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1.
Elife ; 82019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31090542

RESUMO

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. We show that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled - which normally localises to opposite cell edges to Strabismus - reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iε. We conclude that Casein Kinase Iε phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Polaridade Celular , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Proteínas de Membrana/metabolismo , Animais , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Pupa/enzimologia , Pupa/fisiologia , Asas de Animais/enzimologia , Asas de Animais/fisiologia
2.
Nat Commun ; 10(1): 1804, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000703

RESUMO

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Domínios PDZ/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Técnicas Biossensoriais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Fosforilação/fisiologia , Análise de Célula Única/métodos , Xenopus laevis
3.
Bioorg Med Chem Lett ; 28(23-24): 3681-3684, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30385160

RESUMO

Our internal casein kinase 1ε lead inhibitor, compound 1 was partially cleared by the polymorphic cytochrome P450 2D6. CYP2D6 involvement in metabolism implies more extensive clinical trials. We therefore wanted to reduce the contribution to clearance by this enzyme. We utilized metabolism reports for compound 1 performed in recombinant CYP2D6 together with structure-metabolism variation in structures of closely related analogs in order to see if we could incorporate similar substitution patterns in our lead compound. In addition, we utilized a previously established docking method using a modified CYP2D6 crystal structure to see if the metabolism patterns in CYP2D6 could be reproduced to afford the metabolites in the metabolism reports as well as those for the compounds used in the structure-metabolism relationship. All three of these steps, the metabolism report, the establishment of structure-metabolism relationships and the docking, lead to compound 10 where CYP2D6 was not involved in the clearance pathways.


Assuntos
Caseína Quinase Iépsilon/antagonistas & inibidores , Citocromo P-450 CYP2D6/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Sítios de Ligação , Caseína Quinase Iépsilon/metabolismo , Cristalografia por Raios X , Citocromo P-450 CYP2D6/genética , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
4.
J Biol Chem ; 293(42): 16337-16347, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30166345

RESUMO

Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro For each of the AXIN1-binding partners tested, i.e. casein kinase 1 ϵ (CK1ϵ); c-Myc; peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1); and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. We functionally validated the specific AXIN1-CK1ϵ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1ϵ and regulates CK1ϵ-induced phosphorylation of DVL and activation of Wnt/ß-catenin signaling.


Assuntos
Proteína Axina/metabolismo , Caseína Quinase Iépsilon/metabolismo , Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Ligação Competitiva , Proteínas Desgrenhadas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
5.
Int J Exp Pathol ; 99(3): 113-120, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-30073722

RESUMO

Precartilaginous stem cells (PSCs) are adult stem cells which could self-renew or differentiate into chondrocytes to promote bone growth. In this study, we aimed to understand the role of transforming growth factor-ß1 (TGF-ß1) in precartilaginous stem cell (PSC) differentiation and to study the mechanisms that underlie this role. We purified PSCs from the neonatal murine perichondrial mesenchyme using immunomagnetic beads, and primary cultured them. Their phenotype was confirmed by the PSC marker fibroblast growth factor receptor-3 (FGFR-3) overexpression. TGF-ß1 was added to induce PSCs differentiation. TGF-ß1 increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9 and aggrecan) in the cultured PSCs. This was abolished by TGF-ß receptor II (TGFRII) and Casein kinase 1 epsilon (CK1ε) lentiviral shRNA depletion. Meanwhile, we found that TGF-ß1 induced CK1ε activation, glycogen synthase kinase-3ß (GSK3ß) phosphorylation and ß-catenin nuclear translocation in the mouse PSCs, which was almost completely blocked by TGFRII and CK1ε shRNA knockdown. Based on these results, we suggest that TGF-ß1 induces CK1ε activation to promote ß-catenin nuclear accumulation, which then regulates chondrogenesis-related gene transcription to eventually promote mouse PSC differentiation.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Caseína Quinase Iépsilon/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Receptor do Fator de Crescimento Transformador beta Tipo II/agonistas , Fator de Crescimento Transformador beta1/farmacologia , beta Catenina/metabolismo , Células-Tronco Adultas/enzimologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Animais Recém-Nascidos , Caseína Quinase Iépsilon/genética , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/enzimologia , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Fenótipo , Fosforilação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 115(32): E7522-E7531, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30038030

RESUMO

The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/ß-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/ß-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/ß-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic ß-catenin. Moreover, TPA increased the association of ß-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/ß-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/ß-catenin signaling.


