RESUMEN
Phosphorylation of Chk1 by ataxia telangiectasia-mutated and Rad3-related (ATR) is critical for checkpoint activation upon DNA damage. However, how phosphorylation activates Chk1 remains unclear. Many studies suggest a conformational change model of Chk1 activation in which phosphorylation shifts Chk1 from a closed inactive conformation to an open active conformation during the DNA damage response. However, no structural study has been reported to support this Chk1 activation model. Here we used FRET and bimolecular fluorescence complementary techniques to show that Chk1 indeed maintains a closed conformation in the absence of DNA damage through an intramolecular interaction between a region (residues 31-87) at the N-terminal kinase domain and the distal C terminus. A highly conserved Leu-449 at the C terminus is important for this intramolecular interaction. We further showed that abolishing the intramolecular interaction by a Leu-449 to Arg mutation or inducing ATR-dependent Chk1 phosphorylation by DNA damage disrupts the closed conformation, leading to an open and activated conformation of Chk1. These data provide significant insight into the mechanisms of Chk1 activation during the DNA damage response.
Asunto(s)
Daño del ADN , Proteínas Quinasas/química , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Fosforilación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/metabolismoRESUMEN
DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Camptotecina/química , Línea Celular Tumoral , Supervivencia Celular , Daño del ADN , Células HEK293 , Humanos , Fosforilación , Pronóstico , Complejo de la Endopetidasa Proteasomal/química , ARN Interferente Pequeño/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina/químicaRESUMEN
Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.
Asunto(s)
Puntos de Control del Ciclo Celular , Replicación del ADN , Regulación de la Expresión Génica , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas Quinasas/metabolismo , Alanina/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromatina/química , Daño del ADN , ADN de Cadena Simple/genética , Células HEK293 , Células HeLa , Humanos , Espectrometría de Masas , Fosforilación , Serina/química , Transducción de SeñalRESUMEN
Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.
Asunto(s)
Daño del ADN , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Quinasas/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Estrés Oxidativo , Fosforilación , Unión ProteicaRESUMEN
Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Represoras/deficiencia , Transactivadores/deficiencia , Animales , Apoptosis/fisiología , Western Blotting , Citometría de Flujo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Transactivadores/genéticaRESUMEN
Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Señales de Exportación Nuclear/fisiología , Proteínas Quinasas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Secuencias de Aminoácidos , Núcleo Celular/genética , Supervivencia Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Citoplasma/genética , Células HEK293 , Células HeLa , Heterocigoto , Humanos , Mutación , Proteínas Quinasas/genéticaRESUMEN
The Wnt pathway is frequently dysregulated in many cancers, underscoring it as a therapeutic target. Wnt inhibitors have uniformly failed in clinical trials. Here, we report a mechanism of WNT pathway activation through the Semaphorin 3 C neurodevelopmental program in glioma stem-like cells. Sema3C directs ß-catenin nuclear accumulation in a Rac1-dependent process, leading to transactivation of Wnt target genes. Sema3C-driven Wnt signaling occurred despite suppression of Wnt ligand secretion, suggesting that Sema3C drives canonical Wnt signaling independent of Wnt ligand binding. In a mouse model of glioblastoma, combined depletion of Sema3C and ß-catenin partner TCF1 extended animal survival more than single target inhibition alone. In human glioblastoma, Sema3C expression and Wnt pathway activation were highly concordant. Since Sema3C is frequently overexpressed in glioblastoma, Sema3C signaling may be a significant mechanism of resistance to upstream Wnt pathway inhibitors. Dual targeting of Sema3C and Wnt pathways may achieve clinically significant Wnt pathway inhibition.
Asunto(s)
Glioblastoma , Semaforinas , Animales , Humanos , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Ligandos , Semaforinas/genética , Vía de Señalización Wnt/genéticaRESUMEN
We identified two S/T-P-S/T domains (2122-2124, 2126-2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963-2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3ß enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3ß phosphorylated S and T residues in NICD S/T-P-S/T domains. These results suggest that the novel S/T-P-S/T domain, phosphorylated by GSK-3ß is also involved in the nuclear localization of NICD as well as classical NLS.
Asunto(s)
Núcleo Celular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Señales de Localización Nuclear/metabolismo , Receptor Notch1/metabolismo , Transporte Activo de Núcleo Celular , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Ratones , Señales de Localización Nuclear/genética , Fosforilación , Estructura Terciaria de Proteína , Receptor Notch1/química , Receptor Notch1/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Piwi proteins are a subfamily of Argonaute proteins that maintain germ cells in eukaryotes. However, the role of their human homologs in cancer stem cells, and more broadly in cancer, is poorly understood. Here, we report that Piwi-like family members are overexpressed in glioblastoma (GBM), with Piwil1 (Hiwi) most frequently overexpressed (88%). Piwil1 is enriched in glioma stem-like cells (GSCs) to maintain self-renewal. Silencing Piwil1 in GSCs leads to global changes in gene expression resulting in cell-cycle arrest, senescence, or apoptosis. Piwil1 knockdown increases expression of the transcriptional co-regulator BTG2 and the E3-ubiquitin ligase FBXW7, leading to reduced c-Myc expression, as well as loss of expression of stem cell factors Olig2 and Nestin. Piwil1 regulates mRNA stability of BTG2, FBXW7, and CDKN1B. In animal models of GBM, Piwil1 knockdown suppresses tumor growth and promotes mouse survival. These findings support a role of Piwil1 in GSC maintenance and glioblastoma progression.
