RESUMEN

The casein kinase II (CK2) complex consists of catalytic (α) and regulatory (ß) subunits and is highly conserved throughout eukaryotes. Plant CK2 plays critical roles in multiple physiological processes; however, its function in plant immunity remains obscure. In this study, we demonstrated that the unique chloroplast-localized CK2 α subunit (CPCK2) is a negative regulator of Arabidopsis thaliana innate immunity. cpck2 mutants displayed enhanced resistance against the fungal pathogen powdery mildew, Golovinomyces cichoracearum and the virulent bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) DC3000. Moreover, the cpck2-1 mutant accumulated higher salicylic acid (SA) levels and mutations that disabled SA biosynthesis or signaling inhibited cpck2-1-mediated disease resistance. CPCK2 interacted with the chloroplast-localized carbonic anhydrase (CA), SA-binding protein 3 (SABP3), which was required for cpck2-mediated immunity. Significantly, CPCK2 phosphorylated SABP3, which promoted S-nitrosylation of this enzyme. It has previously been established that S-nitrosylation of SABP3 reduces both its SA binding function and its CA activity, which compromises the immune-related function of SABP3. Taken together, our results establish CPCK2 as a negative regulator of SA accumulation and associated immunity. Importantly, our findings unveil a mechanism by which CPCK2 negatively regulates plant immunity by promoting S-nitrosylation of SABP3 through phosphorylation, which provides the first example in plants of S-nitrosylation being promoted by cognate phosphorylation.

RESUMEN

Bone homeostasis relies on the delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The casein kinase 2 interacting protein-1 (CKIP-1), a specific CK2α subunit-interacting protein, has been documented as one of the crucial negative regulators of bone formation. CKIP-1 siRNA therapy has constraints that limit its use in clinical applications. Therefore, it is necessary to explore effective targeting strategies for CKIP-1. In this study, we observed an upregulation of CKIP-1 protein expression in the microgravity environment, while its ubiquitination levels decreased. We further investigated the interaction between CKIP-1 and VHL and found that VHL enhanced CKIP-1 degradation through the ubiquitylation-proteasome system (UPS). Additionally, we discovered a small molecule ligand, named C77, through DNA-encoded library (DEL) screening, which binds to CKIP-1 both in vivo and in vitro, as confirmed by Surface Plasmon Resonance (SPR) and the Cellular Thermal shift assay (CETSA), respectively. Our findings demonstrated the potential of VHL and C77 as guiding factors in the development of CKIP-1-based Proteolysis-Targeting Chimeras (PROTACs), which could be future therapeutic interventions in disuse osteoporosis.

Asunto(s)

Osteoporosis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Humanos , Ligandos , Osteoporosis/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/terapia , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Ubiquitinación , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteolisis , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ratones , Péptidos y Proteínas de Señalización Intracelular

RESUMEN

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.

Asunto(s)

Aromatasa , Estradiol , Células de la Granulosa , Ratones Noqueados , Animales , Femenino , Ratones , Aromatasa/metabolismo , Aromatasa/genética , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Estradiol/metabolismo , Células de la Granulosa/metabolismo

RESUMEN

The rise in drug-resistant fungal infections poses a significant public health concern, necessitating the development of new antifungal therapies. We aimed to address this challenge by targeting a yeast casein kinase of Candida albicans for antifungal drug development. The compound library contained 589 chemical structures similar to the previously identified kinase inhibitor GW461484A. Through virtual screening, four compounds with the PubChem IDs 102583821, 12982634, 102487860, and 86260205 were selected based on their binding energies. Hydrophobic bonds and van der Waals interactions stabilised the docked complexes. Comprehensive interaction studies and a 200-nanosecond molecular dynamics simulation suggested that these molecules can maintain stable interactions with the target, as evidenced by satisfactory RMSD and RMSF values. The Rg-RMSD-based Free Energy Landscape of these complexes indicated thermodynamic stability due to the presence of conformers with global minima. These promising findings highlight the potential for developing novel antifungal therapies targeting Yck2 in C. albicans. Further experimental validation is required to assess the efficacy of these compounds as antifungal agents. This research provides a significant step towards combating antifungal resistance and opens up a new avenue for drug discovery.

