Generation of strong casein kinase 1 inhibitor of Arabidopsis thaliana

AbstractCasein kinase 1 (CK1) is an evolutionarily conserved protein kinase among eukaryotes. Studies on yeast, fungi, and animals have revealed that CK1 plays roles in divergent biological processes. By contrast, the collective knowledge regarding the biological roles of plant CK1 lags was behind those of animal CK1. One of reasons for this is that plants have more multiple genes encoding CK1 than animals. To accelerate the research for plant CK1, a strong CK1 inhibitor that efficiently inhibits multiple members of CK1 proteins in vivo (in planta) is required. Here, we report a novel strong CK1 inhibitor of Arabidopsis (AMI-331). Using a circadian period-lengthening activity as estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship (SAR) study of PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride), a potent CK1 inhibitor of Arabidopsis, and found that PHA767491 analogues bearing a propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) enhance the period-lengthening activity. The period lengthening activity of a hybrid molecule of AMI-212 and AMI-23 (AMI-331) is about 100-fold stronger than that of PHA767491. An in vitro assay indicated a strong inhibitory activity of CK1 kinase by AMI-331. Also, affinity proteomics using an AMI-331 probe showed that targets of AMI-331 are mostly CK1 proteins. As such, AMI-331 is a strong potent CK1 inhibitor that shows promise in the research of CK1 in plants.

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Casein Kinase 1 Delta Regulates the Pace of the Mammalian Circadian Clock

Molecular and Cellular Biology ◽

10.1128/mcb.00338-09 ◽

2009 ◽

Vol 29 (14) ◽

pp. 3853-3866 ◽

Cited By ~ 131

Author(s):

Jean-Pierre Etchegaray ◽

Kazuhiko K. Machida ◽

Elizabeth Noton ◽

Cara M. Constance ◽

Robert Dallmann ◽

...

Keyword(s):

Circadian Clock ◽

Perinatal Period ◽

Casein Kinase ◽

Gene Encoding ◽

Exon 2 ◽

Casein Kinase 1 ◽

Functional Differences ◽

Clock Proteins ◽

Genes Encoding

ABSTRACT Both casein kinase 1 delta (CK1δ) and epsilon (CK1ε) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1δ and CK1ε in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1δΔ2). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1δ. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by ∼20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1δ-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1ε did not alter these circadian endpoints. These results reveal important functional differences between CK1δ and CK1ε: CK1δ plays an unexpectedly important role in maintaining the 24-h circadian cycle length.

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Casein Kinase 1D Encodes a Novel Drug Target in Hedgehog—GLI-Driven Cancers and Tumor-Initiating Cells Resistant to SMO Inhibition

Cancers ◽

10.3390/cancers13164227 ◽

2021 ◽

Vol 13 (16) ◽

pp. 4227

Author(s):

Elisabeth Peer ◽

Sophie Karoline Aichberger ◽

Filip Vilotic ◽

Wolfgang Gruber ◽

Thomas Parigger ◽

...

Keyword(s):

Critical Role ◽

Treatment Strategies ◽

Casein Kinase ◽

Tumor Initiating Cells ◽

Casein Kinase 1 ◽

Hh Signaling ◽

Novel Drug ◽

Smo Inhibitor

(1) Background: Aberrant activation of the hedgehog (HH)—GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH—GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH—GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.

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Ex vivo Pretreatment of Islets with Mitomycin C

Cell Transplantation ◽

10.1177/0963689717721233 ◽

2017 ◽

Vol 26 (8) ◽

pp. 1392-1404 ◽

Cited By ~ 3

Author(s):

Naoya Sato ◽

Junichiro Haga ◽

Takayuki Anazawa ◽

Akira Kenjo ◽

Takashi Kimura ◽

...

