RESUMO
Further analysis has revealed that the signal reported in Extended Data Fig. 1c of this Letter is attributed to phosphorylethanolamine, not carbamoyl phosphate. A newly developed derivatization method revealed that the level of carbamoyl phosphate in these NSCLC extracts is below the detection threshold of approximately 10 nanomoles. These findings do not alter the overall conclusions of the Letter; see associated Amendment for full details. The Letter has not been corrected online.
RESUMO
Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.
Assuntos
Ataxia Telangiectasia , Proteína Quinase Ativada por DNA , Humanos , Proteína Quinase Ativada por DNA/genética , Reparo do DNA , Treonina/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA por Junção de Extremidades , DNA/genéticaRESUMO
Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.
Assuntos
Proteínas de Transporte/metabolismo , Caspase 2/metabolismo , Ciclo Celular , Cisteína Endopeptidases/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Caspase 2/química , Linhagem Celular , Cisteína Endopeptidases/química , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Fibroblastos/metabolismo , Raios gama , Humanos , Camundongos , Mitose , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/metabolismo , DNA/biossíntese , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Amônia/metabolismo , Animais , Bicarbonatos/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Morte Celular , Proliferação de Células , Dano ao DNA/efeitos dos fármacos , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Inativação Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Metabolômica , Camundongos , Mitocôndrias/metabolismo , Nitrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Purinas/metabolismo , Pirimidinas/farmacologia , Fase S , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.
Assuntos
Benzaldeídos/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Raios gama/efeitos adversos , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Mutação com Perda de Função , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Lesões Experimentais por Radiação/patologia , Protetores contra Radiação/uso terapêuticoRESUMO
This review focuses on DNA-dependent protein kinase (DNA-PK), which is the key regulator of canonical non-homologous end-joining (NHEJ), the predominant mechanism of DNA double-strand break (DSB) repair in mammals. DNA-PK consists of the DNA-binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs. They assemble at DNA ends, forming the active DNA-PK complex, which initiates NHEJ-mediated DSB repair. Paradoxically, both Ku and DNA-PKcs are associated with telomeres, and they play crucial roles in protecting the telomere against fusions. Herein, we discuss possible mechanisms and contributions of Ku and DNA-PKcs in telomere regulation.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Reparo do DNA por Junção de Extremidades/genética , DNA Topoisomerases Tipo II/metabolismo , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/genética , Humanos , Autoantígeno Ku/metabolismo , Complexo Shelterina , Telômero/genéticaRESUMO
The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Proteína Quinase Ativada por DNA/química , Humanos , Autoantígeno Ku/fisiologia , Proteínas Nucleares/química , Pontos de Checagem da Fase S do Ciclo Celular , Transdução de Sinais , Estresse Fisiológico , Raios UltravioletaRESUMO
BACKGROUND/AIMS: Hyperglycemia has been shown to increase the incidence and metastasis in various types of cancers. However, the correlation between hyperglycemia and lymphatic metastasis in prostate cancer (PCa) remains unclear. Our previous study demonstrated that lysophosphatidic acid (LPA) enhances vascular endothelial growth factor-C (VEGF-C) expression, a lymphangiogenic factor, through activating it receptors LPA1/3 in prostate cancer (PCa) cells. Moreover, hyperglycemia up-regulates autotaxin (ATX) expression, a LPA-generating enzyme. Therefore, we propose that high glucose promotes VEGF-C expression through LPA signaling in PCa cells. METHODS: Pharmacological inhibitors and siRNAs were utilized to investigate the molecular mechanism of high glucose-induced VEGF-C expression. Real-time PCR and Western blot were used to determine the mRNA and protein expressions, respectively. Cellular bioenergetics analysis was performed to determine the glycolysis levels. RESULTS: We demonstrated that the expressions of VEGF-C, ATX, and calreticulin were increased upon high glucose treatments in PC-3 cells. Moreover, high glucose-induced VEGF-C expression was mediated through the LPA1/3, PLC, Akt, ROS and LEDGF-dependent pathways. Additionally, high glucose enhanced the aerobic glycolysis via LPA1/3. CONCLUSION: These results indicated that hyperglycemia leads to LPA synthesis, and subsequent promoting pathological consequence of PCa. These novel findings could potentially provide new strategies for PCa treatments.
