Casein kinase 1α mediates estradiol secretion via CYP19A1 expression in mouse ovarian granulosa cells.

BACKGROUND: Casein kinase 1α (CK1α), expressed in both ovarian germ and somatic cells, is involved in the initial meiosis and primordial follicle formation of mouse oocytes. Using in vitro and in vivo experiments in this study, we explored the function and mechanism of CK1α in estrogen synthesis in mice ovarian granulosa cells. METHODS: A CK1α knockout (cKO) mouse model, targeted specifically to ovarian granulosa cells (GCs), was employed to establish the influence of CK1α on in vivo estrogen synthesis. The influence of CK1α deficiency on GCs was determined in vivo and in vitro by immunofluorescence analysis and Western blot assay. Transcriptome profiling, differentially expressed genes and gene functional enrichment analyses, and computation protein-protein docking, were further employed to assess the CK1α pathway. Furthermore, wild-type female mice were treated with the CK1α antagonist D4476 to elucidate the CK1α's role in estrogen regulation. RESULTS: Ovarian GCs CK1α deficiency impaired fertility and superovulation of female mice; also, the average litter size and the estradiol (E2) level in the serum of cKO female mice were decreased by 57.3% and 87.4% vs. control mice, respectively. This deficiency disrupted the estrous cycle and enhanced the apoptosis in the GCs. We observed that CK1α mediated the secretion of estradiol in mouse ovarian GCs via the cytochrome P450 subfamily 19 member 1 (CYP19A1). CONCLUSIONS: These findings improve the existing understanding of the regulation mechanism of female reproduction and estrogen synthesis. TRIAL REGISTRATION: Not applicable.

Casein kinase 1α mediates phosphorylation of the Merkel cell polyomavirus large T antigen for ß-TrCP destruction complex interaction and subsequent degradation.

Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF ß-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through in situ proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased ß-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the CSNK1A1 gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection. IMPORTANCE: Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.

Exploring the role of casein kinase 1α splice variants across cancer cell lines.

Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.

Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes.

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability.

The duration of the transcription-repression cycles that give rise to mammalian circadian rhythms is largely determined by the stability of the PERIOD (PER) protein, the rate-limiting components of the molecular clock. The degradation of PERs is tightly regulated by multisite phosphorylation by casein kinase 1 (CK1δ/ε). In this phosphoswitch, phosphorylation of a PER2 degron [degron 2 (D2)] causes degradation, while phosphorylation of the PER2 familial advanced sleep phase (FASP) domain blocks CK1 activity on the degron, stabilizing PER2. However, this model and many other studies of PER2 degradation do not include the second degron of PER2 that is conserved in PER1, termed degron 1 (D1). We examined how these two degrons contribute to PER2 stability, affect the balance of the phosphoswitch, and how they are differentiated by CK1. Using PER2-luciferase fusions and real-time luminometry, we investigated the contribution of both D2 and of CK1-PER2 binding. We find that D1, like D2, is a substrate of CK1 but that D1 plays only a 'backup' role in PER2 degradation. Notably, CK1 bound to a PER1:PER2 dimer protein can phosphorylate PER1 D1 in trans. This scaffolded phosphorylation provides additional levels of control to PER stability and circadian rhythms.

Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway.

Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/ß-catenin pathway, which promotes the degradation of ß-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/ß-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of ß-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/ß-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate ß-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/ß KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-ß-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/ß-catenin pathway at the level of ß-catenin and Axin.

Impact of GSK-3ß and CK-1δ on Wnt signaling pathway in alzheimer disease: A dual target approach.

Alzheimer's disease (AD) is an enigmatic neurological illness that offers few treatment options. Recent exploration has highlighted the crucial connection of the Wnt signaling pathway in AD pathogenesis, shedding light on potential therapeutic targets. The present study focuses on the dual targeting of glycogen synthase kinase-3ß (GSK-3ß) and casein kinase-1δ (CK-1δ) within the framework of the Wnt signaling pathway as a possible technique for AD intervention. GSK-3ß and CK-1δ are multifunctional kinases known for their roles in tau hyperphosphorylation, amyloid processing, and synaptic dysfunction, all of which are major hallmarks of Alzheimer's disease. They are intricately linked to Wnt signaling, which plays a pivotal part in sustaining neuronal function and synaptic plasticity. Dysregulation of the Wnt pathway in AD contributes to cognitive decline and neurodegeneration. This review delves into the molecular mechanisms by which GSK-3ß and CK-1δ impact the Wnt signaling pathway, elucidating their roles in AD pathogenesis. We discuss the potential of small-molecule inhibitors along with their SAR studies along with the multi-targetd approach targeting GSK-3ß and CK-1δ to modulate Wnt signaling and mitigate AD-related pathology. In summary, the dual targeting of GSK-3ß and CK-1δ within the framework of the Wnt signaling pathway presents an innovative and promising avenue for future AD therapies, offering new hope for patients and caregivers in the quest to combat this challenging condition.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation.

