RESUMEN
The human "protein interacting with carboxyl terminus 1" (PICT-1), also designated as the "glioma tumor suppressor candidate region 2 gene product", GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.
Asunto(s)
Multimerización de Proteína , Proteínas Supresoras de Tumor/metabolismo , Cromatografía en Gel , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoprecipitación , Técnicas Analíticas Microfluídicas , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
The human glioma tumor suppressor candidate region 2 gene product, GLTSCR2, also called 'protein interacting with carboxyl terminus 1' (PICT-1), has been implicated in the regulation of two major tumor suppressor proteins, PTEN and p53, and reported to bind the membrane-cytoskeleton regulator of cell signaling, Merlin. PICT-1 is a nucleolar protein, conserved among eukaryotes, and its yeast homolog has been functionally associated with ribosomal RNA processing. By means of confocal microscopy of EGFP and myc-tagged PICT-1 fusion proteins, we delineate that the nucleolar localization of PICT-1 is mediated by two independent nucleolar localization sequences (NoLS). Unlike most NoLSs, these NoLSs are relatively long with flexible boundaries and contain arginine and leucine clusters. In addition, we show that PICT-1 exhibits a nucleolar distribution similar to proteins involved in ribosomal RNA processing, yet does not colocalize precisely with either UBF1 or Fibrillarin under normal or stressed conditions. Identification of the precise location of PICT-1 and the signals that mediate its nucleolar localization is an important step towards advancing our understanding of the demonstrated influence of this protein on cell fate and tumorigenesis.
Asunto(s)
Nucléolo Celular/metabolismo , Señales de Localización Nuclear/fisiología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Transporte de Proteínas/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Estrés Fisiológico/fisiología , Proteínas Supresoras de Tumor/genéticaRESUMEN
KS-Bcl-2, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Nucléolo Celular/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Herpesvirus Humano 8/genética , Humanos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Proteínas Supresoras de Tumor/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genéticaRESUMEN
Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations.