Biomedical subjects

M Barbacid

Publications and source records attributed to M Barbacid.

At least 109 records · Page 6Linked to original sources

PubMed1990

Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.

Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.

Amino Acid Sequence↗

PubMed1990

Expression of the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system.

We have examined the expression of the trkB gene, which encodes a member of the family of protein tyrosine kinase (TK) transmembrane receptors, during mouse embryogenesis using in situ hybridization and Northern analysis. Transcripts were first detected in the neuroepithelium and in the neural crest of 9.5 day embryos with regions of high expression in the neural folds and at the lateral neuroepithelium. However, during the process of cephalization and development of the peripheral nervous system, transcripts were detected in most neural tissues, including the brain, spinal cord, cranial and spinal ganglia, and along the pathways of axonal tracts extending peripherally. In the adult brain, expression continues in a complex pattern that is confined to specific regions or neuron types. The expression of trkB, a TK receptor, in early embryogenesis, and specifically in neural tissues, is consistent with the notion that this gene plays a role in the events that regulate the development of the nervous system.

Animals↗

PubMed1990

C127 cells resistant to transformation by tyrosine protein kinase oncogenes.

C127 is a nontumorigenic mouse cell line widely used in in vitro transformation assays due to its normal morphological appearance and its very low levels of spontaneous transformation. We now report that C127 cells are resistant to transformation by tyrosine protein kinase oncogenes derived from growth factor receptors such as the retroviral v-fms and the human trk transforming genes. In contrast, these cells could be efficiently transformed by members of the ras oncogene family and by serine/threonine kinase oncogenes such as v-mos and v-raf. C127 cells were also found to be resistant to transformation by v-src, the prototype of a large family of tyrosine protein kinase oncogenes whose products are associated with the inner side of the plasma membrane. However, morphologically normal C127 cells expressing pp60v-src acquired a transformed phenotype upon continuous passage in vitro. Somatic cell hybrids (neoR, hygroR) obtained by fusion of G418-resistant C127 cells expressing p70trk (neoR) and hygromycin-resistant NIH3T3 cells (hygroR) exhibited transformed properties as determined by their ability to grow in semisolid agar. In contrast, no such growth was observed when these neoR p70trk-containing C127 cells were fused to control hygroR C127 cells. These results indicate that C127 cells may either lack or express insufficient levels of certain critical substrate(s) necessary for the onset of transformation by tyrosine protein kinase oncogenes.

Animals↗

PubMed1989

trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development.

We have isolated a novel member of the tyrosine protein kinase family of cell surface receptors. This gene, designated trkB, is highly related to the human trk proto-oncogene. At the amino acid level, their respective products share a 57% homology in their extracellular regions including 9 of the 11 cysteines present in the trk proto-oncogene. This homology increases to 88% within their respective tyrosine kinase catalytic domains. Both trk and trkB are equally distantly related to the other members of this gene family of receptors. A biologically active cDNA clone of trkB can direct the synthesis of gp145trkB, a glycoprotein of 145 kd of which only 93 kd correspond to its polypeptide backbone. In adult mice, trkB is preferentially expressed in brain tissue, although significant levels of trkB RNA have also been observed in lung, muscle and ovaries. In addition, trkB transcripts can be detected in mid and late gestation embryos. The trkB locus exhibits a complex pattern of transcription. At least seven RNA species ranging in size from approximately 9 kb to 2 kb have been identified in brain. However, only a subset of these transcripts appears to be expressed in the other tissues. In situ hybridization analysis of 14 and 18 day old mouse embryos indicates that trkB transcripts are localized in the central (CNS) and peripheral (PNS) nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways. These results suggest that trkB may code for a novel cell surface receptor involved in neurogenesis.

Amino Acid Sequence↗

PubMed1989

vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells.

A novel human oncogene, designated vav, was generated by a genetic rearrangement during gene transfer assays. The vav oncogene directs the synthesis of a 3.0 kb mRNA from which we isolated a 2.8 kb-long complementary DNA copy. Nucleotide sequence analysis of this vav oncogene cDNA clone revealed that its 5' 167 bp were derived from pSV2neo DNA cotransfected as a selectable marker during gene transfer. The remaining 2597 bp were unrelated to genes included in current data banks, indicating that the vav oncogene is likely to be derived from a novel human locus. The vav oncogene cDNA clone encompasses a 2391 bp long open reading frame (ORF) capable of directing the synthesis of a 797 amino acid long polypeptide. The predicted vav oncogene protein sequence exhibits several motifs reminiscent of transcriptional factors. They include a highly acidic amino-terminal region separated from two putative nuclear localization signals by a proline-rich sequence, presumably a hinge region. In addition, we identified two zinc-finger-like domains, one of which conforms to the canonical pattern Cys-X2-Cys-X13-Cys-X2-Cys previously found to confer trans-activating activity to the adenovirus E1A protein. Transcription of its normal allele, the vav proto-oncogene, has been exclusively observed in cells of hematopoietic origin, including those of erythroid, lymphoid and myeloid lineages. These findings raise the possibility that this novel locus might play an important role in hematopoiesis.

