RESUMEN
Special AT-rich sequence-binding protein 1 (SATB1) is a tissue-restricted genome organizer that provides a key link between DNA loop organization, chromatin modification/remodeling, and transcription factor association at matrix attachment regions (MARs). The SUMO E3 ligase PIAS1 enhances SUMO conjugation to SATB1 lysine-744, and this modification regulates caspase-6 mediated cleavage of SATB1 at promyelocytic leukemia nuclear bodies (PML NBs). Since this regulated caspase cleavage occurs on only a subset of SATB1, and the products are relatively stable, proteolysis likely mediates cellular processes other than programmed cell death. However, the mechanism for the spatial and temporal regulation of SATB1 sumoylation and caspase cleavage is not known. Here we report that these processes are controlled by SATB1 phosphorylation; specifically, PIAS1 interaction with SATB1 is inhibited by phosphorylation. Mutagenesis studies identified interaction of the PIAS SAP (scaffold attachment factor-A/B/acinus/PIAS) motif with SATB1 N-terminal sequences. Notably, phosphorylation of SATB1 at threonine-188 regulates its interaction with PIAS1. Sequences near this phosphorylation site, LXXLL (residues 193 to 197), appear to be conserved among a subset of SUMO substrate proteins. Thus, this motif may be commonly involved in interaction with the PIAS SAP, and phosphorylation may similarly inhibit some of these substrates by preventing their interaction with the ligase.
Asunto(s)
Caspasas/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Caspasas/genética , Línea Celular , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Datos de Secuencia Molecular , Fosforilación , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
SATB1 (special AT-rich sequence-binding protein-1) provides a key link between DNA loop organization, chromatin modification/remodeling, and association of transcription factors at matrix attachment regions (MARs). To investigate the role of SATB1 in cellular events, we performed a yeast two-hybrid screen that identified SUMO-1, Ubc9, and protein inhibitor of activated STAT (PIAS) family members as SATB1 interaction partners. These proteins, working in concert, enhanced SUMO conjugation to lysine-744 of SATB1. Overexpression of SUMO or PIAS in Jurkat cells, which express high levels of endogenous SATB1, exhibited enhanced caspase cleavage of this MAR-associating protein. Sumoylation-deficient SATB1 (SATB1(K744R)) failed to display the characteristic caspase cleavage pattern; however, fusion of SUMO in-frame to SATB1(K744R) restored cleavage. A SUMO-independent interaction of inactive caspase-6 and SATB1 was noted. A subset of total cellular SATB1 localized into promyelocytic leukemia nuclear bodies where enhanced SATB1 cleavage was detected subsequent to caspase activation. These results reveal a novel sumoylation-directed caspase cleavage of this key regulatory molecule. The role of regulated proteolysis of SATB1 may be to control transcription in immune cells during normal cell functions or to assist in efficient and rapid clearance of nonfunctional or potentially damaging immune cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/química , Proteína SUMO-1/metabolismo , Apoptosis , Sitios de Unión , Núcleo Celular/metabolismo , Células HeLa , Humanos , Células Jurkat , Células K562 , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Microscopía Confocal , Modelos Biológicos , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The bcl-2 major breakpoint region (mbr), located within the 3'-UTR of the bcl-2 gene, is the site of the most common chromosomal translocation, t(14;18) (q32;q21), which occurs in follicular lymphoma. The mbr forms a triplex DNA structure under physiological conditions and the transcription factor special AT-rich sequence-binding protein 1 (SATB1) binds immediately downstream of the mbr. These observations raise the possibility that the mbr may be involved in regulation of bcl-2 gene expression. We investigated the role of the bcl-2 mbr on reporter gene activity and the relevance of SATB1 to this function in a variety of cell lines. We found that the mbr up-regulated reporter gene expression. Deletion of the 37-bp AT-rich SATB1 binding site abolished the bcl-2 mbr regulation of reporter gene expression. Overexpression of SATB1 enhanced bcl-2 mbr up-regulation of the reporter gene activity. Our data strongly demonstrated that the bcl-2 mbr possessed regulatory function that was related to SATB1.
Asunto(s)
Proteínas Proto-Oncogénicas c-bcl-2/genética , Transcripción Genética , Regiones no Traducidas 3'/química , Sitios de Unión , Línea Celular Tumoral , Femenino , Genes Reporteros , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Linfocitos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , TransfecciónRESUMEN
Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions.
Asunto(s)
Proteína SUMO-1/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia de Consenso , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Electricidad EstáticaRESUMEN
PML bodies play an important role in multiple pathways of growth control, such as transformation suppression, apoptosis, and Ras-induced senescence. However, the molecular and biological bases for these physiological phenomena are not well understood. The findings that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery have suggested that PML bodies are transcription-permissible domains. To investigate the role of PML bodies in regulation of cell transformation and apoptosis-related gene transcription, we employed the immuno-FISH method to examine the relationship between PML bodies and the TP53 and BCL2 gene loci. PML bodies were found to localize specifically with the TP53 locus in about 50% of Jurkat interphase nuclei, but never in proximity with the BCL2 locus. We speculate that PML bodies may interact directly with the TP53 DNA sequence to regulate TP53 gene expression.
Asunto(s)
Expresión Génica , Genes bcl-2/genética , Genes p53/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares , Factores de Transcripción/genética , Apoptosis/genética , Transformación Celular Viral/genética , Humanos , Hibridación Fluorescente in Situ , Células Jurkat , Proteína de la Leucemia Promielocítica , Proteínas Supresoras de TumorRESUMEN
A yeast two-hybrid screen of a Jurkat (T cell) derived cDNA library, using SATB1 (a matrix attachment region binding protein) as the bait, yielded four independent isolates of a novel variant of the DNA directed RNA polymerase II, subunit 11 (RPB11). Absence of lysine-17 from the amino terminus of this variant cannot be explained by alternative mRNA splicing. Instead, allele-specific PCR, combined with GenBank database searches, suggests that a recent gene duplication event has resulted in distinct loci encoding three variant forms of RPB11. Differential splicing of mRNA transcripts accounts for unique carboxy termini among the RPB11 proteins. The exclusive association of SATB1 with one form of RPB11 is influenced primarily by the N-terminal amino acid disparity, as deletion of the entire C terminus does not alter interaction affinity. Association of RPB11 with SATB1 maps between amino acids 58 and 222 of SATB1, a region that includes a PDZ-like dimerization motif.