RESUMEN
Excessive activation of CD4+ T cells and T helper type (Th) 17/Th1 cell differentiation are critical events in psoriasis pathogenesis, but the associated molecular mechanism is still unclear. Here, using quantitative proteomics analysis, we found that cyclin-dependent kinase 7 (CDK7) expression was markedly increased in CD4+ T cells from patients with psoriasis compared with healthy controls and was positively correlated with psoriasis severity. Meanwhile, genetic or pharmacological inhibition of CDK7 ameliorated the severity of psoriasis in the imiquimod-induced psoriasis-like mouse model and suppressed CD4+ T-cell activation as well as Th17/Th1 cell differentiation in vivo and in vitro. Furthermore, the CDK7 inhibitor also reduced the enhanced glycolysis of CD4+ T cells from patients with psoriasis. Proinflammatory cytokine IL-23 induced increased CDK7 expression in CD4+ T cells and activated the protein kinase B/mTOR/HIF-1α signaling pathway, enhancing glycolytic metabolism. Correspondingly, CDK7 inhibition significantly impaired IL-23-induced glycolysis via the protein kinase B/mTOR/HIF-1α pathway. In summary, this study shows that CDK7 promotes CD4+ T-cell activation and Th17/Th1 cell differentiation by regulating glycolysis, thus contributing to the pathogenesis of psoriasis. Targeting CDK7 might be a promising immunosuppressive strategy to control skin inflammation mediated by IL-23.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Glucólisis , Psoriasis/inmunología , Células TH1/citología , Células Th17/citología , Animales , Diferenciación Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Interleucina-23/fisiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Psoriasis/etiología , Psoriasis/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
PURPOSE: Among human cancers that harbor mutant (mt) KRas, some, but not all, are dependent on mt KRas. However, little is known about what drives KRas dependency. EXPERIMENTAL DESIGN: Global phosphoproteomics, screening of a chemical library of FDA drugs, and genome-wide CRISPR/Cas9 viability database analysis were used to identify vulnerabilities of KRas dependency. RESULTS: Global phosphoproteomics revealed that KRas dependency is driven by a cyclin-dependent kinase (CDK) network. CRISPR/Cas9 viability database analysis revealed that, in mt KRas-driven pancreatic cancer cells, knocking out the cell-cycle regulators CDK1 or CDK2 or the transcriptional regulators CDK7 or CDK9 was as effective as knocking out KRas. Furthermore, screening of a library of FDA drugs identified AT7519, a CDK1, 2, 7, and 9 inhibitor, as a potent inducer of apoptosis in mt KRas-dependent, but not in mt KRas-independent, human cancer cells. In vivo AT7519 inhibited the phosphorylation of CDK1, 2, 7, and 9 substrates and suppressed growth of xenografts from 5 patients with pancreatic cancer. AT7519 also abrogated mt KRas and mt p53 primary and metastatic pancreatic cancer in three-dimensional (3D) organoids from 2 patients, 3D cocultures from 8 patients, and mouse 3D organoids from pancreatic intraepithelial neoplasia, primary, and metastatic tumors. CONCLUSIONS: A link between CDK hyperactivation and mt KRas dependency was uncovered and pharmacologically exploited to abrogate mt KRas-driven pancreatic cancer in highly relevant models, warranting clinical investigations of AT7519 in patients with pancreatic cancer.
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Quinasas Ciclina-Dependientes/fisiología , Neoplasias Pancreáticas/etiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Animales , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Ratones , Fosforilación , ProteomaRESUMEN
Besides its prominent role in cell proliferation, cyclin-dependent kinases (CDKs) are key players in viral infections as both DNA and RNA viruses modify CDK function to favor viral replication. Recently, a number of specific pharmacological CDK inhibitors have been developed and approved for cancer treatment. The repurposing of these specific CDK inhibitors for the treatment of viral infections may represent a novel effective therapeutic strategy to combat old and emergent viruses. In this review, we describe the role, mechanisms of action, and potential of CDKs as antiviral drug targets. We also discuss the current clinical state of novel specific CDK inhibitors, focusing on their putative use as antivirals, especially against new emerging viruses.
