RESUMEN
A human B-lymphoblastoid lambda gt11 expression library was screened using anti-phosphotyrosine antibodies yielding complementary DNAs encoding active tyrosine kinases. The resulting clones were used to obtain the sequence of a novel 984 amino acid transmembrane tyrosine kinase. Analysis of the complementary DNA revealed extracellular immunoglobulin and fibronectin type III domains and the unusual kinase signature sequence KWIAIES; all are characteristic of the axl family of tyrosine kinases. The novel tyrosine kinase was not expressed in normal B- and T-lymphocytes but, unlike axl, was expressed in numerous neoplastic B- and T-cell lines. Transcripts for the novel receptor-like tyrosine kinase were detected in normal peripheral blood monocytes and bone marrow. One alternatively spliced transcript was detected which contained an insert in the membrane proximal region that could encode for a truncated, soluble receptor. Sequence comparison shows that the kinase may be the human protooncogene for the recently isolated chicken retroviral oncogene v-ryk (recently renamed v-eyk), a truncated tyrosine kinase whose expression by retroviral infection produced sarcomas in chickens. The intracellular domain of the human kinase shows 83% similarity and 71% identity to v-ryk. Since the ryk designation has been used to name another tyrosine kinase and an analysis of RNA expression demonstrated that this novel human kinase is expressed in monocytes and tissues of epithelial and reproductive origin, we have designated our novel protooncogene c-mer.
Asunto(s)
Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Tirosina Quinasas Receptoras , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Médula Ósea/química , Línea Celular Transformada , ADN Complementario/química , Células Epiteliales , Epitelio/química , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Monocitos/química , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Análisis de Secuencia de ADN , Tirosina Quinasa c-MerRESUMEN
The expression of the yeast ADH2 gene is controlled by the transcriptional activator ADR1, a zinc-finger protein that binds to an upstream activating sequence (UAS1) in the ADH2 promoter. We report here the isolation of seven mutations in the ADR1-5c allele, defining five different amino acid changes, that suppress the enhanced ADH2 expression caused by the ADR1-5c allele. Each of the mutations was shown to reduce the activation of ADH2 by a wild-type ADR1 gene, suggesting the mutations disrupt a domain important to the function of both the ADR1 and ADR1-5c proteins. All five amino acid changes occurred within the DNA-binding domain of ADR1 and were shown to severely inhibit the ability of ADR1 to bind UAS1 in vitro. These mutations were found, however, to also affect the ability of ADR1 to activate transcription independent of its ability to bind DNA. These results indicate that the DNA-binding region of ADR1 is involved in both transactivation and DNA binding.