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1.
J Med Chem ; 67(11): 8609-8629, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38780468

ABSTRACT

Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.


Subject(s)
Protein Kinase Inhibitors , Protein Serine-Threonine Kinases , Pteridines , Humans , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pteridines/pharmacology , Pteridines/chemistry , Pteridines/chemical synthesis , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Cell Proliferation/drug effects , Structure-Activity Relationship , Casein Kinase Idelta/antagonists & inhibitors , Casein Kinase Idelta/metabolism , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase 1 epsilon/metabolism , Cell Line, Tumor
2.
Sci Adv ; 10(19): eadj5185, 2024 May 10.
Article in English | MEDLINE | ID: mdl-38728403

ABSTRACT

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.


Subject(s)
Schizosaccharomyces pombe Proteins , Humans , Amino Acid Sequence , Casein Kinase 1 epsilon/metabolism , Casein Kinase 1 epsilon/genetics , Catalytic Domain , Mutation , Peptides/metabolism , Peptides/chemistry , Phosphorylation , Protein Binding , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Substrate Specificity
3.
Neurobiol Dis ; 196: 106516, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38677657

ABSTRACT

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.


Subject(s)
Amyotrophic Lateral Sclerosis , Casein Kinase 1 epsilon , Casein Kinase Idelta , DNA-Binding Proteins , Phosphorylation , DNA-Binding Proteins/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Humans , Casein Kinase Idelta/metabolism , Casein Kinase 1 epsilon/metabolism , HEK293 Cells
4.
EMBO Mol Med ; 15(9): e17451, 2023 09 11.
Article in English | MEDLINE | ID: mdl-37578019

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon , Parkinson Disease , Mice , Animals , Transcription Factors/genetics , DNA-Binding Proteins/genetics , alpha-Synuclein/genetics , Parkinson Disease/genetics , Casein Kinase 1 epsilon/genetics , Mutation
5.
J Biol Chem ; 298(8): 102191, 2022 08.
Article in English | MEDLINE | ID: mdl-35753345

ABSTRACT

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.


Subject(s)
Amyotrophic Lateral Sclerosis , Casein Kinase 1 epsilon , Casein Kinase Idelta , Drosophila Proteins , Frontotemporal Lobar Degeneration , Sarcoma , Amyotrophic Lateral Sclerosis/metabolism , Animals , Casein Kinase 1 epsilon/genetics , Casein Kinase Idelta/genetics , Casein Kinase Idelta/metabolism , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Frontotemporal Lobar Degeneration/metabolism , Humans , Mutation , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism
6.
PLoS Genet ; 18(2): e1010035, 2022 02.
Article in English | MEDLINE | ID: mdl-35139068

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon/physiology , Circadian Rhythm/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Neurons/physiology , Sleep/physiology , Animals , Animals, Genetically Modified , Brain/cytology , Brain/physiology , Casein Kinase 1 epsilon/genetics , Circadian Rhythm/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Female , Gene Expression Regulation , Mutation , Period Circadian Proteins/genetics
7.
Eur J Neurosci ; 55(9-10): 2939-2954, 2022 05.
Article in English | MEDLINE | ID: mdl-34514665

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon , Circadian Clocks , Animals , Casein Kinase 1 epsilon/genetics , Circadian Rhythm/genetics , Female , Male , Mice , Motor Activity , Photoperiod
8.
Nat Commun ; 12(1): 5386, 2021 09 10.
Article in English | MEDLINE | ID: mdl-34508104

