RESUMEN
Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.
Asunto(s)
Antineoplásicos , Caseína Quinasa Ialfa , Proliferación Celular , Quinasa 9 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes , Ensayos de Selección de Medicamentos Antitumorales , Leucemia Mieloide Aguda , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Apoptosis/efectos de los fármacos , Descubrimiento de Drogas , Línea Celular Tumoral , Proteolisis/efectos de los fármacos , Células Tumorales Cultivadas , Quimera Dirigida a la Proteólisis , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.
Asunto(s)
Caseína Quinasa Ialfa , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Inhibidores de Fosfodiesterasa , Humanos , Sitios de Unión , Caseína Quinasa Ialfa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Inmunoterapia , Cinética , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacologíaRESUMEN
Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Caseína Quinasa Ialfa/metabolismo , Glioma/metabolismo , Tolerancia a Radiación , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Caseína Quinasa Ialfa/antagonistas & inhibidores , Caseína Quinasa Ialfa/genética , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Glioma/patología , Glioma/radioterapia , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.
Asunto(s)
Caseína Quinasa Ialfa/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Ritmo Circadiano/efectos de los fármacos , Cronoterapia de Medicamentos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/ultraestructura , Quinasa Idelta de la Caseína/metabolismo , Línea Celular Tumoral , Trastornos Cronobiológicos/tratamiento farmacológico , Relojes Circadianos/efectos de la radiación , Evaluación Preclínica de Medicamentos , Pruebas de Enzimas , Humanos , Luz , Ratones , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Fotoperiodo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/efectos de la radiación , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Disturbance of protein kinase activity may result in dramatic consequences that often lead to cancer development and progression. In tumors of blood origin, both tyrosine kinases and serine/threonine kinases are altered by different types of mutations, critically regulating cancer hallmarks. CK1α and CK2 are highly conserved, ubiquitously expressed and constitutively active pleiotropic kinases, which participate in multiple biological processes. The involvement of these kinases in solid and blood cancers is well documented. CK1α and CK2 are overactive in multiple myeloma, leukemias and lymphomas. Intriguingly, they are not required to the same degree for the viability of normal cells, corroborating the idea of "druggable" kinases. Different to other kinases, mutations on the gene encoding CK1α and CK2 are rare or not reported. Actually, these two kinases are outside the paradigm of oncogene addiction, since cancer cells' dependency on these proteins resembles the phenomenon of "non-oncogene" addiction. In this review, we will summarize the general features of CK1α and CK2 and the most relevant oncogenic and stress-related signaling nodes, regulated by kinase phosphorylation, that may lead to tumor progression. Finally, we will report the current data, which support the positioning of these two kinases in the therapeutic scene of hematological cancers.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Caseína Quinasa Ialfa/metabolismo , Neoplasias Hematológicas/metabolismo , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Caseína Quinasa Ialfa/antagonistas & inhibidores , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Terapia Molecular Dirigida , Estrés FisiológicoRESUMEN
Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of the cis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.