Amino Acid Sequence↗

PubMed1989

Molecular analysis of O6-substituted guanine-induced mutagenesis of ras oncogenes.

We have designed an Ha-ras/thymidine kinase (TK) cassette that permits the incorporation of chemically synthesized adducts within specific domains of the rat Ha-ras protooncogene. This cassette has been used to evaluate the mutagenicity of O6-substituted guanine residues, including O6-methylguanine and O6-benzylguanine, incorporated within the 12th codon of this locus. Mutations were monitored by the ability of these modified Ha-ras DNAs to transform Rat4 TK-cells. Our results indicate that both types of O6-substituted guanines are substantially mutagenic, although the methyl substituent induced a 2-fold higher percentage of transformed Rat4 TK+ colonies than its bulkier benzyl analogue. Interestingly, the mutagenicity of both O6-substituted guanines was found to be independent of their relative position within codon 12, therefore suggesting that the specific activation of Ha-ras oncogenes by GGA----GAA mutations in tumors induced by methylating carcinogens might be due to differences in the accessibility of these guanine residues to the carcinogen rather than to a differential rate of repair. Molecular analysis of the mutations induced by these O6-substituted guanines indicated that O6-methylguanine exclusively induced G----A transitions. In contrast, O6-benzylguanine produced G----C and G----T transversions in addition to G----A transitions. These results suggest that O6-methylguanine and its bulkier analogue O6-benzylguanine may induce mutagenesis by different mechanisms.

Alkylation↗

PubMed1989

Mechanism of activation of the human trk oncogene.

The human trk oncogene was generated by a genetic rearrangement that replaced the extracellular domain of the normal trk tyrosine kinase receptor by sequences coding for the 221 amino-terminal residues of a nonmuscle tropomyosin. Molecular dissection of a cDNA clone of the trk oncogene indicated that both the tropomyosin and tyrosine kinase domains were required for proper transforming activity. Replacement of nonmuscle tropomyosin sequences with those of other tropomyosin isoforms had no deleterious effect. However, when tropomyosin sequences were replaced with those of another cytoskeletal gene, such as beta-actin or beta-globin, transforming activity was completely abolished. These results illustrate the important role of tropomyosin sequences in endowing the trk kinase with transforming properties. Functionally unrelated subdomains of the tropomyosin molecule were equally efficient in activating the trk gene. Moreover, the transforming activity of the trk oncogene was not affected when its subcellular localization was drastically altered. Therefore, tropomyosin sequences are likely to contribute to the malignant activation of the trk oncogene not by facilitating its interaction with defined cytoskeletal structures as initially suspected, but by allowing its kinase domain to fold into a constitutively active configuration.

Amino Acid Sequence↗

PubMed1989

Molecular and biochemical characterization of the human trk proto-oncogene.

Molecular analysis of the human trk oncogene, a transforming gene isolated from a colon carcinoma biopsy, revealed the existence of a novel member of the tyrosine kinase gene family. This locus, which we now designate the trk proto-oncogene, codes for a protein of 790 amino acid residues that has several features characteristic of cell surface receptors. They include (i) a 32-amino-acid-long putative signal peptide, (ii) an amino-terminal moiety (residues 33 to 407) rich in consensus sites for N-glycosylation, (iii) a transmembrane domain, (iv) a kinase catalytic region highly related to that of other tyrosine kinases, and (v) a very short (15 residue) carboxy-terminal tail. Residues 1 to 392 were absent in the trk oncogene, as they were replaced by tropomyosin sequences. However, no other differences were found between the transforming and nontransforming trk alleles (residues 392 to 790), suggesting that no additional mutations are required to activate the transforming potential of this gene. The human trk proto-oncogene codes for a 140,000-dalton glycoprotein, designated gp140proto-trk. However, its primary translational product is a 110,000-dalton glycoprotein which becomes immediately glycosylated, presumably during its translocation into the endoplasmic reticulum. This molecule, designated gp110proto-trk, is further glycosylated to yield the mature form, gp140proto-trk. Both gp110proto-trk and gp140proto-trk proteins possess in vitro kinase activity specific for tyrosine residues. Finally, iodination of intact NIH 3T3 cells expressing trk proto-oncogene products indicated that only the mature form, gp140proto-trk, cross the plasma membrane, becoming exposed to the outside of the cell. These results indicate that the product of the human trk locus is a novel tyrosine kinase cell surface receptor for an as yet unknown ligand.