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.

Combinatorial regulation by ERK1/2 and CK1δ protein kinases leads to HIF-1α association with microtubules and facilitates its symmetrical distribution during mitosis.

Hypoxia-inducible factor-1 (HIF-1) is the key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. We have previously reported that phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes HIF-1α nuclear accumulation and stimulates HIF-1 activity while lack of this modification triggers HIF-1α nuclear export and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ impairs HIF-1 activity by obstructing the formation of a HIF-1α/ARNT heterodimer. Investigation of these two antagonistic events by expressing double phospho-site mutants in HIF1A-/- cells under hypoxia revealed independent and additive phosphorylation effects that can create a gradient of HIF-1α subcellular localization and transcriptional activity. Furthermore, modification by CK1δ caused mitochondrial release of the non-nuclear HIF-1α form and binding to microtubules via its N-terminal domain. In agreement, endogenous HIF-1α could be shown to co-localize with mitotic spindle microtubules and interact with tubulin, both of which were inhibited by CK1δ silencing or inhibition. Moreover, CK1δ expression was necessary for equal partitioning of mother cell-produced HIF-1α to the daughter cell nuclei at the end of mitosis. Overall, our results suggest that phosphorylation by CK1δ stimulates the association of non-nuclear HIF-1α with microtubules, which may serve as a means to establish a symmetric distribution of HIF-1α during cell division under low oxygen conditions.

Apoptosis and eryptosis: similarities and differences.

Eryptosis is a regulated cell death (RCD) of mature erythrocytes initially described as a counterpart of apoptosis for enucleated cells. However, over the recent years, a growing number of studies have emphasized certain differences between both cell death modalities. In this review paper, we underline the hallmarks of eryptosis and apoptosis and highlight resemblances and dissimilarities between both RCDs. We summarize and critically discuss differences in the impact of caspase-3, Ca2+ signaling, ROS signaling pathways, opposing roles of casein kinase 1α, protein kinase C, Janus kinase 3, cyclin-dependent kinase 4, and AMP-activated protein kinase to highlight a certain degree of divergence between apoptosis and eryptosis. This review emphasizes the crucial importance of further studies that focus on deepening our knowledge of cell death machinery and identifying novel differences between cell death of nucleated and enucleated cells. This might provide evidence that erythrocytes can be defined as viable entities capable of programmed cell destruction. Additionally, the revealed cell type-specific patterns in cell death can facilitate the development of cell death-modulating therapeutic agents.

Targeting casein kinase 1 for cancer therapy: current strategies and future perspectives.

Casein Kinase 1 (CK1) is a family of serine/threonine protein kinases that play a crucial role in various cellular processes, including cell proliferation, survival, and metabolism. The dysregulation of CK1 expression has been implicated in the development and progression of several types of cancer, making it an attractive target for anticancer therapy. In this review, we provide an overview of the current strategies employed to target CK1 for cancer therapy and discuss the future perspectives in this field. We highlight the different approaches, including small molecule inhibitors, RNA interference, genome editing, and immunotherapies, which hold immense potential for targeted modulation of CK1 activity in cancer cells. Furthermore, we discuss the challenges associated with targeting CK1 and propose potential strategies to overcome these hurdles. Overall, targeting CK1 holds great promise as a therapeutic strategy for cancer treatment, and further research in this area is warranted.

Data-independent acquisition-based global phosphoproteomics reveal the diverse roles of casein kinase 1 in plant development.

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.

Casein kinase 1 controls the shuttling of epidermal growth factor receptor and estrogen receptor in endometrial carcinoma induced by breast cancer hormonal therapy: Relevance of GPER1/Src.

Casein kinase 1 plays a crucial role in carcinogenesis. 4-Hydroxytamoxifen (4-OHT), which is widely used to treat breast cancer, often leads to the development of endometrial carcinoma with poor prognosis, particularly among women who receiving long-term treatment. This study was performed to elucidate whether specific inhibition of casein kinase 1 (CK1) controls 4-OHT-mediated Ishikawa cell carcinogenesis. 4-OHT significantly stimulated the activity of estrogen receptor alpha (ERα) and nuclear translocation and expression of epidermal growth factor receptor (EGFR) from the plasma membrane to perinuclear or nuclear regions, as well as the activities of G-protein-coupled estrogen receptor 1 (GPER1) and Src in Ishikawa cells. However, inhibition of EGFR by Gefitinib blocked all these events, and inhibition of GPER1 or Src produced a partial block. GPER1 and Src controlled Ishikawa cell carcinogenesis in different manners: GPER1 accelerated EGFR mobility without affecting ERα activity, while Src activated ERα and EGFR without any change in GPER1 expression. EGFR and GPER1 performed reciprocal regulation in endometrial cell carcinogenesis via direct interaction in 4-OHT-treated Ishikawa cells, implying a possible key role of GPER1 in these events. Inhibition of CK1 by CKI-7 and IC261, however, impeded all changes beginning with EGFR translocation and activity in 4-OHT-treated Ishikawa cells. These findings indicate that inhibition of CK1 could control 4-OHT-mediated activation and translocation of ER/EGFR and GPER1/Src expression, inhibiting 4-OHT-triggered endometrial carcinogenesis. Therefore, targeting of CK1 by CKI-7 and IC261 could be a prospective adjuvant therapy for breast cancer patients taking tamoxifen.