Asunto(s)
Compuestos Azo/farmacología , Caseína Quinasa Ialfa/antagonistas & inhibidores , Ritmo Circadiano/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Línea Celular Tumoral , Humanos , Isomerismo , Luz , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/efectos de la radiación , Purinas/química , Purinas/efectos de la radiación , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/efectos de la radiación , Relación Estructura-ActividadRESUMEN
Enhancement of autophagy serves as a promising therapeutic strategy for cancer, including acute myeloid leukemia (AML). Casein kinase 1α (CK1α), encoded by CSNK1A1, regulates Wnt/ßcatenin, p53 and other key signaling pathways, and is critically involved in tumor progression. However, the relationship and mechanism of CK1α with autophagy in AML still remain unclear. In the present study, it was found that AML patients had higher expression of CSNK1A1 mRNA than healthy donors. Furthermore, we analyzed 163 cases of AML patients in the LAML database of TCGA and found that AML patients with high CSNK1A1 had shorter overall survival than those with low or medium CSNK1A1 expression. Furthermore, we demonstrated that CK1α was a negative regulator of autophagy and apoptosis. Pharmacologic inhibition of CK1α using D4476 or CK1α knockdown via lentivirusmediated shRNA suppressed proliferation and the clone formation by enhancing autophagic flux and apoptosis in AML cell lines as well as in patient blast cells. Intriguingly, D4476induced cell death was aggravated in combination with an autophagy inhibitor, Spautin1, suggesting that autophagy may be a prosurvival signaling. CK1α interacted with murine double minute 2 (MDM2) and p53, and CK1α inhibitor D4476 significantly upregulated p53 and phosphorylated 5' AMPactivated protein kinase (AMPK), and substantially inhibited the phosphorylation of mammalian target of rapamycin (mTOR). Our findings indicate that CK1α promotes AML by suppressing p53 downstream of MDM2mediated autophagy and apoptosis, suggesting that targeting CK1α provides a therapeutic opportunity to treat AML.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Apoptosis/fisiología , Autofagia/fisiología , Benzamidas/farmacología , Caseína Quinasa Ialfa/antagonistas & inhibidores , Caseína Quinasa Ialfa/genética , Línea Celular Tumoral , Humanos , Imidazoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.
Asunto(s)
Caseína Quinasa Ialfa/fisiología , Relojes Circadianos/genética , Ritmo Circadiano , Eritrocitos/metabolismo , Eritrocitos/fisiología , Temperatura , Caseína Quinasa Ialfa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Estaurosporina/farmacologíaRESUMEN
Circadian rhythms are controlled by transcriptional feedback loops of clock genes and proteins. The stability of clock proteins is regulated by post-translational modification, such as phosphorylation by kinases. In particular, casein kinase I (CKI) phosphorylates the PER protein to regulate proteasomal degradation and nuclear localization. Therefore, CKI inhibition can modulate mammalian circadian rhythms. In the present study, we have developed novel CKIα and CKIδ dual inhibitors by extensive structural modification of N9 and C2 position of longdaysin. We identified NCC007 that showed stronger period effects (0.32 µM for 5 h period lengthening) in a cell-based circadian assay. The following in vitro kinase assay showed that NCC007 inhibited CKIα and CKIδ with an IC50 of 1.8 and 3.6 µM. We further demonstrated that NCC007 lengthened the period of mouse behavioral rhythms in vivo. Thus, NCC007 is a valuable tool compound to control circadian rhythms through CKI inhibition.
Asunto(s)
Adenina/análogos & derivados , Caseína Quinasa Ialfa/antagonistas & inhibidores , Ritmo Circadiano/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Adenina/síntesis química , Adenina/metabolismo , Adenina/farmacología , Animales , Sitios de Unión , Caseína Quinasa Ialfa/química , Caseína Quinasa Ialfa/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Unión ProteicaAsunto(s)
Caseína Quinasa Ialfa/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Hematopoyesis/efectos de los fármacos , Humanos , Ratones , Células Tumorales Cultivadas , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
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
Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.
Asunto(s)
Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Quinasa Ialfa/antagonistas & inhibidores , Riñón/efectos de los fármacos , Enfermedades Renales Poliquísticas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Piridinas/farmacología , Roscovitina/farmacología , Animales , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Caseína Quinasa Ialfa/genética , Caseína Quinasa Ialfa/metabolismo , Catálisis , Cromatografía de Afinidad/métodos , Modelos Animales de Enfermedad , Humanos , Riñón/enzimología , Riñón/patología , Ratones Transgénicos , Enfermedades Renales Poliquísticas/enzimología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Evasion of forkhead box O (FOXO) family of longevity-related transcription factors-mediated growth suppression is necessary to promote cancer development. Since somatic alterations or mutations and transcriptional dysregulation of the FOXO genes are infrequent in human cancers, it remains unclear how these tumour suppressors are eliminated from cancer cells. The protein stability of FOXO3A is regulated by Casein Kinase 1 alpha (CK1α) in an oncogenic RAS-specific manner, but whether this mode of regulation extends to related FOXO family members is unknown. Here we report that CK1α similarly destabilizes FOXO4 in RAS-mutant cells by phosphorylation at serines 265/268. The CK1α-dependent phosphoregulation of FOXO4 is primed, in part, by the PI3K/AKT effector axis of oncogenic RAS signalling. In addition, mutant RAS coordinately elevates proteasome subunit expression and proteolytic activity to eradicate nuclear FOXO4 proteins from RAS-mutant cancer cells. Importantly, dual inhibition of CK1α and the proteasome synergistically inhibited the growth of multiple RAS-mutant human cancer cell lines of diverse tissue origin by blockade of nuclear FOXO4 degradation and induction of caspase-dependent apoptosis. Our findings challenge the current paradigm that nuclear export regulates the proteolysis of FOXO3A/4 tumour suppressors in the context of cancer and illustrates how oncogenic RAS-mediated degradation of FOXOs, via post-translational mechanisms, blocks these important tumour suppressors.