ABSTRACT

Although inhibitors targeting CDK4/6 kinases (CDK4/6i) have shown promising clinical prospect in treating ER+/HER2- breast cancers, acquired drug resistance is frequently observed and mechanistic knowledge is needed to harness their full clinical potential. Here, we report that inhibition of CDK4/6 promotes ßTrCP1-mediated ubiquitination and proteasomal degradation of RB1, and facilitates SP1-mediated CDK6 transcriptional activation. Intriguingly, suppression of CK1ε not only efficiently prevents RB1 from degradation, but also prevents CDK4/6i-induced CDK6 upregulation by modulating SP1 protein stability, thereby enhancing CDK4/6i efficacy and overcoming resistance to CDK4/6i in vitro. Using xenograft and PDX models, we further demonstrate that combined inhibition of CK1ε and CDK4/6 results in marked suppression of tumor growth in vivo. Altogether, these results uncover the molecular mechanisms by which CDK4/6i treatment alters RB1 and CDK6 protein abundance, thereby driving the acquisition of CDK4/6i resistance. Importantly, we identify CK1ε as an effective target for potentiating the therapeutic efficacy of CDK4/6 inhibitors.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/drug therapy , Casein Kinase 1 epsilon/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Protein Kinase Inhibitors/therapeutic use , Protein Stability/drug effects , Proteolysis/drug effects , Retinoblastoma Binding Proteins/metabolism , Sp1 Transcription Factor/metabolism , Transcriptional Activation/drug effects , Ubiquitin-Protein Ligases/metabolism , Xenograft Model Antitumor Assays
9.
Molecules ; 26(15)2021 Jul 29.
Article in English | MEDLINE | ID: mdl-34361750

ABSTRACT

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.


Subject(s)
Acacia/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Apoptosis/genetics , Binding Sites , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Cell Proliferation/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Humans , Janus Kinase 3/antagonists & inhibitors , Janus Kinase 3/genetics , Janus Kinase 3/metabolism , K562 Cells , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Models, Molecular , NIMA-Related Kinases/antagonists & inhibitors , NIMA-Related Kinases/genetics , NIMA-Related Kinases/metabolism , Plant Bark/chemistry , Plant Extracts/chemistry , Protein Binding , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/isolation & purification , Proto-Oncogene Proteins c-abl/chemistry , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Signal Transduction , Triterpenes/chemistry , Triterpenes/isolation & purification , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
10.
Drugs ; 81(7): 857-866, 2021 May.
Article in English | MEDLINE | ID: mdl-33797740

ABSTRACT

Umbralisib (UKONIQ™) is an oral, first-in-class dual phosphatidylinositol 3-kinase delta (PI3Kδ) and casein kinase 1 epsilon (CK1ε) inhibitor being developed by TG Therapeutics for the treatment of various haematological malignancies. In February 2021, umbralisib received its first approval in the USA for the treatment of adults with relapsed or refractory marginal zone lymphoma (MZL) who have received ≥ 1 prior anti-CD20-based regimen, and relapsed or refractory follicular lymphoma (FL) who have received ≥ 3 prior lines of systemic therapy. Clinical studies in various haematological malignancies, including chronic lymphocytic leukaemia and non-Hodgkin's lymphoma, are underway in multiple countries. This article summarizes the milestones in the development of umbralisib leading to this first approval.


Subject(s)
Hematologic Neoplasms/drug therapy , Heterocyclic Compounds, 4 or More Rings/pharmacology , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Area Under Curve , Casein Kinase 1 epsilon/antagonists & inhibitors , Drug Approval , Drug Interactions , Heterocyclic Compounds, 4 or More Rings/adverse effects , Heterocyclic Compounds, 4 or More Rings/pharmacokinetics , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lymphoma, B-Cell, Marginal Zone/drug therapy , Lymphoma, Follicular/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Metabolic Clearance Rate , Phosphoinositide-3 Kinase Inhibitors/therapeutic use , Randomized Controlled Trials as Topic , United States , United States Food and Drug Administration
11.
Cancer Med ; 10(9): 3139-3152, 2021 05.
Article in English | MEDLINE | ID: mdl-33818013

ABSTRACT

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.


Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression , Liver Neoplasms/genetics , Liver , Neoplasm Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Adult , Aged , Aged, 80 and over , Baculoviral IAP Repeat-Containing 3 Protein/genetics , Baculoviral IAP Repeat-Containing 3 Protein/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/mortality , Casein Kinase 1 epsilon/genetics , Casein Kinase 1 epsilon/metabolism , Databases, Genetic , Female , Gene Expression Profiling , Hippo Signaling Pathway , Humans , Liver/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/mortality , Male , Middle Aged , Neoplasm Proteins/metabolism , Neurofibromin 2/genetics , Neurofibromin 2/metabolism , Nomograms , Prognosis , Proportional Hazards Models , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , ROC Curve , Risk Factors , Transcriptome , Young Adult
12.
Theranostics ; 11(9): 4421-4435, 2021.
Article in English | MEDLINE | ID: mdl-33754069

ABSTRACT

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.


