RESUMEN
The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína Axina/genética , beta Catenina/genética , Proteína de la Poliposis Adenomatosa del Colon/ultraestructura , Proteína Axina/química , Proteína Axina/ultraestructura , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Fosforilación/genética , Multimerización de Proteína/genética , Proteolisis , Ubiquitinación/genética , Vía de Señalización WntRESUMEN
The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-methoxybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield, respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [11C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([11C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-[11C]methoxybenzamide ([11C]5c), were prepared from their O-desmethylated precursor 6a or 6b with [11C]CH3OTf through O-[11C]methylation and isolated by HPLC combined with SPE in 40-45% radiochemical yield, based on [11C]CO2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370-740â¯GBq/µmol with a total synthesis time of â¼40-min from EOB.
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Bencimidazoles/síntesis química , Benzoatos/síntesis química , Quinasa de la Caseína I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Radiofármacos/síntesis química , Enfermedad de Alzheimer/diagnóstico por imagen , Benzamidas/síntesis química , Benzamidas/química , Bencimidazoles/química , Benzoatos/química , Radioisótopos de Carbono , Interacciones Hidrofóbicas e Hidrofílicas , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/química , Radiofármacos/químicaRESUMEN
Several neurodegenerative disorders including Alzheimer's disease (AD) have been connected with deregulation of casein kinase 1 (CK1) activity. Inhibition of CK1 therefore presents a potential therapeutic strategy against such pathologies. Recently, novel class of CK1-specific inhibitors with N-(benzo[d]thiazol-2-yl)-2-phenylacetamide structural scaffold has been discovered. 1-(benzo[d]thiazol-2-yl)-3-phenylureas, on the other hand, are known inhibitors amyloid-beta binding alcohol dehydrogenase (ABAD), an enzyme also involved in pathophysiology of AD. Based on their tight structural similarity, we decided to evaluate series of previously published benzothiazolylphenylureas, originally designed as ABAD inhibitors, for their inhibitory activity towards CK1. Several compounds were found to be submicromolar CK1 inhibitors. Moreover, two compounds were found to inhibit both, ABAD and CK1. Such dual-activity could be of advantage for AD treatment, as it would simultaneously target two distinct pathological processes involved in disease's progression. Based on PAMPA testing both compounds were suggested to permeate the blood-brain barrier, which makes them, together with their unique dual activity, interesting lead compounds for further development.
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3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Quinasa de la Caseína I/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Quinasa de la Caseína I/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Enfermedades Neurodegenerativas/metabolismo , Compuestos de Fenilurea/química , Relación Estructura-ActividadRESUMEN
Cellular signal transduction components are usually regulated not only on transcriptional or translational level, but also by posttranslational modifications. Among these, reversible phosphorylation represents the most abundant modification. In general, phosphorylation events are essential for regulating the activity of central signal transduction proteins, also including kinases itself. Members of the CK1 family can be found as central signal transduction proteins in numerous cellular pathways. Due to its wide variety of cellular functions the activity of CK1 family members has to be tightly regulated. We previously reported that PKA and Chk1 are able to phosphorylate CK1δ within its C-terminal regulatory domain, consequently resulting in altered CK1 kinase activity. In the present study, we show by several methods that protein kinase C α (PKCα) as well is able to phosphorylate CK1δ at its C-terminally located residues S328, T329, and S370. Furthermore, we analyze the functional consequences of PKCα-mediated phosphorylation on CK1δ kinase activity. Mutation of S328, T329, or S370 to alanine dramatically alters the kinetic parameters of CK1δ. By using the PKCα-specific inhibitor Go-6983 in a selected cell culture model, we finally show that the in vitro detected regulatory connection between PKCα and CK1δ is also relevant in the cellular context. Taken together, these data contribute to a deeper understanding of cellular signal transduction networks thereby helping to form a basis for the development of future therapeutic concepts.
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Quinasa Idelta de la Caseína/química , Quinasa Idelta de la Caseína/metabolismo , Proteína Quinasa C-alfa/metabolismo , Secuencias de Aminoácidos , Quinasa Idelta de la Caseína/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Dominios ProteicosRESUMEN
Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1-specific inhibitors. Furthermore, expression of CK1δ and É is changed in colorectal tumors compared to normal bowel epithelium, and high CK1É expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and É expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients' survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer.
