RESUMEN
CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Humanos , Secuencia de Aminoácidos , Caseína Cinasa 1 épsilon/metabolismo , Caseína Cinasa 1 épsilon/genética , Dominio Catalítico , Mutación , Péptidos/metabolismo , Péptidos/química , Fosforilación , Unión Proteica , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Especificidad por SustratoRESUMEN
Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.
Asunto(s)
Caseína Cinasa 1 épsilon , Enfermedad de Parkinson , Ratones , Animales , Factores de Transcripción/genética , Proteínas de Unión al ADN/genética , alfa-Sinucleína/genética , Enfermedad de Parkinson/genética , Caseína Cinasa 1 épsilon/genética , MutaciónRESUMEN
Aberrant cytoplasmic accumulation of an RNA-binding protein, fused in sarcoma (FUS), characterizes the neuropathology of subtypes of ALS and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. Here, we show that casein kinase 1δ (CK1δ) phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. We also show that phosphorylation by CK1δ or CK1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wt or P525L ALS-mutant human FUS in the retina or in neurons, we found coexpression of human CK1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human CK1δ extended fly life span. Taken together, our data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through CK1δ/CK1ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.
Asunto(s)
Esclerosis Amiotrófica Lateral , Caseína Cinasa 1 épsilon , Quinasa Idelta de la Caseína , Proteínas de Drosophila , Degeneración Lobar Frontotemporal , Sarcoma , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Caseína Cinasa 1 épsilon/genética , Quinasa Idelta de la Caseína/genética , Quinasa Idelta de la Caseína/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Mutación , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, dbt plays a central role in both circadian rhythms and development. We investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discuss the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, we uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, we demonstrate that the PER protein is involved in DBT mediated sleep regulation.
Asunto(s)
Caseína Cinasa 1 épsilon/fisiología , Ritmo Circadiano/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Neuronas/fisiología , Sueño/fisiología , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/fisiología , Caseína Cinasa 1 épsilon/genética , Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Femenino , Regulación de la Expresión Génica , Mutación , Proteínas Circadianas Period/genéticaRESUMEN
Affective behaviours and mental health are profoundly affected by disturbances in circadian rhythms. Casein kinase 1 epsilon (CSNK1E) is a core component of the circadian clock. Mice with tau or null mutation of this gene have shortened and lengthened circadian period respectively. Here, we examined anxiety-like, fear, and despair behaviours in both male and female mice of these two different mutants. Compared with wild-type mice, we found reductions in fear and anxiety-like behaviours in both mutant lines and in both sexes, with the tau mutants exhibiting the greatest phenotypic changes. However, the behavioural despair had distinct phenotypic patterns, with markedly less behavioural despair in female null mutants, but not in tau mutants of either sex. To determine whether abnormal light entrainment of tau mutants to 24-h light-dark cycles contributes to these phenotypic differences, we also examined these behaviours in tau mutants on a 20-h light-dark cycle close to their endogenous circadian period. The normalized entrainment restored more wild-type-like behaviours for fear and anxiety, but it induced behavioural despair in tau mutant females. These data show that both mutations of Csnk1e broadly affect fear and anxiety-like behaviours, while the effects on behavioural despair vary with genetics, photoperiod, and sex, suggesting that the mechanisms by which Csnk1e affects fear and anxiety-like behaviours may be similar, but distinct from those affecting behavioural despair. Our study also provides experimental evidence in support of the hypothesis of beneficial outcomes from properly entrained circadian rhythms in terms of the anxiety-like and fear behaviours.
Asunto(s)
Caseína Cinasa 1 épsilon , Relojes Circadianos , Animales , Caseína Cinasa 1 épsilon/genética , Ritmo Circadiano/genética , Femenino , Masculino , Ratones , Actividad Motora , FotoperiodoRESUMEN
The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.