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Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Virosis/tratamiento farmacológico , Animales , Antivirales/uso terapéutico , Quinasas Ciclina-Dependientes/fisiología , Reposicionamiento de Medicamentos , Humanos , Virosis/enzimologíaRESUMEN
Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zebrafish overexpression analyses also showed that the p.G28A and p.N156S alleles were hypomorphic alleles. Importantly, the inhibition of CDK8 kinase activity in zebrafish embryos using a specific chemical inhibitor induced craniofacial and heart defects similar to the patients' phenotype. Taken together, zebrafish studies showed that non-synonymous variants in the kinase domain of CDK8 act as hypomorphic alleles causing human congenital disorder.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/genética , Mutación Missense , Trastornos del Neurodesarrollo/genética , Mutación Puntual , Anomalías Múltiples/genética , Animales , Niño , Anomalías Craneofaciales/genética , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/deficiencia , Quinasa 8 Dependiente de Ciclina/fisiología , Quinasas Ciclina-Dependientes/fisiología , Embrión no Mamífero/anomalías , Embrión no Mamífero/enzimología , Femenino , Cardiopatías Congénitas/genética , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Discapacidad Intelectual/genética , Mutación con Pérdida de Función , Masculino , Dominios Proteicos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/fisiologíaRESUMEN
Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Cromatina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Células HEK293 , Humanos , Mutación , Fosforilación , ARN/biosíntesis , ARN Polimerasa II/química , Análisis de Secuencia de ARN , Serina/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
Cyclin-dependent kinases (CDKs) are a group of serine/threonine protein kinases and play crucial roles in various cellular processes by regulating cell cycle and gene transcription. Cyclin-dependent kinase 12 (CDK12) is an important transcription-associated CDK. It shows versatile roles in regulating gene transcription, RNA splicing, translation, DNA damage response (DDR), cell cycle progression and cell proliferation. Recently, increasing evidence demonstrates the important role of CDK12 in various human cancers, illustrating it as both a biomarker of cancer and a potential target for cancer therapy. Here, we summarize the current knowledge of CDK12, and review the research advances of CDK12's biological functions, especially its role in human cancers and as a potential target and biomarker for cancer therapy.
Asunto(s)
Biomarcadores de Tumor/fisiología , Quinasas Ciclina-Dependientes/fisiología , Neoplasias/enzimología , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Daño del ADN , Reparación del ADN , Femenino , Expresión Génica , Humanos , Masculino , Neoplasias/genética , Neoplasias/terapia , Biosíntesis de Proteínas , Empalme del ARN , Transcripción GenéticaRESUMEN
BACKGROUND: CDK12 is a promising therapeutic target in breast cancer with an effective ability of maintaining cancer cell stemness. OBJECTIVE: We aim to investigate the mechanism of CDK12 in maintaining breast cancer stemness. METHODS: CDK12 expression level was accessed by using RT-qPCR and IHC. CDK12-altered breast cancer cell lines MDA-MB-231-shCDK12 and SkBr-3-CDK12 were then established. CCK8, colony formation assays, and xenograft model were used to value the effect of CDK12 on tumorigenicity. Transwell assay, mammosphere formation, FACS, and lung metastasis model in vivo were determined. Western blot further characterized the mechanism of CDK12 in breast cancer stemness through the c-myc/ß-catenin pathway. RESULTS: Our results showed a higher level of CDK12 exhibited in breast cancer samples. Tumor formation, cancer cell mobility, spheroid forming, and the epithelial-mesenchymal transition will be enhanced in the CDK12high group. In addition, CDK12 was associated with lung metastasis and maintained breast cancer cell stemness. CDK12high cancer cells presented higher tumorigenicity and a population of CD44+ subset compared with CDK12low cells. Our study demonstrated c-myc positively expressed with CDK12. The c-myc/ß-catenin signaling was activated by CDK12, which is a potential mechanism to initiate breast cancer stem cell renewal and may serve as a potential biomarker of breast cancer prognosis. CONCLUSION: CDK12 overexpression promotes breast cancer tumorigenesis and maintains the stemness of breast cancer by activating c-myc/ß-catenin signaling. Inhibiting CDK12 expression may become a potential therapy for breast cancer.