Assuntos
Carcinógenos/toxicidade , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidade , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Proteína Axina/metabolismo , Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fibroblastos , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Fosforilação , Estabilidade Proteica/efeitos dos fármacos , Purinas/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Fator de Transcrição 4 , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
7.
Cell Death Dis ; 9(6): 627, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29795381

RESUMO

Renal cell carcinoma (RCC) is the most common malignant disease of kidney in adults. The proteasome activator REGγ was previously reported to promote the degradation of multiple important regulatory proteins and involved in the progression and development of numerous human cancers. Here, we first reported that REGγ was upregulated in RCC and its upregulation was correlated with a poor prognosis in RCC patients. REGγ depletion obviously suppressed RCC cells proliferation in vitro and in vivo. Notably, casein kinase 1ε (CK1ε) was identified as a novel target of REGγ and knockdown of CK1ε effectively abolished the effect of REGγ depletion on RCC cells growth. Importantly, we also observed that REGγ depletion activated Hippo signaling pathway via stabilizing CK1ε in RCC, indicating the cross-talk between REGγ/CK1ε axis and Hippo pathway during RCC development. In conclusion, our findings suggested that REGγ played a pivotal role in the development of RCC and maybe helpful to identify new therapeutic strategies in the treatment of RCC.


Assuntos
Carcinoma de Células Renais/patologia , Caseína Quinase Iépsilon/metabolismo , Progressão da Doença , Neoplasias Renais/patologia , Complexo de Endopeptidases do Proteassoma/deficiência , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Carcinogênese/patologia , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Estabilidade Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Análise Multivariada , Invasividade Neoplásica , Prognóstico , Modelos de Riscos Proporcionais , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima/genética
8.
Proc Natl Acad Sci U S A ; 115(23): 5986-5991, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29784789

RESUMO

Multisite phosphorylation of the PERIOD 2 (PER2) protein is the key step that determines the period of the mammalian circadian clock. Previous studies concluded that an unidentified kinase is required to prime PER2 for subsequent phosphorylation by casein kinase 1 (CK1), an essential clock component that is conserved from algae to humans. These subsequent phosphorylations stabilize PER2, delay its degradation, and lengthen the period of the circadian clock. Here, we perform a comprehensive biochemical and biophysical analysis of mouse PER2 (mPER2) priming phosphorylation and demonstrate, surprisingly, that CK1δ/ε is indeed the priming kinase. We find that both CK1ε and a recently characterized CK1δ2 splice variant more efficiently prime mPER2 for downstream phosphorylation in cells than the well-studied splice variant CK1δ1. While CK1 phosphorylation of PER2 was previously shown to be robust to changes in the cellular environment, our phosphoswitch mathematical model of circadian rhythms shows that the CK1 carboxyl-terminal tail can allow the period of the clock to be sensitive to cellular signaling. These studies implicate the extreme carboxyl terminus of CK1 as a key regulator of circadian timing.


Assuntos
Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Ritmo Circadiano/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Proteínas Circadianas Period/genética , Fosforilação
9.
J Med Chem ; 61(9): 4087-4102, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29630366

RESUMO

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.


Assuntos
Trifosfato de Adenosina/metabolismo , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Desenho de Drogas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Wnt/biossíntese , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Ligação Competitiva , Caseína Quinase Idelta/química , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/química , Caseína Quinase Iépsilon/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo
10.
Elife ; 72018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29611807

RESUMO

In the Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. Here we show that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Relógios Circadianos , Proteínas de Drosophila/metabolismo , Proteínas Circadianas Period/metabolismo , Ativação Transcricional , Animais , Drosophila , Retroalimentação Fisiológica , Regulação da Expressão Gênica
11.
Am J Physiol Renal Physiol ; 315(1): F57-F73, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29537311

RESUMO

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.