Asunto(s)
Acacia/química , Antineoplásicos Fitogénicos/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Triterpenos/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Apoptosis/genética , Sitios de Unión , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Células K562 , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Quinasas Relacionadas con NIMA/antagonistas & inhibidores , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Corteza de la Planta/química , Extractos Vegetales/química , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Transducción de Señal , Triterpenos/química , Triterpenos/aislamiento & purificación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant disease worldwide. Although the diagnosis and treatment of HCC have greatly improved in the recent years, there is still a lack of accurate methods to predict the prognosis of patients. Evidence has shown that Hippo signaling in tissues adjacent to HCC plays a significant role in HCC development. In the present study, we aimed to construct a model based on the expression of Hippo-related genes (HRGs) in tissues adjacent to HCC to predict the prognosis of HCC patients. METHODS: Gene expression data of paired normal tissues adjacent to HCC (PNTAH) and clinical information were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The HRG signature was constructed using four canonical Hippo-related pathways. Univariate Cox regression analysis was used to screen survival-related HRGs. LASSO and multivariate Cox regression analyses were used to construct the prognostic model. The true and false positive rates of the model were confirmed using receiver operating characteristic (ROC) analysis. RESULTS: The prognostic model was constructed based on the expression levels of five HRGs (NF2, MYC, BIRC3, CSNK1E, and MINK1) in PNTAH. The mortality rate of HCC patients increased as the risk score determined by the model increased. Furthermore, the risk score was found to be an independent risk factor for the survival of patients. ROC analysis showed that the prognostic model had a better predictive value than the other conventional clinical parameters. Moreover, the reliability of the prognostic model was confirmed in TCGA-LIHC cohort. A nomogram was generated to predict patient survival. An exploration of the predictive value of the model in HCC tissues indicated that the model is PNTAH-specific. CONCLUSIONS: We developed and validated a prognostic model based on the expression levels of five HRGs in PNTAH, and this model should be helpful in predicting the prognosis of patients with HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Expresión Génica , Neoplasias Hepáticas/genética , Hígado , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Vía de Señalización Hippo , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Nomogramas , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Curva ROC , Factores de Riesgo , Transcriptoma , Adulto JovenRESUMEN
Background: Amino-terminal enhancer of split (AES) has been identified as a tumor and metastasis suppressor in some cancers including colorectal cancer (CRC), but very little is known about the regulation of AES expression. Methods: Bioinformatics analysis was used to investigate the expression patterns of AES, CK1δ and CK1ε. The co-immunoprecipitation, GST pull-down, Western Blot, real-time PCR and immunohistochemistry were performed to study the mechanism underlying the regulation of AES expression by CK1δ/ε. The biological function was assessed by in vitro colony formation, transwell, sphere formation, tumor organoids, in vivo tumor metastasis model and patient-derived colorectal tumor xenografts (PDTX) model. Results: A strong inverse relationship was observed between the expression of AES and the expression of CK1δ/ε. Mechanically, AES could interact with CK1δ/ε and SKP2 using its Q domain. SKP2 mediated the ubiquitination and degradation of AES in a CK1δ/ε-dependent manner. CK1δ/ε phosphorylated AES at Ser121 and accelerated the SKP2-mediated ubiquitination and degradation of AES. In colon cancer cells, CK1δ/ε antagonized the effect of wild-type AES but not that of its mutant (S121A) on Wnt and Notch signaling, leading to an increase in the expression of Wnt target genes and Notch target genes. By downregulating the expression of AES, CK1δ/ε enhanced anchorage-independent growth, migration, invasion and sphere formation in colon cancer cells. CK1δ/ε also promoted the growth of APCmin/+ colorectal tumor organoids and liver metastasis in colon cancer mouse models through the regulation of AES degradation. Furthermore, CK1 inhibitor SR3029 treatment suppressed tumor growth via stabilizing AES in APCmin/+ colorectal tumor organoids and patient-derived colorectal tumor xenografts (PDTX). Conclusions: Our results revealed that the CK1δ/ε-AES axis is important for CRC tumorigenesis and metastasis, and targeted inhibition of this axis may be a potential therapeutic strategy for CRC.