Asunto(s)
Caseína Quinasa Ialfa/genética , Núcleo Celular/patología , Neoplasias/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Transcripción/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Caseína Quinasa Ialfa/antagonistas & inhibidores , Caseína Quinasa Ialfa/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteína Forkhead Box O3/metabolismo , Factores de Transcripción Forkhead , Humanos , Imidazoles/farmacología , Mutación , Neoplasias/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/metabolismo , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore is subject to regulation by cell signalling; however, the kinase(s) responsible for this event have not been definitively identified. Here using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Corteza Cerebral/enzimología , Cuerpo Estriado/enzimología , Neuronas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Red trans-Golgi/metabolismo , Animales , Benzamidas/farmacología , Caseína Quinasa Ialfa/antagonistas & inhibidores , Caseína Quinasa Ialfa/genética , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Cuerpo Estriado/citología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Imidazoles/farmacología , Indoles/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Floroglucinol/análogos & derivados , Floroglucinol/farmacología , Fosforilación , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Red trans-Golgi/efectos de los fármacosRESUMEN
UNLABELLED: Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle. IMPORTANCE: Mechanisms regulating NS5A phosphorylation and its exact function in the HCV life cycle have not been clearly defined. By using a high-throughput screening system targeting host protein kinases, we identified CKI-α as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-α in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-α-dependent hyperphosphorylation of NS5A plays a role in recruiting NS5A to low-density membrane structures around LDs and facilitating its interaction with the core for new virus particle formation. By using proteomic approach, we identified the region within the low-complexity sequence I of NS5A that is involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of infectious virus production. These findings will provide novel mechanistic insights into the roles of NS5A-associated kinases and NS5A phosphorylation in the HCV life cycle.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Hepacivirus/fisiología , Hepatitis C/virología , Proteínas no Estructurales Virales/metabolismo , Virión/fisiología , Secuencia de Aminoácidos , Western Blotting , Caseína Quinasa Ialfa/antagonistas & inhibidores , Caseína Quinasa Ialfa/genética , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Hepatitis C/metabolismo , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Fosforilación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas no Estructurales Virales/genéticaRESUMEN
Wnt signaling plays key roles in embryonic development and various human diseases. Activity-guided testing to isolate Wnt signaling inhibitors from the methanol extract of Calotropis gigantea (Asclepiadaceae) exudutes identified six Wnt inhibitory cardenolides (1-6), of which 1, 3, 5, and 6 exhibited potent TCF/ß-catenin inhibitory activities (IC50 0.7-3.6 nM). Calotropin (1) inhibited Wnt signaling by decreasing both nuclear and cytosolic ß-catenin in a dose-dependent manner, and promoted degradation of ß-catenin by increasing the phosphorylation of ß-catenin at Ser45 through casein kinase 1α (CK1α). Moreover, 1 significantly increased CK1α protein and mRNA levels. The results suggest that 1 inhibits the Wnt signaling pathway by increasing CK1α protein levels. To the best of our knowledge, calotropin is the first small molecule to increase CK1α levels.