Subject(s)
Carcinogenesis/genetics , Casein Kinase 1 epsilon/genetics , Casein Kinase Idelta/genetics , Co-Repressor Proteins/genetics , Colorectal Neoplasms/genetics , Cell Line , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Colorectal Neoplasms/pathology , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic/genetics , HCT116 Cells , HEK293 Cells , Humans , Organoids/pathology , Phosphorylation/genetics , Ubiquitination/genetics , Wnt Signaling Pathway/genetics
13.
Sci Rep ; 11(1): 4746, 2021 02 26.
Article in English | MEDLINE | ID: mdl-33637777

ABSTRACT

Time-of-day effects have been noted in a wide variety of cognitive behavioral tests, and perturbation of the circadian system, either at the level of the master clock in the SCN or downstream, impairs hippocampus-dependent learning and memory. A number of kinases, including the serine-threonine casein kinase 1 (CK1) isoforms CK1δ/ε, regulate the timing of the circadian period through post-translational modification of clock proteins. Modulation of these circadian kinases presents a novel treatment direction for cognitive deficits through circadian modulation. Here, we tested the potential for PF-670462, a small molecule inhibitor of CK1δ/ε, to improve cognitive performance in C57BL/6J mice in an array of behavioral tests. Compared to vehicle-treated mice tested at the same time of the circadian day, mice treated with PF-670462 displayed better recall of contextual fear conditioning, made fewer working memory errors in the radial arm water maze, and trained more efficiently in the Morris Water Maze. These benefits were accompanied by increased expression of activity-regulated cytoskeleton-associated protein (Arc) in the amygdala in response to an acute learning paradigm. Our results suggest the potential utility of CK1δ/ε inhibition in improving time-of-day cognitive performance.


Subject(s)
Casein Kinase 1 epsilon/drug effects , Casein Kinase Idelta/drug effects , Cognition , Pyrimidines/pharmacology , Amygdala/metabolism , Animals , CLOCK Proteins/metabolism , Conditioning, Psychological , Cytoskeletal Proteins/metabolism , Learning , Maze Learning , Memory , Mice, Inbred C57BL , Nerve Tissue Proteins/metabolism
14.
In Vivo ; 34(5): 2905-2911, 2020.
Article in English | MEDLINE | ID: mdl-32871831

ABSTRACT

BACKGROUND/AIM: Casein kinase 1 epsilon (CK1ε) is a member of the casein kinase 1 family, which includes highly conserved and ubiquitous serine/threonine protein kinases. Recent research has revealed that CK1ε plays an important role in a variety of human cancer types; however, its role in human melanoma remains unclear. The aim of this study was to elucidate the clinical role of CK1ε in patients with melanoma. PATIENTS AND METHODS: Samples from 34 patients with melanoma were analyzed by immunohistochemical staining. Formalin-fixed paraffin-embedded tissue microarrays were also examined by two histopathologists to assess CK1ε protein expression in humans. RESULTS: Cytoplasmic CK1ε protein expression was significantly lower in tumor tissue than in normal tissue. Lack of cytoplasmic CK1ε protein was significantly correlated with distant metastasis (p=0.022) and poorer survival (p=0.030). However, Kaplan-Meier survival analysis revealed that elevated expression of cytoplasmic CK1ε protein was not significantly associated with the overall survival of patients with melanoma. Univariate and multivariate analyses demonstrated that lack of cytoplasmic CK1ε protein expression was related to distant metastasis (p<0.001 and p=0.004), showing that CK1ε was a prognostic factor. CONCLUSION: CK1ε protein expression might serve as a prognostic indicator in the treatment of patients with melanoma.