Asunto(s)
Carcinogénesis/genética , Caseína Cinasa 1 épsilon/genética , Quinasa Idelta de la Caseína/genética , Proteínas Co-Represoras/genética , Neoplasias Colorrectales/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Células HEK293 , Humanos , Organoides/patología , Fosforilación/genética , Ubiquitinación/genética , Vía de Señalización Wnt/genéticaRESUMEN
Casein Kinase 1 epsilon (CK1ε) is a member of the serine (Ser)/threonine (Thr) CK1 family, known to have crucial roles in several biological scenarios and, ever more frequently, in pathological contexts, such as cancer. Recently, the human DEAD-box RNA helicase 3 X-linked (DDX3X), involved in cancer proliferation and viral infections, has been identified as one of CK1ε substrates and its positive regulator in the Wnt/ß-catenin network. However, the way by which these two proteins influence each other has not been fully clarified. In order to further investigate their interplay, we defined the kinetic parameters of CK1ε towards its substrates: ATP, casein, Dvl2 and DDX3X. CK1ε affinity for ATP depends on the nature of the substrate: increasing of casein concentrations led to an increase of KmATP, while increasing DDX3X reduced it. In literature, DDX3X is described to act as an allosteric activator of CK1ε. However, when we performed kinase reactions combining DDX3X and casein, we did not find a positive effect of DDX3X on casein phosphorylation by CK1ε, while both substrates were phosphorylated in a competitive manner. Moreover, CK1ε positively stimulates DDX3X ATPase activity. Our data provide a more detailed kinetic characterization on the functional interplay of these two proteins.
Asunto(s)
Adenosina Trifosfato/metabolismo , Caseína Cinasa 1 épsilon/metabolismo , Caseínas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Caseína Cinasa 1 épsilon/genética , ARN Helicasas DEAD-box/genética , Humanos , Cinética , Fosforilación , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.
Asunto(s)
Caseína Cinasa 1 épsilon/metabolismo , Polaridad Celular , Proteínas de Drosophila/metabolismo , Morfogénesis , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Caseína Cinasa 1 épsilon/genética , Proteínas Dishevelled/genética , Proteínas Dishevelled/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutación con Pérdida de Función , Unión Proteica , Transporte de Proteínas , Proteínas de Unión a Tacrolimus/genética , Alas de Animales/citología , Alas de Animales/crecimiento & desarrolloRESUMEN
Maple syrup urine disease (MSUD) is an inherited error in the metabolism of branched-chain amino acids (BCAAs) caused by a severe deficiency of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which ultimately leads to neurological disorders. The limited therapies, including protein-restricted diets and liver transplants, are not as effective as they could be for the treatment of MSUD due to the current lack of molecular insights into the disease pathogenesis. To address this issue, we developed a Drosophila model of MSUD by knocking out the dDBT gene, an ortholog of the human gene encoding the dihydrolipoamide branched chain transacylase (DBT) subunit of BCKDH. The homozygous dDBT mutant larvae recapitulate an array of MSUD phenotypes, including aberrant BCAA accumulation, developmental defects, poor mobile behavior and disrupted L-glutamate homeostasis. Moreover, the dDBT mutation causes neuronal apoptosis during the developmental progression of larval brains. The genetic and functional evidence generated by in vivo depletion of dDBT expression in the eye indicates severe impairment of retinal rhabdomeres. Further, the dDBT mutant shows elevated oxidative stress and higher lipid peroxidation accumulation in the larval brain. Therefore, we conclude from in vivo evidence that the loss of dDBT results in oxidative brain damage that may lead to neuronal cell death and contribute to aspects of MSUD pathology. Importantly, when the dDBT mutants were administrated with Metformin, the aberrances in BCAA levels and motor behavior were ameliorated. This intriguing outcome strongly merits the use of the dDBT mutant as a platform for developing MSUD therapies.This article has an associated First Person interview with the joint first authors of the paper.