenylalanine or serine, and (c) a revertant FSV, genotypically identical to wt FSV, in which the codon for tyrosine-1073 had been restored. Latency periods from cell inoculation to tumor formation were 12-29 weeks with Rat-2 cells, 6-8 weeks with mutant-transformed Rat-2 cells, and 2-4 weeks with wt FSV- and revertant FSV-transformed Rat-2 cells. Untransfected Rat-2 cells formed tumors that histologically resembled low grade fibrosarcomas or fibromas and were characterized by uniform fusiform cells in parallel arrays with a prominent collagenous stroma. The growth pattern of tumors produced by mutant FSV-transformed cells was generally similar, although cellular forms and intercellular organization were less uniform. In contrast, Rat-2 cells transformed with either wt FSV or revertant FSV produced tumors that resembled highly malignant sarcomas and were composed of diffuse sheets of pleomorphic, disorganized cells and stroma rich in hyaluronate but poor in fibrous components. Local invasion occurred in 25% of tumors produced by Rat-2 cells and in 53 and 36% of tumors formed by mutant FSV- and wt FSV-transformed cells, respectively. In culture, Rat-2 cells and mutant FSV-transformed cells produced fibrillar pericellular matrices of collagen I and fibronectin. From 5 to 15% of protein secreted by these cells was collagen. Cultures of wt FSV- and revertant FSV-transformed cells lacked collagen and fibronectin matrices and collagen secretion was reduced to 0-2%. These results show that clinically relevant histological characteristics of malignant tumors can correlate with single amino acid substitutions previously shown to affect the enzymatic activity and transforming ability of an oncogenic protein tyrosine kinase. The mechanisms underlying some of the histological differences in this system may be related to differences in the production of extracellular matrix components among the transformed cells.

Animals↗
PubMed1986

Protein kinase activity of FSV (Fujinami sarcoma virus) P130gag-fps shows a strict specificity for tyrosine residues.

A number of oncogenic viruses encode transforming proteins with protein kinase activities apparently specific for tyrosine residues. Recent evidence has raised questions as to the substrate specificity of these kinases in general and the physiological relevance of tyrosine phosphorylation in particular. The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) is strongly phosphorylated at 2 tyrosine residues in FSV-transformed cells of which 1 (Tyr-1073) is also the major site of P130gag-fps intermolecular autophosphorylation in vitro. We have investigated the specificity of the protein kinase activity intrinsic to FSV P130gag-fps by using site-directed mutagenesis to change the codon for Tyr-1073 to those for the other commonly phosphorylated hydroxyamino acids, serine and threonine. This approach has some advantages over the use of synthetic peptides to define protein kinase recognition sites in that the protein containing the altered target site can be expressed in intact cells. In addition it allows higher order as well as primary structure of the enzyme recognition site to be considered. Neither serine nor threonine were phosphorylated when substituted for tyrosine at position 1073 of P130gag-fps indicating a stringent specificity for tyrosine as a substrate of the P130gag-fps protein kinase autophosphorylating activity. Consistent with the suggestion that tyrosine phosphorylation is of functional significance we find that these and other FSV Tyr-1073 mutants have depressed enzymatic and oncogenic capacities.

Amino Acid Sequence↗
PubMed1986

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.

Adenosine Triphosphate↗
PubMed1984

Mutagenesis of Fujinami sarcoma virus: evidence that tyrosine phosphorylation of P130gag-fps modulates its biological activity.

The 130 kd transforming protein of Fujinami sarcoma virus (FSV P130gag -fps) possesses a tyrosine-specific protein kinase activity and is itself phosphorylated at several tyrosine and serine residues in FSV-transformed cells. We have used oligonucleotide-directed mutagenesis of the FSV genome to change the TAT codon for tyrosine (1073), the major site of P130gag -fps phosphorylation, to a TTT codon for phenylalanine that cannot be phosphorylated. This mutant FSV induces the transformation of rat-2 cells but with a long latent period as compared with wild-type FSV. The P130gag -fps protein encoded by the mutant retains the ability to phosphorylate tyrosine, but is five times less active as a kinase in vitro than wild-type FSV P130gag -fps. These data indicate that tyrosine phosphorylation stimulates the biochemical and biological activities of FSV P130gag -fps, and they set a precedent for the ability of this amino acid modification to modulate protein function.

Amino Acid Sequence↗
PubMed1983

Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein.

The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.

Avian Sarcoma Viruses↗
PubMed1982

Localization and characterization of phosphorylation sites of the Fujinami avian sarcoma virus and PRCII virus transforming proteins.

Fujinami sarcoma virus (FSV) and PRCII are avian sarcoma viruses which share cellularly derived v-fps transforming sequences. The FSV P140gag-fps gene product is phosphorylated on three distinct tyrosine residues in transformed cells or in an in vitro kinase reaction. Three variants of FSV, and the related virus PRCII which lacks about half of the v-fps sequence found in FSV, encode gene products which are all phosphorylated at tyrosine residues contained within identical tryptic peptides. This indicates a stringent conservation of amino acid sequence at the tyrosine phosphorylation sites which presumably reflects the importance of these sites for the biologic activity of the transforming proteins. Under suitable conditions the proteolytic enzymes p15 and V8 protease each introduce one cut into FSV P140, p15 in the N-terminal gag-encoded region and V8 protease in the middle of the fps-encoded region. Using these enzymes we have mapped the major site of tyrosine phosphorylation to the C-terminal end of the fps region of FSV P140gag-fps. A second tyrosine phosphorylation site is found in the fps region of FSV P140 isolated from transformed cells, and a minor tyrosine phosphorylation site is found in the N-terminal gag-encoded region. Our results suggest that the C-terminal fps-encoded region is required for expression of the tyrosine-specific protein kinase activity.

Avian Sarcoma Viruses↗
PubMed1982

Herpesvirus-induced "early" glycoprotein: characterization and possible role in immune cytolysis.

Glycoprotein GVP-11 (molecular weight, 71,500), induced by bovine herpesvirus type 1, was detected on the external surface of infected cells. It could be categorized as an "early" or "beta" class protein since it was synthesized early in the infectious process and its expression was not dependent upon prior viral DNA replication in the infected cells. Monoclonal antibodies directed against GVP-11 immunoprecipitated that glycoprotein and some low-molecular-weight polypeptides from infected cells labeled with either [35S]methionine or [3H]glucosamine. Immunoprecipitation of extracts from cells surface labeled with 125I yielded an additional 138,000-molecular-weight polypeptide. Tunicamycin- or bromovinyl deoxyuridine-treated infected cells yielded polypeptides that were smaller in size than corresponding glycoproteins in untreated cells. Tunicamycin-sensitive glycosylation appeared to be necessary for the expression of the glycoproteins on the surface of the infected cells. The monoclonal antibodies directed against GVP-11 and serum from an immune cow could participate in antibody- and complement-mediated immunocytolysis of infected cells, and this immunocytolysis could be enhanced by arresting cells in the early phase of viral gene expression by treatment with inhibitors of viral DNA synthesis.

Animals↗