RESUMEN

Sodium and potassium channels, especially Nav1.5 and Kir2.1, play key roles in the formation of action potentials in cardiomyocytes. These channels interact with, and are regulated by, synapse-associated protein 97 (SAP97). However, the regulatory role of SAP97 in myocyte remains incompletely understood. Here, we investigate the function of SAP97 phosphorylation in the regulation of Nav1.5 and Kir2.1 channel complexes and the upstream regulation of SAP97. We found that SAP97 is phosphorylated by casein kinase II (CK2) in vitro. In addition, transfection of casein kinase 2 interacting protein-1 (CKIP-1) into cardiomyocytes to drive CK2 from the nucleus to the cytoplasm, increased SAP97 phosphorylation and Nav1.5 and Kir2.1 current activity. These findings demonstrated that CKIP-1 modulates the subcellular translocation of CK2, which regulates Nav1.5 and Kir2.1 channel complex formation and activity in cardiomyocytes.

Asunto(s)

Quinasa de la Caseína II , Miocitos Cardíacos , Canal de Sodio Activado por Voltaje NAV1.5 , Canales de Potasio de Rectificación Interna , Miocitos Cardíacos/metabolismo , Quinasa de la Caseína II/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Animales , Ratas , Fosforilación , Transporte de Proteínas , Humanos , Proteínas Portadoras/metabolismo , Ratas Sprague-Dawley

RESUMEN

The androgen receptor (AR) is a key driver of prostate cancer (PCa) and, as such, current mainstay treatments target this molecule. However, resistance commonly arises to these therapies and, therefore, additional targets must be evaluated to improve patient outcomes. Consequently, alternative approaches for indirectly targeting the AR are sought. AR crosstalk with other signalling pathways, including several protein kinase signalling cascades, has been identified as a potential route to combat therapy resistance. The casein kinase 1 (CK1) family of protein kinases phosphorylate a multitude of substrates, allowing them to regulate a diverse range of pathways from the cell cycle to DNA damage repair. As well as its role in several signalling pathways that are de-regulated in PCa, mutational data suggest its potential to promote prostate carcinogenesis. CK1α is one isoform predicted to regulate AR activity via phosphorylation and has been implicated in the progression of several other cancer types. In this review, we explore how the normal biological function of CK1 is de-regulated in cancer, the impact on signalling pathways and how this contributes towards prostate tumourigenesis, with a particular focus on the CK1α isoform as a novel therapeutic target for PCa.

RESUMEN

Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix αD could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the αD targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.

Asunto(s)

Quinasa de la Caseína II , Inhibidores de Proteínas Quinasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Humanos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Adenosina Trifosfato/metabolismo , Sitios de Unión

RESUMEN

Casein kinase II (CK2) has recently emerged as a pivotal mediator in the propagation of inflammation across various diseases. Nevertheless, its role in the pathogenesis of sepsis remains unexplored. Here, we investigated the involvement of CK2 in sepsis progression and the potential beneficial effects of silmitasertib, a selective and potent CK2α inhibitor, currently under clinical trials for COVID-19 and cancer. Sepsis was induced by caecal ligation and puncture (CLP) in four-month-old C57BL/6OlaHsd mice. One hour after the CLP/Sham procedure, animals were assigned to receive silmitasertib (50 mg/kg/i.v.) or vehicle. Plasma/organs were collected at 24 h for analysis. A second set of experiments was performed for survival rate over 120 h. Septic mice developed multiorgan failure, including renal dysfunction due to hypoperfusion (reduced renal blood flow) and increased plasma levels of creatinine. Renal derangements were associated with local overactivation of CK2, and downstream activation of the NF-ĸB-iNOS-NO axis, paralleled by a systemic cytokine storm. Interestingly, all markers of injury/inflammation were mitigated following silmitasertib administration. Additionally, when compared to sham-operated mice, sepsis led to vascular hyporesponsiveness due to an aberrant systemic and local release of NO. Silmitasertib restored sepsis-induced vascular abnormalities. Overall, these pharmacological effects of silmitasertib significantly reduced sepsis mortality. Our findings reveal, for the first time, the potential benefits of a selective and potent CK2 inhibitor to counteract sepsis-induced hyperinflammatory storm, vasoplegia, and ultimately prolonging the survival of septic mice, thus suggesting a pivotal role of CK2 in sepsis and silmitasertib as a novel powerful pharmacological tool for drug repurposing in sepsis.