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Apoptosis , Encéfalo/enzimología , Caseína Cinasa 1 épsilon/deficiencia , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/enzimología , Enfermedad de la Orina de Jarabe de Arce/enzimología , Neurogénesis , Neuronas/enzimología , Animales , Animales Modificados Genéticamente , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Caseína Cinasa 1 épsilon/genética , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Larva/enzimología , Larva/genética , Peroxidación de Lípido , Masculino , Enfermedad de la Orina de Jarabe de Arce/tratamiento farmacológico , Enfermedad de la Orina de Jarabe de Arce/genética , Enfermedad de la Orina de Jarabe de Arce/patología , Metformina/farmacología , Actividad Motora , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo , FenotipoRESUMEN
Human casein kinase 1 delta (CK1δ) and epsilon (CK1ε) are members of a conserved family of abundant, ubiquitously expressed serine/threonine kinases that regulate multiple cellular processes including circadian rhythm and endocytosis. Here, we have investigated the localization and interactomes of endogenously tagged CK1δ and CK1ε during interphase and mitosis. CK1δ and CK1ε localize to centrosomes throughout the cell cycle, and in interphase cells to the nucleus, and in both a diffuse and punctate pattern in the cytoplasm. Also, for the first time, they were detected at the midbody during cell division. Mass spectrometry analysis identified a total of 181 proteins co-purifying with a Venus multifunctional (VM)-tagged CK1δ and/or CK1ε. GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), a protein required for efficient endocytosis, was consistently one of the most abundant interacting partners. We demonstrate that GAPVD1 is a substrate of CK1δ/ε with up to 38 phosphorylated residues in vitro and in vivo. Wildtype and a phosphomimetic mutant of GAPVD1, but not a phospho-ablating mutant, were able to rescue defects in transferrin and EGF internalization caused by loss of endogenous GAPVD1. Our results indicate that GAPVD1 is an important interacting partner and substrate of CK1δ/ε for endocytosis.
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Sistemas CRISPR-Cas , Caseína Cinasa 1 épsilon/genética , Quinasa Idelta de la Caseína/genética , Endocitosis/genética , Marcación de Gen/métodos , Fosfoproteínas/genética , Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Factores de Intercambio de Guanina Nucleótido , Células HEK293 , Células HeLa , Humanos , Interfase/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Especificidad por SustratoRESUMEN
Osteoarthritis (OA) is a high-morbidity skeletal disease worldwide and the exact mechanisms underlying OA pathogenesis are not fully understood. Casein kinase 1 epsilon (CK1ε) is a serine/threonine protein kinase, but its relationship with OA is still unknown. We demonstrated that CK1ε was upregulated in articular cartilage of human patients with OA and mice with experimentally induced OA. Activity of CK1ε, demonstrated by analysis of phosphorylated substrates, was significantly elevated in interleukin (IL)-1ß-induced OA-mimicking chondrocytes. CK1ε inhibitor or CK1ε short hairpin RNA (shRNA) partially blocked matrix metalloproteinase (MMP) expression by primary chondrocytes induced by IL-1ß, and also inhibited cartilage destruction in knee joints of experimental OA model mice. Conversely, overexpression of CK1ε promoted chondrocyte catabolism. Previous studies indicated that CK1ε was involved in canonical Wnt/ß-catenin signaling and noncanonical Wnt/c-Jun N-terminal kinase (JNK) signaling pathway. Interestingly, the activity of JNK but not ß-catenin decreased after CK1ε knockdown in IL-1ß-treated chondrocytes in vitro, and JNK inhibition reduced MMP expression in chondrocytes overexpressing CK1ε, which illustrated that CK1ε-mediated OA was based on JNK pathway. In conclusion, our results demonstrate that CK1ε promotes OA development, and inhibition of CK1ε could be a potential strategy for OA treatment in the future.