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Neoplasias de la Mama/etiología , Quinasas Ciclina-Dependientes/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , beta Catenina/fisiología , Adulto , Anciano , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Quinasas Ciclina-Dependientes/análisis , Quinasas Ciclina-Dependientes/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Persona de Mediana Edad , Vía de Señalización Wnt/fisiologíaRESUMEN
Sae2 functions in the DNA damage response by controlling Mre11-Rad50-Xrs2 (MRX)-catalyzed end resection, an essential step for homology-dependent repair of double-strand breaks (DSBs), and by attenuating DNA damage checkpoint signaling. Phosphorylation of Sae2 by cyclin-dependent kinase (CDK1/Cdc28) activates the Mre11 endonuclease, while the physiological role of Sae2 phosphorylation by Mec1 and Tel1 checkpoint kinases is not fully understood. Here, we compare the phenotype of sae2 mutants lacking the main CDK (sae2-S267A) or Mec1 and Tel1 phosphorylation sites (sae2-5A) with sae2Δ and Mre11 nuclease defective (mre11-nd) mutants. The phosphorylation-site mutations confer DNA damage sensitivity, but not to the same extent as sae2Δ. The sae2-S267A mutation is epistatic to mre11-nd for camptothecin (CPT) sensitivity and synergizes with sgs1Δ, whereas sae2-5A synergizes with mre11-nd and exhibits epistasis with sgs1Δ. We find that attenuation of checkpoint signaling by Sae2 is mostly independent of Mre11 endonuclease activation but requires Mec1 and Tel1-dependent phosphorylation of Sae2. These results support a model whereby CDK-catalyzed phosphorylation of Sae2 activates resection via Mre11 endonuclease, whereas Sae2 phosphorylation by Mec1 and Tel1 promotes resection by the Dna2-Sgs1 and Exo1 pathways indirectly by dampening the DNA damage response.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Daño del ADN/fisiología , Endonucleasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Catálisis , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Organismos Modificados Genéticamente , Fosforilación/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/genéticaRESUMEN
CDK8 and CDK19 Mediator kinases are transcriptional co-regulators implicated in several types of cancer. Small-molecule CDK8/19 inhibitors have recently entered or are entering clinical trials, starting with breast cancer and acute myeloid leukemia (AML). To identify other cancers where these novel drugs may provide benefit, we queried genomic and transcriptomic databases for potential impact of CDK8, CDK19, or their binding partner CCNC. sgRNA analysis of a panel of tumor cell lines showed that most tumor types represented in the panel, except for some central nervous system tumors, were not dependent on these genes. In contrast, analysis of clinical samples for alterations in these genes revealed a high frequency of gene amplification in two highly aggressive subtypes of prostate cancer and in some cancers of the GI tract, breast, bladder, and sarcomas. Analysis of survival correlations identified a group of cancers where CDK8 expression correlated with shorter survival (notably breast, prostate, cervical cancers, and esophageal adenocarcinoma). In some cancers (AML, melanoma, ovarian, and others), such correlations were limited to samples with a below-median tumor mutation burden. These results suggest that Mediator kinases are especially important in cancers that are driven primarily by transcriptional rather than mutational changes and warrant an investigation of their role in additional cancer types.
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Ciclina C/fisiología , Quinasa 8 Dependiente de Ciclina/fisiología , Quinasas Ciclina-Dependientes/fisiología , Neoplasias/metabolismo , Línea Celular Tumoral , Ciclina C/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Bases de Datos Genéticas , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Proper wound healing is dynamic in order to maintain the corneal integrity and transparency. Impaired or delayed corneal epithelial wound healing is one of the most frequently observed ocular defect and difficult to treat. Cyclin dependen kinase (cdk), a known cell cycle regulator, required for proper proliferating and migration of cell. We therefore investigated the role of cell cycle regulator cdk10, member of cdk family and its functional association with transcriptional factor (ETS2) at active phase of corneal epithelial cell migration. Our data showed that cdk10 was associated with ETS2, while its expression was upregulated at the active phase (18 hours) of cell migration and gradually decrease as the wound was completely closed. Topical treatment with anti-cdk10 and ETS2 antibodies delayed the wound closure time at higest concentration (10 µg/ml) compared to control. Further, our results also showed increased mRNA expression of cdk10 and ETS2 at active phase of migration at approximately 2 fold. Collectively, our data reveals that cdk10 and ETS2 efficiently involved during corneal wound healing. Further studies are warranted to better understand the mechanism and safety of topical cdk10 and ETS2 proteins in corneal epithelial wound-healing and its potential role for human disease treatment.
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Lesiones de la Cornea/patología , Quinasas Ciclina-Dependientes/fisiología , Epitelio Corneal/patología , Proteína Proto-Oncogénica c-ets-2/fisiología , Cicatrización de Heridas , Lesiones de la Cornea/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Epitelio Corneal/enzimología , Epitelio Corneal/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Proteína Proto-Oncogénica c-ets-2/metabolismoRESUMEN
Kindler syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering and photosensitivity. KS is caused by loss of function mutations in FERMT1, which encodes Kindlin-1. Kindlin-1 is a FERM domain containing adaptor protein that is found predominantly at cell-extracellular matrix adhesions where it binds to integrin ß subunits and is required for efficient integrin activation. Using keratinocytes derived from a patient with KS, into which wild-type Kindlin-1 (Kin1WT) has been expressed, we show that Kindlin-1 binds to cyclin-dependent kinase (CDK)1 and CDK2. CDK1 and CDK2 are key regulators of cell cycle progression, however, cell cycle analysis showed only small differences between the KS and KS-Kin1WT keratinocytes. In contrast, G2/M cell cycle arrest in response to oxidative stress induced by hydrogen peroxide (H2 O2 ) was enhanced in KS keratinocytes but not KS-Kin1WT cells, following inhibition of CDK activity. Furthermore, KS keratinocytes were more sensitive to DNA damage in response to H2 O2 and this was exacerbated by treatment with the CDK inhibitor roscovitine. Thus, in Kindlin-1 deficient keratinocytes, CDK activity can further regulate oxidative damage induced cell cycle arrest and DNA damage. This provides further insight into the key pathways that control sensitivity to oxidative stress in KS patients.