Subject(s)
Casein Kinase 1 epsilon , Melanoma , Biomarkers, Tumor/genetics , Cytoplasm , Humans , Kaplan-Meier Estimate , Melanoma/genetics , Prognosis
15.
Int J Mol Sci ; 21(17)2020 Sep 03.
Article in English | MEDLINE | ID: mdl-32899434

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/metabolism , Casein Kinase 1 epsilon/metabolism , Caseins/metabolism , DEAD-box RNA Helicases/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Casein Kinase 1 epsilon/genetics , DEAD-box RNA Helicases/genetics , Humans , Kinetics , Phosphorylation , Wnt Proteins/genetics , beta Catenin/genetics
16.
Biochem Biophys Res Commun ; 532(3): 406-413, 2020 11 12.
Article in English | MEDLINE | ID: mdl-32888647

ABSTRACT

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of ß-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of ß-catenin transcriptional activity. Consistent with this, wild-type Daple promoted ß-catenin transcriptional activity, following dissociation of ß-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated ß-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.


Subject(s)
Casein Kinase 1 epsilon/metabolism , Dishevelled Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Microfilament Proteins/metabolism , Wnt Signaling Pathway/physiology , Animals , Carrier Proteins/metabolism , Dishevelled Proteins/chemistry , Gene Knockdown Techniques , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , L Cells , Mice , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/genetics , Phosphorylation , Wnt3A Protein/metabolism , beta Catenin/metabolism
17.
PLoS Genet ; 16(8): e1008820, 2020 08.
Article in English | MEDLINE | ID: mdl-32750048

ABSTRACT

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.


Subject(s)
Casein Kinase 1 epsilon/metabolism , Cell Polarity , Drosophila Proteins/metabolism , Morphogenesis , Tacrolimus Binding Proteins/metabolism , Animals , Casein Kinase 1 epsilon/genetics , Dishevelled Proteins/genetics , Dishevelled Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster , Loss of Function Mutation , Protein Binding , Protein Transport , Tacrolimus Binding Proteins/genetics , Wings, Animal/cytology , Wings, Animal/growth & development
18.
Dis Model Mech ; 13(8)2020 08 27.
Article in English | MEDLINE | ID: mdl-32680850

ABSTRACT

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.


Subject(s)
Amino Acids, Branched-Chain/metabolism , Apoptosis , Brain/enzymology , Casein Kinase 1 epsilon/deficiency , Drosophila Proteins/deficiency , Drosophila melanogaster/enzymology , Maple Syrup Urine Disease/enzymology , Neurogenesis , Neurons/enzymology , Animals , Animals, Genetically Modified , Brain/drug effects , Brain/embryology , Casein Kinase 1 epsilon/genetics , Disease Models, Animal , Drosophila Proteins/genetics , Drosophila melanogaster/drug effects , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Genetic Predisposition to Disease , Larva/enzymology , Larva/genetics , Lipid Peroxidation , Male , Maple Syrup Urine Disease/drug therapy , Maple Syrup Urine Disease/genetics , Maple Syrup Urine Disease/pathology , Metformin/pharmacology , Motor Activity , Neurons/drug effects , Neurons/pathology , Oxidative Stress , Phenotype
19.
Sci Rep ; 10(1): 6797, 2020 04 22.
Article in English | MEDLINE | ID: mdl-32321936

ABSTRACT

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.


Subject(s)
CRISPR-Cas Systems , Casein Kinase 1 epsilon/genetics , Casein Kinase Idelta/genetics , Endocytosis/genetics , Gene Targeting/methods , Phosphoproteins/genetics , Casein Kinase 1 epsilon/metabolism , Casein Kinase Idelta/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Centrosome/metabolism , Guanine Nucleotide Exchange Factors , HEK293 Cells , HeLa Cells , Humans , Interphase/genetics , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Substrate Specificity
20.
FASEB J ; 34(5): 6466-6478, 2020 05.
Article in English | MEDLINE | ID: mdl-32175635

ABSTRACT

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.


Subject(s)
Cartilage, Articular/pathology , Casein Kinase 1 epsilon/metabolism , Chondrocytes/pathology , JNK Mitogen-Activated Protein Kinases/metabolism , Osteoarthritis/pathology , Animals , Cartilage, Articular/metabolism , Case-Control Studies , Casein Kinase 1 epsilon/genetics , Cells, Cultured , Chondrocytes/metabolism , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , Male , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , Middle Aged , Osteoarthritis/genetics , Osteoarthritis/metabolism , Phosphorylation , Wnt Signaling Pathway , beta Catenin/genetics , beta Catenin/metabolism
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