Keyword(s):

Mitomycin C ◽

Validation Studies ◽

Ex Vivo ◽

Chemotactic Activity ◽

Vitro Assay ◽

Proinflammatory Mediators ◽

Genes Encoding ◽

Matrix Metallopeptidase

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

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Casein kinase 1D encodes a novel drug target in Hedgehog-GLI driven cancers and tumor-initiating cells resistant to SMO inhibition

10.1101/2021.06.17.448773 ◽

2021 ◽

Author(s):

Elisabeth Peer ◽

Sophie K Aichberger ◽

Filip Vilotic ◽

Gruber Wolfgang ◽

Thomas Parigger ◽

...

Keyword(s):

Cancer Cells ◽

Critical Role ◽

Casein Kinase ◽

Tumor Initiating Cells ◽

Casein Kinase 1 ◽

Hh Signaling ◽

Novel Drug ◽

Smo Inhibitor

Abstract: (1) Background: Aberrant activation of the Hedgehog (HH)/GLI pathway in stem-like tumor initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector Smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH/GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH/GLI driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity and monitored oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO-inhibitor sensitive and resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent tumor-initiating cancer cells in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor sensitive and resistant tumors.

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Altered synaptic adaptation and gain in sensory circuits of the casein kinase 1 delta (CK1dT44A) mouse model of migraine

10.1101/719039 ◽

2019 ◽

Cited By ~ 1

Author(s):

Pratyush Suryavanshi ◽

Punam Sawant Pokam ◽

KC Brennan

Keyword(s):

Steady State ◽

Cortical Neurons ◽

Casein Kinase ◽

Local Network ◽

Casein Kinase 1 ◽

Thermal Pain ◽

Readily Releasable Pool ◽

Short Stimulus

AbstractMigraine is a very common and disabling neurological disorder that remains poorly understood at the cellular and circuit level. Transgenic mice harboring a mutation in casein kinase 1 delta (CK1dT44A) represent the first animal model of non-hemiplegic migraine. These mice have decreased sensory thresholds to mechanical and thermal pain after treatment with the migraine trigger nitroglycerin; and an increased susceptibility to cortical spreading depression (CSD), which models the migraine aura. In this study, we investigated cellular and synaptic mechanisms within sensory cortical circuits that might underlie the migraine relevant phenotypes of CK1dT44A mice, using in vitro and in vivo whole cell electrophysiology. Surprisingly we found that at resting state, CK1dT44A neurons exhibited hyperpolarized membrane potentials, due to increased tonic inhibition. Despite this reduction in baseline excitability, CK1dT44A neurons fired action potentials more frequently in response to current injection. And despite similar synaptic and dendritic characteristics to wild type neurons, excitatory but not inhibitory CK1dT44A synapses failed to adapt to high frequency short-stimulus trains, resulting in elevated steady state excitatory currents. The increased steady state currents were attributable to an increased replenishment rate of the readily releasable pool, providing a presynaptic mechanism for the CK1dT44A phenotype. Finally, during in vivo experiments, CK1dT44A animals showed increased duration and membrane potential variance at ‘cortical up states’, showing that the intrinsic and synaptic changes we observed have excitatory consequences at the local network level. In conclusion excitatory sensory cortical neurons and networks in CK1dT44A animals appear to exhibit decreased adaptation and increased gain that may inform the migraine phenotype.

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Identification of Domains Responsible for Ubiquitin-Dependent Degradation of dMyc by Glycogen Synthase Kinase 3β and Casein Kinase 1 Kinases

Molecular and Cellular Biology ◽

10.1128/mcb.01535-08 ◽

2009 ◽

Vol 29 (12) ◽

pp. 3424-3434 ◽

Cited By ~ 21

Author(s):

Margherita Galletti ◽

Sara Riccardo ◽

Federica Parisi ◽

Carlina Lora ◽

Mahesh Kumar Saqcena ◽

...

Keyword(s):

Compound Eye ◽

Glycogen Synthase ◽

Physiological Role ◽

Casein Kinase ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3Β ◽

Casein Kinase 1 ◽

The One

ABSTRACT In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3β (GSK3β). Using Drosophila S2 cells, we demonstrate that CK1α promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3β in vertebrates. Mutation of ck1α or -ε or sgg/gsk3β in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3β, and members of the CK1 family. We demonstrate that mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1α and GSK3β kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation.