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Vesícula/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Daño del ADN/efectos de los fármacos , Epidermólisis Ampollosa/patología , Queratinocitos/enzimología , Estrés Oxidativo/efectos de los fármacos , Enfermedades Periodontales/patología , Trastornos por Fotosensibilidad/patología , Roscovitina/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Genes Reporteros , Humanos , Peróxido de Hidrógeno/toxicidad , Cadenas beta de Integrinas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in ArabidopsisArabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Quinasas Ciclina-Dependientes/fisiología , Recombinación Genética , Alelos , Arabidopsis/citología , Proteínas de Arabidopsis/fisiología , Cromosomas de las Plantas , Intercambio Genético , MeiosisRESUMEN
Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. Most of our understanding of their functions has been obtained from studies in single-cell organisms and mitotically proliferating cultured cells. In mammals, there are more than 20 cyclins and 20 CDKs. Although genetic ablation studies in mice have shown that most of these factors are dispensable for viability and fertility, uncovering their functional redundancy, CCNA2, CCNB1, and CDK1 are essential for embryonic development. Cyclin/CDK complexes are known to regulate both mitotic and meiotic cell cycles. While some mechanisms are common to both types of cell divisions, meiosis has unique characteristics and requirements. During meiosis, DNA replication is followed by two successive rounds of cell division. In addition, mammalian germ cells experience a prolonged prophase I in males or a long period of arrest in prophase I in females. Therefore, cyclins and CDKs may have functions in meiosis distinct from their mitotic functions and indeed, meiosis-specific cyclins, CCNA1 and CCNB3, have been identified. Here, we describe recent advances in the field of cyclins and CDKs with a focus on meiosis and early embryogenesis.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Ciclinas/fisiología , Gametogénesis/genética , Células Germinativas/fisiología , Animales , Embrión de Mamíferos , Femenino , Humanos , Masculino , Mamíferos , Meiosis , RatonesRESUMEN
BACKGROUND: Cyclin-dependent kinase-like 1 (CDKL1) is a member of the cell division control protein 2-related serine-threonine protein kinase family. It is known to occur in various malignant tumors, but its role in neuroblastoma (NB) remains unclear. METHODS: We constructed a CDKL1-silenced NB cell strain (SH-SY5Y) and used real-time PCR and western blotting to confirm the silencing. Functional analyses were performed using the MTT, colony-formation, FACS, wound-healing and transwell invasion assays. RESULTS: The expression of CDKL1 was significantly upregulated in NB tissue as compared to the adjacent normal tissue. CDKL1 knockdown significantly suppressed cell viability and colony formation ability. It also induced cell cycle G0/G1 phase arrest and apoptosis, and suppressed the migration and invasion ability of SH-SY5Y cells. CDKL1 knockdown decreased the CDK4, cyclin D1 and vimentin expression levels, and increased the caspase-3, PARP and E-cadherin expression levels in SH-SY5Y cells. CONCLUSIONS: Our findings suggest that CDKL1 plays an important role in NB cell proliferation, migration and invasion. It might serve as a potential target for NB therapy.