Assuntos
Caseína Quinase Ialfa/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Rim/efeitos dos fármacos , Doenças Renais Policísticas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Roscovitina/farmacologia , Animais , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Catálise , Cromatografia de Afinidade/métodos , Modelos Animais de Doenças , Humanos , Rim/enzimologia , Rim/patologia , Camundongos Transgênicos , Doenças Renais Policísticas/enzimologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Blood ; 131(11): 1206-1218, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29317454

RESUMO

Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.


Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
13.
Bioorg Med Chem ; 26(3): 590-602, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29289448

RESUMO

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.


Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Sítios de Ligação , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Permeabilidade/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Ratos , Relação Estrutura-Atividade , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
14.
J Biol Chem ; 293(1): 163-176, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29109149

RESUMO

Oxidative and endoplasmic reticulum (ER) stresses are hallmarks of the pathophysiology of ALS and other neurodegenerative diseases. In these stresses, different kinases phosphorylate eukaryotic initiation factor eIF2α, enabling the translation of stress response genes; among these is GADD34, the protein product of which recruits the α-isoform of protein phosphatase 1 catalytic subunit (PP1α) and eIF2α to assemble a phosphatase complex catalyzing eIF2α dephosphorylation and resumption of protein synthesis. Aberrations in this pathway underlie the aforementioned disorders. Previous observations indicating that GADD34 is induced by arsenite, a thiol-directed oxidative stressor, in the absence of eIF2α phosphorylation suggest other roles for GADD34. Here, we report that arsenite-induced oxidative stress differs from thapsigargin- or tunicamycin-induced ER stress in promoting GADD34 transcription and the preferential translation of its mRNA in the absence of eIF2α phosphorylation. Arsenite also stabilized GADD34 protein, slowing its degradation. In response to oxidative stress, but not ER stress, GADD34 recruited TDP-43, and enhanced cytoplasmic distribution and cysteine modifications of TDP-43 promoted its binding to GADD34. Arsenite also recruited a TDP-43 kinase, casein kinase-1ϵ (CK1ϵ), to GADD34. Concomitant with TDP-43 aggregation and proteolysis after prolonged arsenite exposure, GADD34-bound CK1ϵ catalyzed TDP-43 phosphorylations at serines 409/410, which were diminished or absent in GADD34-/- cells. Our findings highlight that the phosphatase regulator, GADD34, also functions as a kinase scaffold in response to chronic oxidative stress and recruits CK1ϵ and oxidized TDP-43 to facilitate its phosphorylation, as seen in TDP-43 proteinopathies.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse Oxidativo/fisiologia , Proteína Fosfatase 1/metabolismo , Proteinopatias TDP-43/metabolismo , Animais , Arsenitos/farmacologia , Caseína Quinase Iépsilon/metabolismo , Proteínas de Ciclo Celular/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Proteína Fosfatase 1/deficiência
15.
Sci Rep ; 7(1): 13478, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29044200

RESUMO

Hyperphosphorylation of tau and imbalanced expression of 3R-tau and 4R-tau as a result of dysregulation of tau exon 10 splicing are believed to be pivotal to the pathogenesis of tau pathology, but the molecular mechanism leading to the pathologic tau formation in Alzheimer's disease (AD) brain is not fully understood. In the present study, we found that casein kinase 1ε (CK1ε) was increased significantly in AD brains. Overexpression of CK1ε in cultured cells led to increased tau phosphorylation at many sites. Moreover, we found that CK1ε suppressed tau exon 10 inclusion. Levels of CK1ε were positively correlated to tau phosphorylation, 3R-tau expression and tau pathology, and negatively correlated to 4R-tau in AD brains. Overexpression of CK1ε in the mouse hippocampus increased tau phosphorylation and impaired spontaneous alternation behavior. These data suggest that CK1ε is involved in the regulation of tau phosphorylation, the alternative splicing of tau exon 10, and cognitive performance. Up-regulation of CK1ε might contribute to tau pathology by hyperphosphorylating tau and by dysregulating the alternative splicing of tau exon 10 in AD.