Asunto(s)
Calotropis/química , Cardenólidos/química , Cardenólidos/farmacología , Caseína Quinasa Ialfa/antagonistas & inhibidores , Calotropis/metabolismo , Cardenólidos/aislamiento & purificación , Caseína Quinasa Ialfa/genética , Caseína Quinasa Ialfa/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Fosforilación/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Despite extensive insights into the underlying genetics and biology of acute myeloid leukemia (AML), overall survival remains poor and new therapies are needed. We found that casein kinase 1 α (Csnk1a1), a serine-threonine kinase, is essential for AML cell survival in vivo. Normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected by shRNA-mediated knockdown of Csnk1a1. To identify downstream mediators of Csnk1a1 critical for leukemia cells, we performed an in vivo pooled shRNA screen and gene expression profiling. We found that Csnk1a1 knockdown results in decreased Rps6 phosphorylation, increased p53 activity, and myeloid differentiation. Consistent with these observations, p53-null leukemias were insensitive to Csnk1a1 knockdown. We further evaluated whether D4476, a casein kinase 1 inhibitor, would exhibit selective antileukemic effects. Treatment of leukemia stem cells (LSCs) with D4476 showed highly selective killing of LSCs over normal HSPCs. In summary, these findings demonstrate that Csnk1a1 inhibition causes reduced Rps6 phosphorylation and activation of p53, resulting in selective elimination of leukemia cells, revealing Csnk1a1 as a potential therapeutic target for the treatment of AML.
Asunto(s)
Caseína Quinasa Ialfa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Caseína Quinasa Ialfa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Silenciador del Gen/efectos de los fármacos , Humanos , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína S6 Ribosómica/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Resultado del TratamientoRESUMEN
Casein kinase I alpha (CK1α) is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1) extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP), an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Segregación Cromosómica , Desarrollo Embrionario , Meiosis , Oocitos/citología , Regulación Alostérica , Animales , Benzamidas/farmacología , Western Blotting , Caseína Quinasa Ialfa/antagonistas & inhibidores , Femenino , Técnica del Anticuerpo Fluorescente , Imidazoles/farmacología , Ratones , Compuestos de Pirvinio/farmacología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Protein kinase CK1 (casein kinase 1) isoforms are involved in the regulation of various physiological functions including apoptosis. The specific CK1 inhibitor D4476 may either inhibit or foster apoptosis. Similar to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity following energy depletion (removal of glucose) or oxidative stress (exposure to the oxidant tert-butyl hydroperoxide [TBOOH]). Western blotting was utilized to verify that erythrocytes express the protein kinase CK1α, and FACS analysis to determine whether the CK1 inhibitor D4476 and CK1α activator pyrvinium pamoate modify forward scatter (reflecting cell volume), annexin V binding (reflecting phosphatidylserine exposure), and Fluo3 fluorescence (reflecting cytosolic Ca(2+) activity). As a result, both, human and murine erythrocytes express CK1 isoform α. Glucose depletion (48 hours) and exposure to 0.3 mM TBOOH (30 minutes) both decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence. CK1 inhibitor D4476 (10 µM) significantly blunted the decrease in forward scatter, the increase in annexin V binding and the increase in Fluo 3 fluorescence. (R)-DRF053, another CK1 inhibitor, similarly blunted the increase in annexin V binding upon glucose depletion. The CK1α specific activator pyrvinium pamoate (10 µM) significantly enhanced the increase in annexin V binding and Fluo3 fluorescence upon glucose depletion and TBOOH exposure. In the presence of glucose, pyrvinium pamoate slightly but significantly increased Fluo3 fluorescence. In conclusion, CK1 isoform α participates in the regulation of erythrocyte programmed cell death by modulating cytosolic Ca(2+) activity.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Eritrocitos/efectos de los fármacos , Compuestos de Anilina/química , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas/farmacología , Calcio/metabolismo , Caseína Quinasa Ialfa/antagonistas & inhibidores , Tamaño de la Célula/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/metabolismo , Glucosa/farmacología , Humanos , Imidazoles/farmacología , Ratones , Fosfatidilserinas/farmacología , Compuestos de Pirvinio/farmacología , Xantenos/química , terc-Butilhidroperóxido/farmacologíaRESUMEN
Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5'-3' exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.