Asunto(s)
Movimiento Celular , Proliferación Celular , Quinasas Ciclina-Dependientes/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuroblastoma/genética , Apoptosis , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/fisiopatologíaRESUMEN
Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/metabolismo , Biología Computacional/métodos , Ciclina C/genética , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasa 8 Dependiente de Ciclina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitosis , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cyclins and cyclin-dependent protein kinases (CDKs) are important proteins that are required for the regulation and expression of the large number of components necessary for the passage through the cell cycle. The concentrations of the CDKs are generally constant, but their activities are controlled by the oscillation of the cyclin levels during each cell cycle. Additional CDK family members play significant roles in a wide range of activities including gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1-phase of the cell cycle produce D type cyclins that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein. Subsequently, CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factor, which in turn lead to the biosynthesis of dozens of proteins required for cell cycle progression. Consequently, cells pass the G1-restriction point and are committed to complete cell division in the absence of mitogenic stimulation. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S-, G2-, and M-phase progression. A crucial mechanism in controlling cell cycle progression is the precise timing of more than 32,000 phosphorylation and dephosphorylation reactions catalyzed by a network of protein kinases and phosphoprotein phosphatases as determined by mass spectrometry. Increased cyclin or CDK expression or decreased levels of endogenous CDK modulators/inhibitors such as INK4 or CIP/KIP have been observed in a wide variety of carcinomas, hematological malignancies, and sarcomas. The pathogenesis of neoplasms because of mutations in the CDKs are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Palbociclib, ribociclib, and abemaciclib are FDA-approved CDK4/6 inhibitors used in the treatment of breast cancer. These drugs have IC50 values for CKD4/6 in the low nanomolar range. These inhibitors bind in the cleft between the N-terminal and C-terminal lobes of the CDKs and they inhibit ATP binding. Like ATP, these agents form hydrogen bonds with hinge residues that connect the small and large lobes of protein kinases. Like the adenine base of ATP, these antagonists interact with catalytic spine residues CS6, CS7, and CS8. These and other CDK antagonists are in clinical trials for the treatment of a wide variety of malignancies. As inhibitors of the cell cycle, it is not surprising that one of their most common toxicities is myelosuppression with decreased neutrophil production.
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Antineoplásicos/uso terapéutico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Ciclo Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/fisiología , Humanos , Ligandos , Neoplasias/tratamiento farmacológicoRESUMEN
CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.
Asunto(s)
Caseína Quinasa Ialfa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Caseína Quinasa Ialfa/fisiología , Proliferación Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/fisiología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/fisiología , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos/genética , Hematopoyesis , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Proteína p53 Supresora de Tumor/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: SEVEN IN ABSENTIA (SINA) is a RING domain-containing ubiquitin ligase involved in Drosophila eye formation. SINA-like proteins in plants are involved in several signaling pathways. Of the 18 SINA-like proteins identified in Arabidopsis, SEVEN IN ABSENTIA 2 (SINA2) lacks a canonical RING domain and is thought to lack ubiquitin ligase activity. RESULTS: Our results show that SINA2 has E3 ligase activity in vitro, raising the possibility that a modified B-box domain may compensate for its lack of a RING domain. SINA2 physically interacts with the nuclear protein CYCLIN-DEPENDENT KINASE G1 (CDKG1), which acts as a positive regulator of plant responses to abiotic stress. CDKG1 is expressed in multiple tissues and its expression increased in response to abscisic acid (ABA) and osmotic stress. Transgenic Arabidopsis plants that ectopically express CDKG1 exhibit increased tolerance to ABA and osmotic stress treatments during seed germination and cotyledon development, while the loss-of-function cdkg1 mutant plants show reduced tolerance to ABA and osmotic stress treatments. Moreover, CDKG1-dependent phosphorylation of SINA2 positively affects its E3 ubiquitin ligase activity. CONCLUSIONS: Based on these results, we propose that CDKG1 modulates SINA2 ubiquitin ligase activity to regulate its effect on plant responses to ABA and osmotic stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/fisiología , Quinasas Ciclina-Dependientes/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Presión Osmótica , Fosforilación , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Quinasas Ciclina-Dependientes/fisiología , Replicación del ADN , Genoma Fúngico/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Organismos Modificados Genéticamente , Fosforilación , Origen de Réplica/genética , Fase S/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Transducción de Señal/genéticaRESUMEN
An uncontrolled cell cycle is an obvious marker of tumor cells. The G1S phase is an important restriction point in the normal cell cycle, but in cancer cells the restriction function is reduced, leading to uncontrolled cell proliferation. Two cyclindependent kinases (CDKs), CDK4 and CDK6, play a crucial role in the G1S phase transition. Inhibitors of CDK4/6 are presently the subjects of numerous studies, and PD 0332991, an inhibitor of CDK4/6, has been used to treat hormone receptor (HR)positive, advancedstage breast cancer. This inhibitor has also been studied in other cancers, such as lung cancer. In this review, we will discuss the regulation of the normal cell cycle transition from G1 to S phase, the most promising inhibitor of CDK4/6, PD 0332991, as applied in different cancers, and finally we propose a mechanism of acquired resistance as well as the incredible potential for CDK4/6 inhibitors in the treatment of cancer. Briefly, we assert that, going forward, a new treatment pattern for cancer may be a combination therapy with a cell cycle inhibitor and a molecular targeted drug.