Asunto(s)

Quinasa de la Caseína II , Sepsis , Animales , Masculino , Ratones , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/tratamiento farmacológico , Insuficiencia Multiorgánica/prevención & control , Naftiridinas , Fenazinas , Inhibidores de Proteínas Quinasas/farmacología , Pteridinas/farmacología , Sepsis/tratamiento farmacológico , Sepsis/complicaciones

Asunto(s)

Arabidopsis , Etilenos , Hojas de la Planta , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Etilenos/metabolismo , Etilenos/biosíntesis , Hojas de la Planta/fisiología , Hojas de la Planta/crecimiento & desarrollo , Senescencia de la Planta , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética

RESUMEN

Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these drugs can cause severe and persistent pain. GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) are implicated in various neurological disorders, including neuropathic pain. It is unclear whether and how constitutive calcineurin, a Ca2+/calmodulin protein phosphatase, controls synaptic CP-AMPARs. In this study, we found that blocking CP-AMPARs with IEM-1460 markedly reduced the amplitude of AMPAR-EPSCs in excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2), but not in inhibitory neurons expressing vesicular GABA transporter, in the spinal cord of FK506-treated male and female mice. FK506 treatment also caused an inward rectification in the current-voltage relationship of AMPAR-EPSCs specifically in VGluT2 neurons. Intrathecal injection of IEM-1460 rapidly alleviated pain hypersensitivity in FK506-treated mice. Furthermore, FK506 treatment substantially increased physical interaction of α2δ-1 with GluA1 and GluA2 in the spinal cord and reduced GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of spinal cords. Correspondingly, inhibiting α2δ-1 with pregabalin, Cacna2d1 genetic knock-out, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide reversed inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons caused by FK506 treatment. In addition, CK2 inhibition reversed FK506 treatment-induced pain hypersensitivity, α2δ-1 interactions with GluA1 and GluA2, and inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons. Thus, the increased prevalence of synaptic CP-AMPARs in spinal excitatory neurons plays a major role in calcineurin inhibitor-induced pain hypersensitivity. Calcineurin and CK2 antagonistically regulate postsynaptic CP-AMPARs through α2δ-1-mediated GluA1/GluA2 heteromeric assembly in the spinal dorsal horn.

Asunto(s)

Calcineurina , Quinasa de la Caseína II , Receptores AMPA , Médula Espinal , Tacrolimus , Animales , Receptores AMPA/metabolismo , Ratones , Calcineurina/metabolismo , Masculino , Femenino , Tacrolimus/farmacología , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratones Endogámicos C57BL , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/fisiología , Inhibidores de la Calcineurina/farmacología , Fenotipo , Canales de Calcio

RESUMEN

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.

Asunto(s)

Antígenos Virales de Tumores , Caseína Quinasa Ialfa , Poliomavirus de Células de Merkel , Proteínas con Repetición de beta-Transducina , Poliomavirus de Células de Merkel/genética , Poliomavirus de Células de Merkel/metabolismo , Humanos , Fosforilación , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Proteínas con Repetición de beta-Transducina/metabolismo , Proteínas con Repetición de beta-Transducina/genética , Antígenos Virales de Tumores/metabolismo , Antígenos Virales de Tumores/genética , Interacciones Huésped-Patógeno , Proteolisis , Replicación Viral , Unión Proteica , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Transformadores de Poliomavirus/genética , Infecciones por Polyomavirus/virología , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/genética

RESUMEN

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.

Asunto(s)

Empalme Alternativo , Caseína Quinasa Ialfa , Animales , Humanos , Ratones , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Línea Celular Tumoral , Proliferación Celular , Células HCT116 , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología

RESUMEN

Osteoarthritis (OA) is a chronic joint disease characterized by cartilage imbalance and disruption of cartilage extracellular matrix secretion. Identifying key genes that regulate cartilage differentiation and developing effective therapeutic strategies to restore their expression is crucial. In a previous study, we observed a significant correlation between the expression of the gene encoding casein kinase-2 interacting protein-1 (CKIP-1) in the cartilage of OA patients and OA severity scores, suggesting its potential involvement in OA development. To test this hypothesis, we synthesized a chondrocyte affinity plasmid, liposomes CKIP-1, to enhance CKIP-1 expression in chondrocytes. Our results demonstrated that injection of CAP-Lipos-CKIP-1 plasmid significantly improved OA joint destruction and restored joint motor function by enhancing cartilage extracellular matrix (ECM) secretion. Histological and cytological analyses confirmed that CKIP-1 maintains altered the phosphorylation of the signal transduction molecule SMAD2/3 of the transforming growth factor-ß (TGF-ß) pathway by promoting the phosphorylation of the 8T, 416S sit. Taken together, this work highlights a novel approach for the precise modulation of chondrocyte phenotype from an inflammatory to a noninflammatory state for the treatment of OA and may be broadly applicable to patients suffering from other arthritic diseases.