Asunto(s)
Cartílago Articular/patología , Caseína Cinasa 1 épsilon/metabolismo , Condrocitos/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Osteoartritis/patología , Animales , Cartílago Articular/metabolismo , Estudios de Casos y Controles , Caseína Cinasa 1 épsilon/genética , Células Cultivadas , Condrocitos/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Masculino , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Osteoartritis/genética , Osteoartritis/metabolismo , Fosforilación , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Necroptosis is a regulated necrotic cell death pathway, mediated by a supermolecular complex called the necrosome, which contains receptor-interacting protein kinase 1 and 3 (RIPK1, RIPK3) and mixed-lineage kinase domain-like protein (MLKL). Phosphorylation of human RIPK3 at serine 227 (S227) has been shown to be required for downstream MLKL binding and necroptosis progression. Tandem immunoprecipitation of RIPK3 reveals that casein kinase 1 (CK1) family proteins associate with the necrosome upon necroptosis induction, and this interaction depends on the kinase activity of RIPK3. In addition, CK1 proteins colocalize with RIPK3 puncta during necroptosis. Importantly, CK1 proteins directly phosphorylate RIPK3 at S227 in vitro and in vivo. Loss of CK1 proteins abolishes S227 phosphorylation and blocks necroptosis. Furthermore, a RIPK3 mutant with mutations in the CK1 recognition motif fails to be phosphorylated at S227, does not bind or phosphorylate MLKL, and is unable to activate necroptosis. These results strongly suggest that CK1 proteins are necrosome components which are responsible for RIPK3-S227 phosphorylation.
Asunto(s)
Caseína Cinasa 1 épsilon/metabolismo , Caseína Quinasa Ialfa/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Serina/metabolismo , Caseína Cinasa 1 épsilon/genética , Caseína Quinasa Ialfa/genética , Quinasa Idelta de la Caseína/genética , Células HeLa , Humanos , Fosforilación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Serina/genéticaRESUMEN
The molecular clock network in mast cells has been shown to be a factor responsible for circadian regulation of allergic inflammation. PF670462 is a selective inhibitor of casein kinase 1δ and ε (CK1δ/ε) that control the posttranslational modification of clock proteins. The aims of this study were to evaluate the effects of PF670462 on gene and protein expression of FcεRI, the high-affinity IgE receptor, in canine mast cells and on IgE-mediated immediate-type cutaneous reactions in dogs. PF670462 decreased mRNA expression of FcεRIα and ß, but not γ, and protein expression of FcεRI in a canine mast cell line. Furthermore, PF670462 suppressed IgE-mediated immediate-type cutaneous erythema in dogs. These findings indicate that CK1δ/ε function as regulators for FcεRI expression and IgE-mediated cutaneous reactions in dogs.
Asunto(s)
Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Enfermedades de los Perros/metabolismo , Inmunoglobulina E/metabolismo , Pirimidinas/farmacología , Receptores de IgE/metabolismo , Anafilaxia , Animales , Caseína Cinasa 1 épsilon/genética , Enfermedades de los Perros/genética , Perros , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Mastocitos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de IgE/genéticaRESUMEN
OBJECTIVE: Prostate cancer, one of the most common malignant tumors in urology, now has become a malignant disease that seriously threatens the health of men in China. Although there are a large number of clinical studies on the treatment of patients with prostate cancer, many patients have entered the advanced stage of diagnosis, and little is known about its pathogenesis. MATERIALS AND METHODS: We identified a series of ncRNA and TF by differential expression analysis, co-expression analysis, enrichment analysis, connectivity analysis, and hypergeometric test strategies for prostate cancer expression genomes. RESULTS: 53 modules related to prostate cancer PC-3 cells were obtained, involving module focusing of 4448 genes. Based on these modules, we predicted that miR-26a-5p, miR-130a-3p, miR-519d-3p, etc. have important regulatory effects on prostate cancer PC-3 cells. At the same time, a series of transcription factors (relating to RELA, SOX10, TP53, and TWIST2, etc.) were obtained and may play a key regulatory role in prostate cancer PC-3 cell-related modules. CONCLUSIONS: These results suggest that FENDRR in prostate cancer may reduce tumor invasion in prostate cancer PC-3 cells by targeting CSNK1E, which may have favourable effort to better understand the underlying pathogenesis of prostate cancer and provide a tough theoretical basis for further studying prostate cancer.