Assuntos
Glucose/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/metabolismo , Calreticulina/antagonistas & inibidores , Calreticulina/genética , Calreticulina/metabolismo , Linhagem Celular Tumoral , Glicólise , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lisofosfolipídeos/metabolismo , Masculino , Consumo de Oxigênio/efeitos dos fármacos , Fosfoinositídeo Fosfolipase C/genética , Fosfoinositídeo Fosfolipase C/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Regulação para Cima/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability.
Assuntos
Pontos de Checagem do Ciclo Celular , Instabilidade Cromossômica , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , Segregação de Cromossomos , Técnicas de Inativação de Genes , Humanos , Camundongos , Mitose/efeitos dos fármacos , Mitose/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Moduladores de Tubulina/farmacologia , Proteínas Ativadoras de ras GTPase/genética , Quinase 1 Polo-LikeRESUMO
High-charge and -energy (HZE) particles comprise space radiation and they pose a challenge to astronauts on deep space missions. While exposure to most HZE particles decreases neurogenesis in the hippocampus-a brain structure important in memory-prior work suggests that 12C does not. However, much about 12C's influence on neurogenesis remains unknown, including the time course of its impact on neurogenesis. To address this knowledge gap, male mice (9â»11 weeks of age) were exposed to whole-body 12C irradiation 100 cGy (IRR; 1000 MeV/n; 8 kEV/µm) or Sham treatment. To birthdate dividing cells, mice received BrdU i.p. 22 h post-irradiation and brains were harvested 2 h (Short-Term) or three months (Long-Term) later for stereological analysis indices of dentate gyrus neurogenesis. For the Short-Term time point, IRR mice had fewer Ki67, BrdU, and doublecortin (DCX) immunoreactive (+) cells versus Sham mice, indicating decreased proliferation (Ki67, BrdU) and immature neurons (DCX). For the Long-Term time point, IRR and Sham mice had similar Ki67+ and DCX+ cell numbers, suggesting restoration of proliferation and immature neurons 3 months post-12C irradiation. IRR mice had fewer surviving BrdU+ cells versus Sham mice, suggesting decreased cell survival, but there was no difference in BrdU+ cell survival rate when compared within treatment and across time point. These data underscore the ability of neurogenesis in the mouse brain to recover from the detrimental effect of 12C exposure.
Assuntos
Giro Denteado/citologia , Giro Denteado/efeitos da radiação , Células Piramidais/metabolismo , Células Piramidais/efeitos da radiação , Irradiação Corporal Total , Animais , Biomarcadores , Isótopos de Carbono , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Proteína Duplacortina , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Camundongos , Neurogênese , Células Piramidais/citologiaRESUMO
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.
Assuntos
Replicação do DNA , Proteína Quinase Ativada por DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Fosforilação , Complexo ShelterinaRESUMO
REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.
Assuntos
Dano ao DNA , Replicação do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Proteínas Nucleares/fisiologia , Nucleotidiltransferases/fisiologia , Camptotecina/toxicidade , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Conversão Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estresse Fisiológico/genética , Inibidores da Topoisomerase I/toxicidade , Ubiquitina-Proteína LigasesRESUMO
Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including ß-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 µm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.
Assuntos
Senescência Celular/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Genoma Humano , Selenoproteínas/metabolismo , Acetilcisteína/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Senescência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Instabilidade Genômica , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo , Paraquat/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Selenoproteínas/antagonistas & inibidores , Selenoproteínas/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.
Assuntos
Ciclo Celular/efeitos dos fármacos , Proteína Quinase Ativada por DNA/metabolismo , Mitose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Proteína Quinase Ativada por DNA/genética , Citometria de Fluxo , Células HCT116 , Células HeLa , Humanos , Immunoblotting , Microtúbulos/metabolismo , Mitose/genética , Nocodazol/farmacologia , Fosforilação/efeitos dos fármacosRESUMO
DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.