Assuntos
Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Caseína Quinase Iépsilon/genética , Regulação da Expressão Gênica , Proteínas tau/metabolismo , Processamento Alternativo , Doença de Alzheimer/patologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Caseína Quinase Iépsilon/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Éxons , Feminino , Imunofluorescência , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Agregados Proteicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Proteínas tau/genética
16.
EMBO J ; 36(20): 3046-3061, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-28963394

RESUMO

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co-ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt-villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε-deficient enterocyte populations, with the exception of Lgr5+ ISCs, which exhibit Dvl2-dependent Wnt signaling attenuation. CKIδ/ε-depleted gut organoids cease proliferating and die rapidly, yet survive and resume self-renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine.


Assuntos
Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Mucosa Intestinal/fisiologia , Células-Tronco/fisiologia , Via de Sinalização Wnt , Animais , Proliferação de Células , Células Epiteliais/fisiologia , Camundongos
17.
Cell Res ; 27(12): 1466-1484, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29056748

RESUMO

Planar cell polarity (PCP) is an evolutionarily conserved essential mechanism that provides directional information to control and coordinate polarized cellular and tissue behavior during embryonic development. Disruption of PCP leads to severe morphological defects in vertebrates and its dysregulation results in a variety of human diseases such as neural tube defects and skeletal dysplasia. PCP is governed by a set of highly conserved core proteins that are asymmetrically localized at the cell surface throughout the polarized tissues. The uniform directionality of PCP is established by global cues, such as Wg/Wnt signaling gradients that break the original symmetrical localization of core PCP proteins including Vang/Vangl and Fz/Fzd. However, the exact mechanism remains elusive. In this study, we found that Vangl2 phosphorylation, which was previously identified to be induced by Wnt5a signaling, is required for Vangl2 functions in mammalian PCP in multiple tissues. The in vivo activities of Vangl2 are determined by its phosphorylation level. Phospho-mutant Vangl2 exhibits dominant negative effects, whereas Vangl2 with reduced phosphorylation is hypomorphic. We show that Vangl2 phosphorylation is essential for its uniform polarization pattern. Moreover, serine/threonine kinases CK1ɛ and CK1δ are redundantly required for Wnt5a-induced Vangl2 phosphorylation. Dvl family members are also required for Wnt5a-induced Vangl2 phosphorylation by enhancing the interaction of CK1 and Vangl2. These findings demonstrate that induction of Vangl protein phosphorylation plays an essential role in transducing Wnt5a signaling to establish PCP in mammalian development, suggesting a phosphorylation-regulated "Vangl activity gradient" model in addition to the well-documented "Fz activity gradient" model in Wnt/PCP signaling.


Assuntos
Polaridade Celular , Proteínas do Tecido Nervoso/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Fosforilação , Via de Sinalização Wnt
18.
J Alzheimers Dis ; 59(2): 615-631, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28671110

