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M Barbacid

Publications and source records attributed to M Barbacid.

At least 19 recordsLinked to original sources
PubMed1992

Steel factor stimulates the tyrosine phosphorylation of the proto-oncogene product, p95vav, in human hemopoietic cells.

Steel factor (SF) (also called stem cell factor, mast cell growth factor, or c-kit ligand) is a recently cloned hemopoietic growth factor that is produced by bone marrow stromal cells, fibroblasts, and hepatocytes. In both mouse and man it acts synergistically with several colony stimulating factors, including interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF), to induce the proliferation and differentiation of primitive hemopoietic precursor cells. In order to study its mechanism of action and to explore the molecular basis for its synergistic activity we have examined the proteins that become tyrosine phosphorylated in response to SF, IL-3, and GM-CSF. We report herein that SF, but not IL-3 or GM-CSF, dramatically stimulates the tyrosine phosphorylation of the product of the recently discovered proto-oncogene, vav, in two SF-responsive human cell lines, M07E and TF-1. Although phosphorylation is very rapid, reaching maximal levels within 2 min at 37 degrees C, co-immunoprecipitation studies suggest that c-kit may either not associate directly with p95vav or bind to it with very low affinity. Nonetheless, our data suggest that c-kit may utilize p95vav to mediate downstream signaling in hemopoietic cells.

Animals↗
PubMed1992

Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells.

Activation of B lymphocytes by engagement of their immunoglobulin M antigen receptors results in phosphorylation of a number of proteins on tyrosine residues. One such protein is p95vav, the product of the vav proto-oncogene. Tyrosine phosphorylation of p95vav occurred within seconds of immunoglobulin M cross-linking and was independent of other events induced during stimulation of B cells, such as protein kinase C activation, guanosine triphosphate-binding protein signaling, and calcium mobilization. Moreover, engagement of antigen receptors induced the rapid (approximately 5 seconds) and transient (approximately 60 seconds) association of p95vav with a 70-kilodalton tyrosine-phosphorylated protein, Vap-1, an interaction mediated by the Src homology 2 domain of p95vav. These results suggest that the vav proto-oncogene participates in the signaling processes that mediate the antigen-induced activation of B lymphocytes.

Amino Acids↗
PubMed1992

Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates.

Several proteins implicated in the regulation of cellular responses to mitogenic stimuli contain a common non-catalytic domain, SH2 (for src-homologous domain 2), that mediates their interaction with activated tyrosine protein kinases. Here we report that p95vav, a proto-oncogene product specifically expressed in cells of the haematopoietic system, contains an SH2 domain and is a substrate for tyrosine protein kinases. Exposure of quiescent NIH3T3 cells ectopically expressing p95vav to either epidermal or platelet-derived growth factors induces the rapid phosphorylation of this protein on tyrosine residues. Activation of the receptors for these growth factors by their cognate ligand results in their association with p95vav, a process mediated by its SH2 domain. In T cells, co-activation of the T-cell receptor and the accessory CD4 cell-surface protein also results in the phosphorylation of the endogenous p95vav protein in tyrosine residues. Phosphorylation of p95vav is rapid, transient and precedes the appearance of most other phosphotyrosine-containing proteins. In addition to the SH2 domain, p95vav contains structural motifs not found in other tyrosine kinase substrates. One such motif is a helix-loop-helix/leucine zipper-like domain which shares some sequence similarity with these motifs in the Myc and Max proteins. Deletion of the helix-loop-helix-like motif causes oncogenic activation of p95vav. These results indicate that p95vav is a new type of signal transduction molecule and suggest a possible role for this protein in the transduction of tyrosine phosphorylation signalling into transcriptional events.

Animals↗
PubMed1992

Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.

Amino Acid Sequence↗
PubMed1992

Nerve growth factor mediates signal transduction through trk homodimer receptors.

We have investigated the molecular nature of the high affinity nerve growth factor (NGF) receptors by using cell lines expressing gp75LNGFR and gp140trk. Our results suggest that gp75LNGFR and gp140trk interact with NGF independently and that only gp140trk mediates NGF signaling. NGF binds to gp140trk with picomolar affinity and induces its phosphorylation on tyrosine residues regardless of the presence of gp75LNGFR. NGF-gp140trk complexes display the slow dissociation rate and rapid internalization characteristics of high affinity NGF receptors. Cross-linking studies reveal the existence of gp75LNGFR and gp140trk homodimers. However, we were unable to detect gp75LNGFR-gp140trk heterodimers. Coexpression in COS cells of wild-type and kinase deficient mutants reveals that gp140trk receptors can undergo intermolecular phosphorylation, indicating the formation of functional homodimers. Moreover, these kinase deficient mutants inhibit NGF-induced signaling through wild-type gp140trk receptors. These results indicate that the functional high affinity NGF receptors consist of gp140trk homodimeric (or oligomeric) complexes.

3T3 Cells↗
PubMed1992

The trkB tyrosine protein kinase is a receptor for neurotrophin-4.

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp145trkB, a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp145trkB receptor.