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Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3

Cell Death and Disease ◽

10.1038/s41419-019-2146-4 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 3

Author(s):

Song-Yi Lee ◽

Hyunjoo Kim ◽

Cathena Meiling Li ◽

Jaemin Kang ◽

Ayaz Najafov ◽

...

Keyword(s):

Ectopic Expression ◽

Specific Inhibitor ◽

Casein Kinase ◽

In Vitro Assays ◽

Serine Threonine Kinase ◽

Casein Kinase 1 ◽

Threonine Kinase ◽

Necrosis Factor

AbstractUpon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.

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T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009588 ◽

2021 ◽

Vol 15 (7) ◽

pp. e0009588

Author(s):

Edio Maldonado ◽

Diego A. Rojas ◽

Fabiola Urbina ◽

Aldo Solari

Keyword(s):

Protein Kinases ◽

Dna Polymerase ◽

Excision Repair ◽

Polymerase Activity ◽

Casein Kinase ◽

Phosphorylation Sites ◽

Casein Kinase 1 ◽

Synthesis Activity

The unicellular protozoan Trypanosoma cruzi is the causing agent of Chagas disease which affects several millions of people around the world. The components of the cell signaling pathways in this parasite have not been well studied yet, although its genome can encode several components able to transduce the signals, such as protein kinases and phosphatases. In a previous work we have found that DNA polymerase β (Tcpolβ) can be phosphorylated in vivo and this modification activates the synthesis activity of the enzyme. Tcpolβ is kinetoplast-located and is a key enzyme in the DNA base excision repair (BER) system. The polypeptide possesses several consensus phosphorylation sites for several protein kinases, however, a direct phosphorylation of those sites by specific kinases has not been reported yet. Tcpolβ has consensus phosphorylation sites for casein kinase 1 (CK1), casein kinase 2 (CK2) and aurora kinase (AUK). Genes encoding orthologues of those kinases exist in T. cruzi and we were able to identify the genes and to express them to investigate whether or no Tcpolβ could be a substrate for in vitro phosphorylation by those kinases. Both CK1 and TcAUK1 have auto-phosphorylation activities and they are able to phosphorylate Tcpolβ. CK2 cannot perform auto-phosphorylation of its subunits, however, it was able to phosphorylate Tcpolβ. Pharmacological inhibitors used to inhibit the homologous mammalian kinases can also inhibit the activity of T. cruzi kinases, although, at higher concentrations. The phosphorylation events carried out by those kinases can potentiate the DNA polymerase activity of Tcpolβ and it is discussed the role of the phosphorylation on the DNA polymerase and lyase activities of Tcpolβ. Taken altogether, indicates that CK1, CK2 and TcAUK1 can play an in vivo role regulating the function of Tcpolβ.

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p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs

Oncogene ◽

10.1038/sj.onc.1201541 ◽

1997 ◽

Vol 15 (14) ◽

pp. 1727-1736 ◽

Cited By ~ 108

Author(s):

U Knippschild ◽

D M Milne ◽

L E Campbell ◽

A J DeMaggio ◽

E Christenson ◽

...

Keyword(s):

Casein Kinase ◽

Casein Kinase 1

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Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection

10.1101/2021.11.16.468817 ◽

2021 ◽

Author(s):

Xiao-Dong Fang ◽

Qiang Gao ◽

Ying Zang ◽

Ji-Hui Qiao ◽

Dong-Min Gao ◽

...

Keyword(s):

Phase Separation ◽

Virus Infection ◽

Casein Kinase ◽

Casein Kinase 1 ◽

N Protein ◽

Intrinsically Disordered ◽

P Protein ◽

L Proteins

Liquid–liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infections is largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host casein kinase 1 (CK1)-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted virus replication through concentrating the N, L proteins, and genome RNA into viroplasms. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state leading to enhanced virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of viral P protein and virus infection.

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