RESUMEN
Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Genes Supresores de Tumor , Células Madre Hematopoyéticas/citología , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Encéfalo/embriología , Química Encefálica , Región Branquial/química , Región Branquial/embriología , Proteínas Portadoras/química , Ciclo Celular , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/química , Cromosomas Humanos Par 8 , Gránulos Citoplasmáticos/química , ADN/análisis , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Desarrollo Embrionario y Fetal , Extremidades/embriología , Ganglios Espinales/química , Ganglios Espinales/embriología , Células HeLa , Humanos , Leucemia Eritroblástica Aguda , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Médula Espinal/química , Médula Espinal/embriología , TransfecciónRESUMEN
A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.