Adrenal Gland Neoplasms↗
PubMed1992

10'-Desmethoxystreptonigrin, a novel analog of streptonigrin.

10'-Desmethoxystreptonigrin, a novel analog of streptonigrin produced by Streptomyces albus, was discovered in a screen for inhibitors of farnesylation of RAS p21 protein. The compound was isolated from the fermentation broth and its structure determined. It is markedly cytotoxic to several human tumor cell lines and also exhibits potent broad-spectrum antibacterial activity.

Animals↗
PubMed1992

K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors.

K252a, an efficient serine/threonine protein kinase inhibitor (IC50s of 10 to 30 nM), has been shown to block the neuronal differentiation of rat pheochromocytoma PC12 cells induced by nerve growth factor (NGF). In this report, we demonstrate that K252a is a potent inhibitor (IC50 of 3 nM) of the tyrosine protein kinase activity of the NGF receptor gp140trk, the product of the trk protooncogene. K252a also inhibits the kinase activity of its transforming alleles, the trk oncogenes, and of the related neurotrophin receptors gp145trkB and gp145trkC, the products of the other known members of the trk gene family, trkB and trkC. In contrast, K252a has no effect (even at micromolar concentrations) on other tyrosine protein kinases such as the receptors for EGF and PDGF and the products of the v-src and v-fms oncogenes. In addition, K252a rapidly reverts the transformed phenotype of NIH3T3 cells transformed by either autocrine stimulation of the trk family of receptors by their cognate ligands or by expression of trk oncogenes isolated from human tumors. The selectivity of K252a for the catalytic activity of the trk family of kinases should help to establish the structural basis for the rational design of highly specific tyrosine protein kinase inhibitors.

3T3 Cells↗
PubMed1991

trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3.

We report the isolation and molecular characterization of trkC, a new member of the trk family of tyrosine protein kinase genes. trkC is preferentially expressed in the brain. In situ hybridization studies revealed trkC transcripts in the hippocampus, cerebral cortex, and the granular cell layer of the cerebellum. The product of the trkC gene has been identified as a glycoprotein of 145,000 daltons, gp145trkC, which is equally related to the previously characterized gp140trk and gp145trkB tyrosine kinases. gp145trkC is a functional receptor for neurotrophin-3 (NT-3). However, gp145trkC does not bind the highly related neurotrophic factors NGF or BDNF. In proliferating cells, the interaction between gp145trkC and NT-3 elicits a more efficient biological response than when NT-3 binds to its other receptors gp140trk and gp145trkB. These results indicate that gp145trkC may play an important role in mediating the neurotrophic effects of NT-3.

Amino Acid Sequence↗
PubMed1991

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.

Animals↗
PubMed1991

The trk proto-oncogene encodes a receptor for nerve growth factor.

Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.

Adrenal Gland Neoplasms↗
PubMed1991

Generation of monoclonal antibodies specific for ras p21 Glu-12 oncoproteins: detection in carcinogen-induced mammary carcinomas.

ras genes have been shown to become oncogenes by single point mutations which result in amino acid substitutions that affect either their GTPase activity (positions 12, 13, 59, 61) or their affinity for GTP and GDP. Ras oncogenes and their corresponding proteins have been described in a variety of human cancers as well as in animal tumors induced by physical and chemical carcinogens. One of these animal tumor systems involves the induction of mammary carcinomas in rats by a single dose of N-nitroso-N-methylurea (NMU), a methylating carcinogen. These NMU-induced mammary carcinomas contain transforming H-ras genes activated by G----A transitions in the second nucleotide of their 12th codon, presumably a consequence of the pre-mutagenic lesions induced by NMU. These G----A mutations result in the replacement of the normal glycine in the 12th position of the ras p21 protein by a glutamic acid residue. In this study, we report the generation of monoclonal antibodies (Mab) reactive with oncogenic ras p21 proteins containing glutamic acid at position 12 (p21 Glu-12). Mab designated E184 specifically recognized activated ras p21 Glu-12 proteins but not normal p21 (Gly-12) or p21 proteins activated by other position 12 substitutions including arginine, aspartic acid, cysteine, valine or serine residues. Western blot analysis of NMU-induced mammary carcinomas demonstrated that Mab E184 recognized p21 proteins expressed in these rat tumors but not p21 present in normal tissues nor in other carcinogen-induced tumors known to carry H-ras oncogenes activated by mutations at position 61.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The trk family of oncogenes and neurotrophin receptors.