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Caseína Cinasa 1 épsilon/genética , Quinasa Idelta de la Caseína/genética , Neoplasias Colorrectales/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Anciano , Animales , Western Blotting , Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Perfilación de la Expresión Génica , Células HT29 , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Carga Tumoral/genéticaRESUMEN
Casein kinase 1 (CK1) is serine/threonine protein kinase highly conserved among eukaryotes, and regulates multiple developmental and signaling events through phosphorylation of target proteins. Arabidopsis early flowering 1 (EL1)-like (AELs) are plant-specific CK1s with varied functions, but identification and validation of their substrates is a major bottleneck in elucidating their physiological roles. Here, we conducted a quantitative phosphoproteomic analysis in data-independent acquisition mode to systematically identify CK1 substrates. We extracted proteins from seedlings overexpressing individual AEL genes (AEL1/2/3/4-OE) or lacking AEL function (all ael single mutants and two triple mutants) to identify the high-confidence phosphopeptides with significantly altered abundance compared to wild-type Col-0. Among these, we selected 3985 phosphopeptides with higher abundance in AEL-OE lines or lower abundance in ael mutants compared with Col-0 as AEL-upregulated phosphopeptides, and defined 1032 phosphoproteins. Eight CK1s substrate motifs were enriched among AEL-upregulated phosphopeptides and verified, which allowed us to predict additional candidate substrates and functions of CK1s. We functionally characterized a newly identified substrate C3H17, a CCCH-type zinc finger transcription factor, through biochemical and genetic analyses, revealing a role for AEL-promoted C3H17 protein stability and transactivation activity in regulating embryogenesis. As CK1s are highly conserved across eukaryotes, we searched the rice, mouse, and human protein databases using newly identified CK1 substrate motifs, yielding many more candidate substrates than currently known, largely expanding our understanding of the common and distinct functions exerted by CK1s in Arabidopsis and humans, facilitating future mechanistic studies of CK1-mediated phosphorylation in different species.
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Proteínas de Arabidopsis , Arabidopsis , Humanos , Animales , Ratones , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Quinasa de la Caseína I/genética , Fosfopéptidos/química , Desarrollo de la Planta/genéticaRESUMEN
Protein kinases play a vital role in biology and deregulation of kinases is implicated in numerous diseases ranging from cancer to neurodegenerative diseases, making them a major target class for the pharmaceutical industry. However, the high degree of conservation that exists between ATP-binding sites among kinases makes it difficult for current inhibitors to be highly specific. In the context of neurodegeneration, several groups including ours, have linked different kinases such as CK1 and Alzheimer's disease for example. Strictly CK1-isoform specific regulators do not exist and known CK1 inhibitors are inhibiting the enzymatic activity, targeting the ATP-binding site. Here we review compounds known to target CK1, as well as other inhibitory types that could benefit CK1. We introduce the DNA-encoded library (DEL) technology that might represent an interesting approach to uncover allosteric modulators instead of ATP competitors. Such a strategy, taking into account known allosteric inhibitors and mechanisms, might help designing modulators that are more specific towards a specific kinase, and in the case of CK1, toward specific isoforms.
RESUMEN
Biological systems operate in constant communication through shared components and feedback from changes in the environment. Casein kinase 1 (CK1) is a family of protein kinases that functions in diverse biological pathways and its regulation is beginning to be understood. The several isoforms of CK1 take part in key steps of processes including protein translation, cell-cell interactions, synaptic dopaminergic signaling and circadian rhythms. While CK1 mutations are rarely the primary drivers of disease, the kinases are often found to play an accessory role in metabolic disorders and cancers. In these settings, the dysregulation of CK1 coincides with increased disease severity. Among kinases, CK1 is unique in that its substrate specificity changes dramatically with its own phosphorylation state. Understanding the process that governs CK1 substrate selection is thus useful in identifying its role in various ailments. An illustrative example is the PERIOD2 (PER2) phosphoswitch, where CK1δ/ε kinase activity can be varied between three different substrate motifs to regulate the circadian clock.