Asunto(s)
Proteínas Argonautas/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glioma/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Argonautas/genética , Neoplasias Encefálicas/genética , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioma/genética , Humanos , Masculino , Ratones , Nestina/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estabilidad del ARNRESUMEN
Danshen is widely used in traditional Chinese medicine, often in combination with other herbs. To check the effect of Danshen on the proliferation of breast cancer cells, Danshen extract was used to treat MCF-7 and MCF-7 HER2 cells, the latter of which overexpresses HER2. HER2 is a receptor tyrosine kinase, and is involved in signal transduction pathways leading to tumor cell proliferation. MTT and cell proliferation assays revealed that Danshen strongly inhibited the proliferation of both MCF-7 vec cells and MCF-7 HER2 cells. Flow cytometry analyses indicated that Danshen induced cell cycle delay in the G1 phase. HER2 expression was shown to confer resistance to Danshen-induced inhibition of proliferation and cell cycle delay, suggesting that HER2 is responsible for the resistance to Danshen. Danshen treatment induced the down-regulation of Akt phosphorylation and an increase in p27 in MCF-7 vec and MCF-7 HER2 cells. Nevertheless, MCF-7 HER2 cells were more resistant to the Danshen-induced inhibition of Akt phosphorylation and p27 up-regulation.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Medicamentos Herbarios Chinos/farmacología , Fenantrolinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Fosforilación , Receptor ErbB-2/metabolismo , Salvia miltiorrhiza/químicaRESUMEN
5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent.
Asunto(s)
Antineoplásicos/uso terapéutico , Floxuridina/uso terapéutico , Neoplasias/tratamiento farmacológico , Uracil-ADN Glicosidasa/metabolismo , Uracilo/metabolismo , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Daño del ADN , Replicación del ADN , Resistencia a Medicamentos , Floxuridina/farmacología , Histonas/metabolismo , Humanos , ARN Interferente Pequeño/genética , Uracil-ADN Glicosidasa/genéticaRESUMEN
Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase, is centrally involved in cell-cycle checkpoints and cellular response to DNA damage. Phosphorylation of Chk1 at 2 Ser/Gln (SQ) sites, Ser-317 and Ser-345, by the upstream kinase ATR is critical for checkpoint activation. However, the precise molecular mechanisms controlling Chk1 phosphorylation and subsequent checkpoint activation are not well understood. Here, we report unique autoregulatory mechanisms that control protein phosphorylation of human Chk1, as well as checkpoint activation and cell viability. Phosphorylation of Ser-317 is required, but not sufficient, for maximal phosphorylation at Ser-345. The N-terminal kinase domain of Chk1 prevents Chk1 phosphorylation at the C-terminus by ATR in the absence of DNA damage. Loss of the inhibitory effect imposed by the N-terminus causes constitutive phosphorylation of Chk1 by ATR under normal growth conditions, which in turn triggers artificial checkpoints that suppress the S-phase progression. Furthermore, two point mutations were identified that rendered Chk1 constitutively active, and expression of the constitutively active mutant form of Chk1 inhibited cancer cell proliferation. Our findings therefore reveal unique regulatory mechanisms of Chk1 phosphorylation and suggest that expression of constitutively active Chk1 may represent a novel strategy to suppress tumor growth. Cancer Res; 72(15); 3786-94. ©2012 AACR.
Asunto(s)
Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Dominio Catalítico/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Células HEK293 , Células HeLa , Homeostasis/fisiología , Humanos , Fosforilación/genética , Mutación Puntual/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas Serina-Treonina Quinasas/fisiología , Serina/genética , Serina/metabolismoRESUMEN
BRCA2 is responsible for familial breast and ovarian cancer, and its gene product is linked to DNA repair and transcriptional regulation. The BRCA2 protein exists mainly in the nucleus. Here, we show that BRCA2 has a centrosomal localization signal (CLS), localizes also to centrosomes during S and early M phases, and may regulate duplication and separation of the centrosomes. Green fluorescent protein (GFP) fused to the CLS peptides from BRCA2 (GFP-CLS) localizes to centrosomes and prevents endogenous BRCA2 from localizing to centrosomes. In addition, expression of GFP-CLS in cells leads to the abnormal duplication and positioning of centrosomes, resulting in the generation of multinuclear cells. These results thus implicate BRCA2 in the regulation of the centrosome cycle, and provide new insight into the aneuploid nature of many breast cancers.