Assuntos
Dano ao DNA , Proteína de Replicação A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Fase G2/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilação/genética , Proteína da Leucemia Promielocítica , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/metabolismo , Fase S/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. RESULTS: In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. CONCLUSIONS: Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.
Assuntos
Caseína Quinase II/metabolismo , Quebras de DNA de Cadeia Dupla , Antígenos Nucleares/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Autoantígeno Ku , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.
Assuntos
Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Animais , Antígenos Nucleares/metabolismo , Células CHO , Cricetinae , Cricetulus , DNA/metabolismo , Proteína Quinase Ativada por DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , Lasers , Fosforilação , FotodegradaçãoRESUMO
Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activation of multiple LPA G protein-coupled receptors. We previously reported that LPA suppresses oxidative stress in premature aging Hutchinson-Gilford progeria syndrome (HGPS) patient fibroblasts via its type 3 receptor (LPA3). Mitochondria have been suggested to be the primary origin of oxidative stress via the overproduction of reactive oxygen species (ROS). Mitochondria are responsible for producing ATP through oxidative phosphorylation (OXPHOS) and have a calcium buffering capacity for the cell. Defects in mitochondria will lead to declined antioxidant capacity and cell apoptosis. Therefore, we aim to demonstrate the regulatory role of LPA3 in mitochondrial homeostasis. siRNA-mediated depletion of LPA3 leads to the depolarization of mitochondrial potential (ΔΨm) and cellular ROS accumulation. In addition, the depletion of LPA3 enhances cisplatin-induced cytochrome C releasing. This indicates that LPA3 is essential to suppress the mitochondrial apoptosis pathway. LPA3 is also shown to improve mitochondrial ADP-ATP exchange by enhancing the protein level of ANT2. On the other hand, LPA3 regulates calcium uptake from the ER to mitochondria via the IP3R1-VDAC1 channel. Moreover, activation of LPA3 by selective agonist OMPT rescues mitochondrial homeostasis of H2O2-induced oxidative stress cells and HGPS patient fibroblasts by improving mitochondrial ΔΨm and OXPHOS. In summary, our findings imply that LPA3 acts as the gatekeeper for mitochondrial healthiness to maintain cell youth. Furthermore, LPA3 can be a promising therapeutic target to prevent mitochondrial oxidative stress in aging and HGPS.
RESUMO
The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), telomeric repeat-containing RNA (TERRA), and protection of telomeres 1 (POT1) have been reported to orchestrate to displace replication protein A (RPA) from telomeric overhangs, ensuring orderly telomere replication and capping. Our previous studies further demonstrated that DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-dependent hnRNPA1 phosphorylation plays a crucial role in the promotion of hnRNPA1 binding to telomeric overhangs and RPA displacement during G2-M phases. However, it is unclear that how the subsequent exchange between hnRNPA1 and POT1 is orchestrated. Here we report that the protein phosphatase 2A (PP2A) depends on its scaffold subunit, which is called PPP2R1A, to interact with and dephosphorylate hnRNPA1 in the late M phase. Furthermore, PP2A-mediated hnRNPA1 dephosphorylation and TERRA accumulation act in concert to promote the hnRNPA1-to-POT1 switch on telomeric single-stranded DNA. Consequently, defective PPP2R1A results in ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage response at telomeres as well as induction of fragile telomeres. Combined inhibition of ATR and PP2A induces entry into a catastrophic mitosis and leads to synthetic lethality of tumor cells. In addition, PPP2R1A levels correlate with clinical stages and prognosis of multiple types of cancers. Taken together, our results indicate that PP2A is critical for telomere maintenance. IMPLICATIONS: This study demonstrates that the PP2A-dependent hnRNPA1 dephosphorylation and TERRA accumulation facilitates the formation of the protective capping structure of newly replicated telomeres, thus exerting essential oncogenic role in tumorigenesis.
Assuntos
Proteína Fosfatase 2 , Proteínas de Ligação a Telômeros , Proteínas de Ligação a DNA , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Fatores de TranscriçãoRESUMO
Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP's interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.