RESUMEN
Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.
Asunto(s)
Factor Apoptótico 1 Activador de Proteasas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/fisiopatología , Citocromos c/metabolismo , Glicoproteínas/metabolismo , Apoptosis , Factor Apoptótico 1 Activador de Proteasas/genética , Neoplasias de la Mama/genética , Citosol/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Células MCF-7 , Unión ProteicaRESUMEN
Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Ratones , Microscopía Confocal , Mutación , Células 3T3 NIH , Proteínas Nucleares/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Proteins that contain the recently described MIF4G and/or MA3 domains function in translation, cell growth, proliferation, transformation, and apoptosis. Examples of MIF4G/MA3 containing proteins and their functions include eIF4G, which serves as a scaffold for assembly of factors required for translation initiation, programmed cell death protein 4 (Pdcd4) that inhibits translation and functions as a tumor suppressor, and NMD2, which is essential for nonsense-mediated mRNA decay. MIF4G and MA3 domains serve as binding sites for one or more isoforms of the eIF4A family of ATP-dependent DEAD-box RNA helicases that are required for translation and for nonsense-mediated decay. In this report, we describe the characterization of a novel MIF4G/MA3 family member called NOM1 (nucleolar protein with MIF4G domain 1) that was identified at the chromosome 7q36 breakpoint involved in 7;12 translocations associated with certain acute leukemias of childhood. NOM1, which includes a previously described EST called c7orf3, encodes a ubiquitously expressed transcript composed of 11 exons and an approximately 3 kb 3' UTR that contains several Alu repeats. The predicted NOM1 protein contains one MIF4G domain and one MA3 domain and, consistent with data obtained with other MIF4G/MA3 proteins, interacts with members of the eIF4A family of helicases. Database searches reveal that NOM1 homologs exist in several organisms and that at least two of these are essential genes. Finally, like its Saccharomyces cerevisiae homolog Sgd1p, NOM1 localizes predominantly to the nucleolus. These data demonstrate that NOM1 is a new member of the MIF4G/MA3 family of proteins and suggest that it may provide an essential function in metazoans.