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BACKGROUND: The casein kinase 1 (CK1) family is involved in regulating many cellular processes, including membrane trafficking, DNA damage repair, cytoskeleton dynamics, cytoskeleton maintenance and apoptosis. CK1 isoforms, especially CK1δ and CK1ε have emerged as important therapeutic targets for severe disorders such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), familial advanced sleep phase syndrome and cancer. Due to the importance of CK1 for the pathogenesis of disorders, there are great interests in the development of CK1 inhibitors. METHODS: Using SciFinder® as a tool, the publications about the biology of CK1 and the recent developments of CK1 inhibitors were surveyed with an exclusion of those published as patents. RESULTS: This review presents the current state of knowledge on the development of CK1 inhibitors, including both synthetic small molecular inhibitors that were divided into 7 categories according to structural features, and the natural compounds. An overview of the advancement of CK1 inhibitors was given, with the introduction of various existing CK1 inhibitors, their inhibitory activities, and the structure-activity relationships. CONCLUSION: Through physicochemical characterization and biological investigations, it is possible to understand the structure-activity relationship of CK1 inhibitors, which will contribute to better design and discovery of potent and selective CK1 inhibitors as potential agents for severe disorders such as AD, ALS and cancer.
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Quinasa de la Caseína I , Neoplasias , Apoptosis , Quinasa de la Caseína I/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Isoformas de Proteínas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Glioma is a type of tumor that usually occurs in the adult central nervous system. Protein kinases have become important targets for oncotherapy since they are closely correlated with signal transduction. The role of the casein kinase 1 (CK1) gene in glioma remains to be fully elucidated. METHODS: The mRNA and protein expression of CK1 were analyzed by Realtime PCR, Western blot and immunohistochemistry. The cell behavior was assayed by MTT, Transwell and cell scratch methods. Cell cycle and cell apoptosis were performed by flow cytometer. Construction of stable cell line was completed by lentivirus infection. The nude mouse model was used for in vivo analysis on the role of CK1 by injecting the cells into subcutaneous tissue, tail vein and cerebral cortex. The prognostic role of CK1 in glioma was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: immunohistochemical staining demonstrated that the expression of CK1 in glioma samples was correlated with the grade of glioma. Survival analysis using Kaplan-Meier and multivariate analysis by Cox regression indicated that CK1 could be used as an independent prognostic marker for glioma. The methyl thiazolyl tetrazolium (MTT), transwell, and cell scratch assays demonstrated that the CK1 gene promoted cell proliferation and invasion through the phosphatidylinositol 3 kinase/matrix metalloproteinase 2 (AKT-MMP2) signaling pathway. In vivo experiments in mice also confirmed the ability of CK1 to enhance tumor proliferation and metastasis, with the metastatic site being the small intestine. CONCLUSIONS: the expression of CK1 was correlated with glioma grade and patient survival and it may enhance glioma proliferation and metastasis via AKT-MMP2 pathway.
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BACKGROUND: Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. METHODS: We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. RESULTS: First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. CONCLUSIONS: In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.
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Células Madre Embrionarias Humanas , Diferenciación Celular , Endodermo , Humanos , Niacinamida/farmacología , PáncreasRESUMEN
BACKGROUND: This study aimed to investigate the effects of high Aß load on the microglial function, neuronal activity, and repressor element-1 silencing transcription factor (REST) expression and nuclear localization and further explore the mechanism underlying nuclear REST deficiency and the correlation between pathology and symptoms in Alzheimer's disease (AD). METHODS: BV2 cells and N2A cells were treated with Aß at different concentrations, and the viability, apoptosis and autophagy of these cells were further evaluated. The expression of major histocompatibility complex class II (MHC-II, a marker of microglial activation), REST in the neurons and REST specific phosphorylase casein kinase 1 (CK1) were detected. RESULTS: Aß at a low concentration (2.5 µmol/L) could alter the microglial morphology, and the proportion of amoebic microglia and protein expression of MHC-II increased in a Aß concentration dependent manner. Aß at a high concentration (10 µmol/L) was able to reduce REST expression, elevate the expression of pro-apoptotic and pro-autophagic genes, inhibit the expression of anti-apoptotic gene, and reduce the neuronal activity. REST expression reduced, but CK1 increased in neurons, and CK1 inhibitor significantly increased REST expression. there was co-expression of REST and CK1 in the brain of AD mice, which was characterized by reduced nuclear REST expression and elevated CK1 expression. CONCLUSIONS: High Aß load may cause microglial cell dysfunction and loss of REST expression in the neurons, resulting in dementia. The reduced nuclear REST might be ascribed to its degradation by ubiquitination. This supports the hypothesis that high plaque load may increase the risk for dementia.