Asunto(s)

Condrocitos , Homeostasis , Liposomas , Osteoartritis , Condrocitos/metabolismo , Osteoartritis/terapia , Osteoartritis/patología , Osteoartritis/metabolismo , Liposomas/química , Humanos , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Masculino , Fosforilación , Cartílago Articular/metabolismo , Cartílago Articular/patología , Factor de Crecimiento Transformador beta/metabolismo , Matriz Extracelular/metabolismo , Proteína smad3/metabolismo , Proteína smad3/genética , Transducción de Señal , Plásmidos/genética , Nanopartículas/química , Nanopartículas/uso terapéutico , Proteína Smad2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética

RESUMEN

Ten-eleven translocation 1 (TET1) is a methylcytosine dioxygenase involved in active DNA demethylation. In our previous study, we demonstrated that TET1 reprogrammed the ovarian cancer epigenome, increased stem properties, and activated various regulatory networks, including metabolic networks. However, the role of TET1 in cancer metabolism remains poorly understood. Herein, we uncovered a demethylated metabolic gene network, especially oxidative phosphorylation (OXPHOS). Contrary to the concept of the Warburg effect in cancer cells, TET1 increased energy production mainly using OXPHOS rather than using glycolysis. Notably, TET1 increased the mitochondrial mass and DNA copy number. TET1 also activated mitochondrial biogenesis genes and adenosine triphosphate production. However, the reactive oxygen species levels were surprisingly decreased. In addition, TET1 increased the basal and maximal respiratory capacities. In an analysis of tricarboxylic acid cycle metabolites, TET1 increased the levels of α-ketoglutarate, which is a coenzyme of TET1 dioxygenase and may provide a positive feedback loop to modify the epigenomic landscape. TET1 also increased the mitochondrial complex I activity. Moreover, the mitochondrial complex I inhibitor, which had synergistic effects with the casein kinase 2 inhibitor, affected ovarian cancer growth. Altogether, TET1-reprogrammed ovarian cancer stem cells shifted the energy source to OXPHOS, which suggested that metabolic intervention might be a novel strategy for ovarian cancer treatment.

RESUMEN

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.

Asunto(s)

Proteínas Circadianas Period , Estabilidad Proteica , Humanos , Quinasa de la Caseína I/metabolismo , Quinasa de la Caseína I/genética , Ritmo Circadiano , Degrones , Células HEK293 , Proteínas Circadianas Period/metabolismo , Proteínas Circadianas Period/genética , Fosforilación , Proteolisis

RESUMEN

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.

Asunto(s)

Caseína Quinasa Ialfa , Vía de Señalización Wnt , beta Catenina , Humanos , Empalme Alternativo , beta Catenina/metabolismo , beta Catenina/genética , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Células HEK293 , Fosforilación , Proteína Wnt3A/metabolismo , Proteína Wnt3A/genética

RESUMEN

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.

RESUMEN

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

Asunto(s)

Quinasa de la Caseína II , Cromatina , Proteína HMGA2 , Células Madre Hematopoyéticas , Ratones Noqueados , Proteína HMGA2/metabolismo , Proteína HMGA2/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Ratones , Humanos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Cromatina/metabolismo , Cromatina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Hematopoyesis , Estrés Fisiológico , Fluorouracilo/farmacología , Regeneración , Fosforilación , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Ratones Endogámicos C57BL

RESUMEN

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.

Asunto(s)

Esclerosis Amiotrófica Lateral , Caseína Cinasa 1 épsilon , Quinasa Idelta de la Caseína , Proteínas de Unión al ADN , Fosforilación , Proteínas de Unión al ADN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Humanos , Quinasa Idelta de la Caseína/metabolismo , Caseína Cinasa 1 épsilon/metabolismo , Células HEK293

RESUMEN

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.

Asunto(s)

Enfermedad de Alzheimer , Glucógeno Sintasa Quinasa 3 beta , Vía de Señalización Wnt , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Humanos , Vía de Señalización Wnt/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Quinasa Idelta de la Caseína/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Estructura Molecular , Animales , Relación Estructura-Actividad