RESUMO

A disruption to circadian rhythmicity and the sleep/wake cycle constitutes a major feature of Alzheimer's disease (AD). The maintenance of circadian rhythmicity is regulated by endogenous clock genes and a number of external Zeitgebers, including light. This study investigated the light induced changes in the expression of clock genes in a triple transgenic model of AD (3×Tg-AD) and their wild type littermates (Non-Tg). Changes in gene expression were evaluated in four brain areas¾suprachiasmatic nucleus (SCN), hippocampus, frontal cortex and brainstem¾of 6- and 18-month-old Non-Tg and 3×Tg-AD mice after 12 h exposure to light or darkness. Light exposure exerted significant effects on clock gene expression in the SCN, the site of the major circadian pacemaker. These patterns of expression were disrupted in 3×Tg-AD and in 18-month-old compared with 6-month-old Non-Tg mice. In other brain areas, age rather than genotype affected gene expression; the effect of genotype was observed on hippocampal Sirt1 expression, while it modified the expression of genes regulating the negative feedback loop as well as Rorα, Csnk1ɛ and Sirt1 in the brainstem. In conclusion, during the early development of AD, there is a disruption to the normal expression of genes regulating circadian function after exposure to light, particularly in the SCN but also in extra-hypothalamic brain areas supporting circadian regulation, suggesting a severe impairment of functioning of the clock gene pathway. Even though this study did not demonstrate a direct association between these alterations in clock gene expression among brain areas with the cognitive impairments and chrono-disruption that characterize the early onset of AD, our novel results encourage further investigation aimed at testing this hypothesis.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Ritmo Circadiano/genética , Regulação da Expressão Gênica/genética , RNA/metabolismo , Fatores Etários , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Análise de Variância , Animais , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Modelos Animais de Doenças , Humanos , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteínas tau/metabolismo
19.
Genes Brain Behav ; 16(7): 725-738, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28594147

RESUMO

Genetic and pharmacological studies indicate that casein kinase 1 epsilon (Csnk1e) contributes to psychostimulant, opioid, and ethanol motivated behaviors. We previously used pharmacological inhibition to demonstrate that Csnk1e negatively regulates the locomotor stimulant properties of opioids and psychostimulants. Here, we tested the hypothesis that Csnk1e negatively regulates opioid and psychostimulant reward using genetic inhibition and the conditioned place preference assay in Csnk1e knockout mice. Similar to pharmacological inhibition, Csnk1e knockout mice showed enhanced opioid-induced locomotor activity with the mu opioid receptor agonist fentanyl (0.2 mg/kg i.p.) as well as enhanced sensitivity to low-dose fentanyl reward (0.05 mg/kg). Interestingly, female knockout mice also showed a markedly greater escalation in consumption of sweetened palatable food - a behavioral pattern consistent with binge eating that also depends on mu opioid receptor activation. No difference was observed in fentanyl analgesia in the 52.5°C hot plate assay (0-0.4 mg/kg), naloxone conditioned place aversion (4 mg/kg), or methamphetamine conditioned place preference (0-4 mg/kg). To identify molecular adaptations associated with increased drug and food behaviors in knockout mice, we completed transcriptome analysis via mRNA sequencing of the striatum. Enrichment analysis identified terms associated with myelination and axon guidance and pathway analysis identified a differentially expressed gene set predicted to be regulated by the Wnt signaling transcription factor, Tcf7l2. To summarize, Csnk1e deletion increased mu opioid receptor-dependent behaviors, supporting previous studies indicating an endogenous negative regulatory role of Csnk1e in opioid behavior.


Assuntos
Bulimia/genética , Caseína Quinase Iépsilon/genética , Transtornos Relacionados ao Uso de Opioides/genética , Receptores Opioides mu/metabolismo , Animais , Caseína Quinase Iépsilon/metabolismo , Condicionamento Clássico , Corpo Estriado/metabolismo , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Recompensa , Transcriptoma
20.
Bioorg Med Chem Lett ; 27(12): 2663-2667, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28487075

RESUMO

Described herein is the design, synthesis and biological evaluation of a series of N-(1H-pyrazol-3-yl)quinazolin-4-amines against a panel of eight disease relevant protein kinases. The kinase inhibition results indicated that two compounds inhibited casein kinase 1δ/ε (CK1δ/ε) with some selectivity over related kinases, namely CDK5/p25, GSK-3α/ß, and DYRK1A. Docking studies with 3c and 3d revealed the key interactions with desired amino acids in the ATP binding site of CK1δ. Furthermore, compound 3c also elicited selective cytotoxic activity against the pancreas ductal adenocarcinoma (PANC-1) cell line. Taken together, the results of this study establish N-(1H-pyrazol-3-yl)quinazolin-4-amines especially 3c and 3d as valuable lead molecules with great potential for CK1δ/ε inhibitor development targeting neurodegenerative disorders and cancer.


Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Quinazolinas/farmacologia , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade
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