Fission yeast CK1 promotes DNA double-strand break repair through both homologous recombination and non-homologous end joining.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Glutaredoxin 1 protects lens epithelial cells from epithelial-mesenchymal transition by preventing casein kinase 1α S-glutathionylation during posterior capsular opacification.

Oxidative stress drives protein S-glutathionylation, which regulates the structure and function of target proteins and is implicated in the pathogenesis of many diseases. Glutaredoxin 1 (Grx1), a cytoplasmic deglutathionylating enzyme, maintains a reducing environment within the cell under various conditions by reversing S-glutathionylation. Grx1 performs a wide range of antioxidant activities in the lens and prevents protein-thiol mixed disulfide accumulation, reducing protein-protein aggregation, insolubilization, and apoptosis of lens epithelial cells. Oxidative stress is related to epithelial-mesenchymal transition (EMT) during posterior capsular opacification (PCO). However, whether Grx1-regulated protein S-glutathionylation plays an essential role in PCO remains unclear. In this study, we revealed that Grx1 expression was decreased in mice following cataract surgery. Furthermore, the absence of Grx1 elevated oxidative stress and protein S-glutathionylation and aggravated EMT in both in vitro and in vivo models. Concurrently, these results could be reversed by Grx1 overexpression. Notably, liquid chromatography-tandem mass spectrometry results showed that casein kinase 1α (CK1α) was susceptible to S-glutathionylation under oxidative stress, and CK1α S-glutathionylation (CK1α-SSG) was mediated at Cys249. The absence of Grx1 upregulated CK1α-SSG, subsequently decreasing the CK1α-induced phosphorylation of ß-catenin at Ser45. The consequential downregulation of degradative ß-catenin and upregulation of its nuclear translocation activated the Wnt/ß-catenin signaling pathway and aggravated EMT. In conclusion, the downregulated expression of Grx1 in mice following cataract surgery aggravated EMT by upregulating the extent of CK1α-SSG. To the best of our knowledge, our study is the first to report how S-glutathionylation regulates CK1α activity under oxidative stress.

Casein Kinase 1δ Phosphorylates TDP-43 and Suppresses Its Function in Tau mRNA Processing.

BACKGROUND: Neurofibrillary tangle aggregated from anomalous hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD). Trans-active response DNA-binding protein of 43 kDa (TDP-43) enhances the instability and exon (E) 10 inclusion of tau mRNA. Cytoplasmic inclusion of hyperphosphorylated TDP-43 in the neurons constitutes the third most prevalent proteinopathy of AD. Casein kinase 1δ (CK1δ) is elevated in AD brain and phosphorylates TDP-43 in vitro. OBJECTIVE: To determine the roles of CK1δ in phosphorylation, aggregation, and function of TDP-43 in the processing of tau mRNA. METHODS: The interaction and colocalization of TDP-43 and CK1δ were analyzed by co-immunoprecipitation and immunofluorescence staining. TDP-43 phosphorylation by CK1δ was determined in vitro and in cultured cells. RIPA-insoluble TDP-43 aggregates obtained by ultracentrifugation were analyzed by immunoblots. The instability and E10 splicing of tau mRNA were studied by using a reporter of green fluorescence protein tailed with 3'-untranslational region of tau mRNA and a mini-tau gene and analyzed by real-time quantitative PCR and reverse transcriptional PCR. RESULTS: We found that CK1δ interacted and co-localized with TDP-43. TDP-43 was phosphorylated by CK1δ at Ser379, Ser403/404, and Ser409/410 in vitro and in cultured cells, which was mutually enhanced. CK1δ overexpression promoted the aggregation of TDP-43 and suppressed its activity in enhancing the instability and E10 inclusion of tau mRNA. CONCLUSION: CK1δ phosphorylates TDP-43, promotes its aggregation, and inhibits its activity in promoting the instability of tau mRNA and inclusion of tau E10. Elevated CK1δ in AD brain may contribute to TDP-43 and tau pathologies directly or indirectly.

Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction.

AIMS: Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). METHODS AND RESULTS: Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI. CONCLUSIONS: Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

Role of casein kinase 1 in the amoeboid migration of B-cell leukemic and lymphoma cells: A quantitative live imaging in the confined environment.

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.