To date more than twenty five different oncogenes have been identified in human neoplasias. One of these oncogenes is trk, a transforming gene originally isolated from a colon carcinoma biopsy by using gene transfer assays. This oncogene encodes a chimeric molecule that contains the 221 amino terminal residues of a non-muscle tropomyosin followed by the transmembrane and cytoplasmic domains of the trk proto-oncogene product, a tyrosine protein kinase receptor. trk oncogenes have also been identified in a significant fraction of thyroid papillary carcinomas. Some of these trk oncogenes contain sequences derived from genes other than tropomyosin. One such gene is tpr, a gene first identified as a component of the human met oncogene. The trk proto-oncogene, non-muscle tropomyosin and tpr map in the long arm of chromosome 1. Therefore, trk oncogenes are likely to result from internal rearrangements or from unequal cross-overs between two chromosome 1s. Recent studies have demonstrated that the product of the trk proto-oncogene, gp140trk, is the functional receptor for nerve growth factor (NGF). NGF elicits the rapid phosphorylation of gp140trk on tyrosine residues. Moreover, addition of NGF to NIH3T3 cells expressing gp140trk induces the transient expression of c-Fos, DNA synthesis and morphologic transformation. Finally, transfection of the trk proto-oncogene into NGF non-responsive PC12 mutant cells restores NGF responsiveness. Two additional genes designated trkB and trkC have recently been isolated in our laboratory. These genes are highly related to the trk proto-oncogene and encode tyrosine protein kinases which serve as functional receptors for the NGF-related neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophic-3 (NT-3), respectively. Whether mutations in these novel members of the trk family of receptor genes are also implicated in human cancer remains to be determined.

Animals↗
PubMed1991

Mechanism of activation of the vav protooncogene.

vav is a human locus that appears to be specifically expressed in cells of hematopoietic origin regardless of their differentiation lineage. This gene was first identified as a result of its malignant activation during the course of gene transfer assays (Katzav, S., Martin-Zanca, D., and Barbacid, M. EMBO J., 8: 2283-2290, 1989). In this study, we report the isolation of complementary DNA clones containing the entire coding sequence of the mouse vav protooncogene. Antisera raised against a peptide corresponding to a predicted hydrophilic domain have allowed us to identify the product of the vav gene as a 95,000 Da protein. Analysis of the deduced amino acid sequence of p95vav revealed an amino-terminal leucine-rich region not present in the activated vav oncogene. This region consists of an amphipathic helix-loop-helix followed by a leucine zipper, a structure reminiscent of the carboxy-terminal region of myc proteins and the steroid binding domain of nuclear receptors. In vitro mutagenicity studies have indicated that removal of the amphipathic helix-loop-helix is sufficient to activate the transforming potential of human and mouse vav protooncogenes. vav proteins also possess a cysteine-rich domain whose sequence predicts the formation of two putative metal binding-like domains, Cys-X2-Cys-X13-Cys-X2-Cys and His-X2-Cys-X6-Cys-X2-His. Replacement of some of these cysteine and histidine residues completely abolished the transforming activity of vav genes. Further examination of the alignment of cysteine residues in this region revealed an alternative structure, Cys-X2-Cys-X13-Cys-X2-Cys-X7-Cys-X6-Cys, which is reminiscent of the phorbol ester binding domain of protein kinase C. A similar domain has been recently identified in a second enzyme, diacylglycerol kinase. These structural similarities, along with its expression pattern, suggest that the vav protooncogene codes for a new type of signal-transducing molecule that may play an important role in controlling hematopoiesis.

Amino Acid Sequence↗
PubMed1990

Activation of ras oncogenes preceding the onset of neoplasia.

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.

Adenofibroma↗
PubMed1990

The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain.

We previously identified two tyrosine protein kinase genes, designated trk and trkB, that code for putative neurogenic cell surface receptors. In this study, we report that the mouse trkB locus codes for at least two classes of receptor-like molecules. These trkB proteins, designated gp145trkB and gp95trkB, have identical extracellular and transmembrane domains, suggesting that they might recognize the same ligand(s). However, only gp145trkB contains a long cytoplasmic region, which includes a catalytic tyrosine protein kinase domain. trkB transcripts coding for this protein were observed in the cerebral cortex and the pyramidal cell layer of the hippocampus. In contrast, transcripts coding for the noncatalytic gp95trkB molecule were found in the ependymal linings of the cerebral ventricles and in the choroid plexus. These results illustrate that a tyrosine protein kinase locus can code for two structurally and functionally distinct cellular receptors.

Amino Acid Sequence↗
PubMed1990

The human VAV proto-oncogene maps to chromosome region 19p12----19p13.2.

A novel human oncogene, designated VAV, has been recently characterized. This oncogene was generated by a rearrangement within the 5' coding sequences of a normal cellular gene, the VAV proto-oncogene. The normal VAV gene is specifically expressed in hematopoietic cells regardless of their differentiation lineage. We now report that the VAV locus has been localized in the human genome at chromosome 19p12----19p13.2 by analysis of its segregation pattern in rodent-human somatic cell hybrids and by chromosomal in situ hybridization. The VAV locus might be closely linked to the insulin receptor (INSR) locus, as suggested by comigration of INSR and VAV high-molecular-weight DNA fragments after pulsed-field gel electrophoresis. The VAV chromosomal assignment is of interest because chromosome region 19p13 is involved in different karyotypic abnormalities in a variety of malignancies including melanomas and leukemias. The identification of a novel proto-oncogene that maps to that region will enable us to define whether VAV is involved in any of the translocations observed.

Animals↗