Amino Acid Sequence↗

PubMed1989

The trk oncogene abrogates growth factor requirements and transforms hematopoietic cells.

Oncogenes may cause transformation by altering the requirements of cells for growth factors which normally regulate growth. This type of transformation is particularly evident in the ability of oncogenes of the tyrosine protein kinase gene family to abrogate the requirement of hematopoietic cells for growth factors such as interleukin-3 (IL-3). We have used this property to study the effect of the human trk oncogene on hematopoietic cells. This oncogene was generated by a genetic rearrangement that fused a non-muscle tropomyosin gene to a tyrosine protein kinase with structural features characteristic of growth factor receptors. The results presented here demonstrate that a replication-defective murine retrovirus expressing the human trk oncogene can abrogate the growth factor requirements of hematopoietic cells through a non-autocrine mechanism. In these trk-transformed cells, many of the cellular proteins which are phosphorylated on tyrosine residues following IL-3 stimulation are constitutively phosphorylated, with the notable exception of a 140 kDa membrane, IL-3-binding protein. The 140 kDa protein becomes phosphorylated only following stimulation with IL-3 in these trk oncogene-transformed hematopoietic cells.

Animals↗

PubMed1988

Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas.

These studies were set up to determine whether those oncogenes participating in the initiation of mammary carcinogenesis (for example, ras oncogenes) play a direct role in the outcome of events associated with the late stages of tumor development such as loss of hormone dependency. Mammary carcinomas induced by a single carcinogenic insult in pubescent rats was selected as an in vivo model system with direct relevance to human breast cancer. Acquisition of hormone-independent growth in these carcinogen-induced tumors was found to be independent of the activation of ras oncogenes during the early stages of carcinogenesis. In agreement with these observations, introduction of a human ras oncogene into human MCF-7 breast carcinoma cells did not abrogate their hormonal dependency for growth in vivo. These findings suggest that those events responsible for the critical stages of breast cancer development occur independently and in an uncoordinated manner.

Animals↗

PubMed1988

Frequent generation of oncogenes by in vitro recombination of TRK protooncogene sequences.

Transfection of NIH 3T3 cells with cDNA clones containing either the entire coding sequences or the tyrosine protein kinase domain of the human TRK protooncogene results in the frequent generation of transforming genes. Activation of most of these TRK oncogenes involves acquisition of DNA sequences. These sequences, unlike those present in the original human TRK oncogene, are not derived from tropomyosin genes. The products of these in vitro-generated TRK oncogenes retain the parental tyrosine protein kinase activity and contain an intact carboxyl terminus. However, they exhibit distinct biochemical properties. Whereas some of them are nonglycosylated cytoplasmic molecules, others were found to be transmembrane glycoproteins. These results suggest that TRK oncogenes may induce malignant transformation by allowing their tyrosine kinase to interact with various substrates depending on the nature of their activating sequences. If so, the TRK kinase may serve as a pleiotropic marker to identify various cellular proteins whose unscheduled phosphorylation on tyrosine residues contributes to neoplastic transformation.

Animals↗

PubMed1988

Oncogene detection at the single cell level.

We describe strategies for the detection of oncogenes at the single-cell level and for the positive identification of under-represented oncogenic alleles in mixed populations of normal and tumor cells. By combining the Polymerase Chain Reaction (PCR) technique with a liquid hybridization and gel retardation assay, we have been able to detect H-ras sequences in single cells, including in one fertilized mouse ovum. We also describe a modification of the PCR protocol involving the use of mismatched primers. This procedure allows for the creation of novel Restriction Fragment Length Polymorphisms (RFLP) diagnostic of specific point mutations. This experimental approach has allowed us to detect ras oncogenes in a single heterozygous cell in the presence of 10(5) normal cells.

Alleles↗

PubMed1987

Identification and biochemical characterization of p70TRK, product of the human TRK oncogene.

TRK is a human transforming gene generated in a colon carcinoma by a somatic rearrangement that fused a nonmuscle tropomyosin gene to sequences that shared extensive homology with members of the tyrosine-protein kinase supergene family. These sequences are likely to be derived from a transmembrane receptor gene whose putative ligand binding domain has been replaced by tropomyosin. In the present studies, we have expressed the entire coding sequences of the TRK oncogene as well as its protein kinase-related carboxyl-terminal domain in Escherichia coli. Antisera raised against these bacteria-synthesized TRK polypeptides has allowed us to identify the gene product of the TRK oncogene as a 70-kDa protein. Immunoprecipitates containing p70TRK have an associated protein kinase activity specific for tyrosine residues. Moreover, p70TRK is phosphorylated in vivo in serine (75%), threonine (20%), and tyrosine (5%) residues. Finally, immunofluorescence and cellular fractionation studies indicate that p70TRK is preferentially located in the cytoplasmic fraction.

Amino Acids↗

PubMed1987