RESUMO
Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. Here, we show that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. We propose that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes.
Assuntos
Cromossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Xenopus/metabolismo , Animais , Anticorpos/imunologia , Aurora Quinases , Extratos Celulares , Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/imunologia , Proteínas Cromossômicas não Histona/metabolismo , Ativação Enzimática , Histonas/metabolismo , Microtúbulos/metabolismo , Óvulo , Fosforilação , Ligação Proteica , Fuso Acromático/química , Estatmina/metabolismo , Proteínas de Xenopus/imunologia , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , DNA Helicases/genética , DNA de Cadeia Simples/genética , Humanos , Mutação , Fosforilação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Xenopus laevisRESUMO
Using a variety of biochemical and cell-based approaches, we show that estrogen receptor alpha (ERalpha) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERalpha (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERalpha acetylated at Lys266 and Lys268. The acetylation of ERalpha by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERalpha in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERalpha in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand-dependent gene regulatory activity of ERalpha. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.
Assuntos
DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Lisina/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Gatos , Compartimento Celular , Células Cultivadas , Sequência Conservada , Cricetinae , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Niacinamida/farmacologia , Coativador 2 de Receptor Nuclear/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Sirtuínas/metabolismo , Ativação TranscricionalRESUMO
Nuclear shape and size vary between species, during development, and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). Whereas microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects, leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Núcleo Celular/ultraestrutura , Replicação do DNA , Microtúbulos/ultraestrutura , Proteínas Nucleares/genética , Polimerização , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.
Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Switching de Imunoglobulina/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina/genética , Translocação Genética/efeitos dos fármacosRESUMO
We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC).
Assuntos
Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Fosfopeptídeos/análise , Fosfopeptídeos/química , Sequência de Aminoácidos , Animais , Aurora Quinases , Cromossomos/química , Cromossomos/metabolismo , Estrutura Molecular , Peso Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Xenopus laevisRESUMO
The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1-Cul1-F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.
Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Ubiquitinação , Animais , Antígenos Nucleares/química , Proteínas de Ligação a DNA/química , Autoantígeno Ku , Modelos Biológicos , Oligonucleotídeos/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Recombinação Genética/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , XenopusRESUMO
Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.
Assuntos
Linfócitos B/fisiologia , Citidina Desaminase/metabolismo , Switching de Imunoglobulina/fisiologia , Mutação Puntual/genética , Animais , Linfócitos B/metabolismo , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , FosforilaçãoRESUMO
Activation-induced cytidine deaminase (AID) initiates Ig class switch recombination and somatic hypermutation by producing U:G mismatches in DNA. These mismatches also have the potential to induce DNA damage including double-stranded breaks and chromosome translocations; therefore, strict regulation of AID is important for maintaining genomic stability. In addition to transcriptional regulation, it has been proposed that phosphorylation can also modulate AID activity. Using a combination of MS and immunochemical approaches we found that 5-15% of the AID expressed in activated B cells was phosphorylated at serine-38 (p38AID). This form of AID was enriched in the chromatin fraction in activated B cells, suggesting a role for phosphorylation in targeting AID to DNA. Consistent with this idea, serine-38 to alanine mutant AID (AID(S38A)) showed diminished somatic hypermutation activity on artificial and physiological DNA targets. We conclude that a small fraction of AID is phosphorylated in activated B cells and that the modified form contributes disproportionately to hypermutation.