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Caseína Cinasa 1 épsilon/genética , Regulación hacia Abajo , Neoplasias de la Próstata/patología , ARN Largo no Codificante/genética , Caseína Cinasa 1 épsilon/metabolismo , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Invasividad Neoplásica , Células PC-3 , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismoRESUMEN
Sleep disturbances and memory impairment are common symptoms of Alzheimer's disease (AD). Given that the circadian clock regulates sleep, hippocampal function, and neurodegeneration, it represents a therapeutic target against AD. Casein kinase 1δ/ε (CK1δ/ε) are clock regulators and overexpressed in AD brains, making them viable targets to improve sleep and cognition. In this study, we evaluated the therapeutic potential of a small molecule CK1δ/ε inhibitor (PF-670462) in a triple transgenic mouse model of AD (3xTg-AD). Mass spectrometry-based proteomic analyses revealed that PF-670462 administration in 3xTg-AD mice reversed hippocampal proteomic alterations in several AD-related and clock-regulated pathways, including synaptic plasticity and amyloid precursor protein processing. Furthermore, PF-670462 administration rescued working memory deficits and normalized behavioral circadian rhythm disturbances in 3xTg-AD mice. Our study provides in vivo proof of concept for CK1δ/ε inhibition against AD-associated hippocampal proteomic changes, memory impairment, and circadian disturbances.
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Enfermedad de Alzheimer/terapia , Caseína Cinasa 1 épsilon/genética , Quinasa Idelta de la Caseína/genética , Trastornos de la Memoria/terapia , Trastornos del Sueño-Vigilia/terapia , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Relojes Circadianos/genética , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Trastornos de la Memoria/complicaciones , Trastornos de la Memoria/genética , Trastornos de la Memoria/patología , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Plasticidad Neuronal/efectos de los fármacos , Proteómica/métodos , Pirimidinas/farmacología , Trastornos del Sueño-Vigilia/complicaciones , Trastornos del Sueño-Vigilia/genética , Trastornos del Sueño-Vigilia/patologíaRESUMEN
It is well recognized that metastasis can occur early in the course of lung adenocarcinoma (LAD) development, and yet the molecular mechanisms driving this capability of rapid metastasis remain incompletely understood. Here we reported that a long noncoding RNA, LINC00673, was up-regulated in LAD cells. Of note, we first found that LINC00673-v4 was the most abundant transcript of LINC00673 in LAD cells and its expression was associated with adverse clinical outcome of LAD. In vitro and in vivo experiments demonstrated that LINC00673-v4 enhanced invasiveness, migration, and metastasis of LAD cells. Mechanistically, LINC00673-v4 augmented the interaction between DDX3 and CK1ε and thus the phosphorylation of dishevelled, which subsequently activated WNT/ß-catenin signaling and consequently caused aggressiveness of LAD. Antagonizing LINC00673-v4 suppressed LAD metastasis in vivo. Together, our data suggest that LINC00673-v4 is a driver molecule for metastasis via constitutively activating WNT/ß-catenin signaling in LAD and may represent a potential therapeutic target against the metastasis of LAD.
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Adenocarcinoma del Pulmón/genética , Caseína Cinasa 1 épsilon/genética , ARN Helicasas DEAD-box/genética , ARN Largo no Codificante/genética , Adenocarcinoma del Pulmón/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Activación Transcripcional , Vía de Señalización Wnt/genéticaRESUMEN
Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 É (CK1É) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1É-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1É in DVL conformational dynamics.
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Caseína Cinasa 1 épsilon/metabolismo , Proteínas Dishevelled/metabolismo , Dominios PDZ/fisiología , Vía de Señalización Wnt/fisiología , Animales , Técnicas Biosensibles , Caseína Cinasa 1 épsilon/genética , Proteínas Dishevelled/genética , Pruebas de Enzimas/métodos , Transferencia Resonante de Energía de Fluorescencia , Receptores Frizzled/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Fosforilación/fisiología , Análisis de la Célula Individual/métodos , Xenopus laevisRESUMEN
Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIε) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. We mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.