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1.
Oncogene ; 37(3): 363-376, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28945225

RESUMO

Evasion of forkhead box O (FOXO) family of longevity-related transcription factors-mediated growth suppression is necessary to promote cancer development. Since somatic alterations or mutations and transcriptional dysregulation of the FOXO genes are infrequent in human cancers, it remains unclear how these tumour suppressors are eliminated from cancer cells. The protein stability of FOXO3A is regulated by Casein Kinase 1 alpha (CK1α) in an oncogenic RAS-specific manner, but whether this mode of regulation extends to related FOXO family members is unknown. Here we report that CK1α similarly destabilizes FOXO4 in RAS-mutant cells by phosphorylation at serines 265/268. The CK1α-dependent phosphoregulation of FOXO4 is primed, in part, by the PI3K/AKT effector axis of oncogenic RAS signalling. In addition, mutant RAS coordinately elevates proteasome subunit expression and proteolytic activity to eradicate nuclear FOXO4 proteins from RAS-mutant cancer cells. Importantly, dual inhibition of CK1α and the proteasome synergistically inhibited the growth of multiple RAS-mutant human cancer cell lines of diverse tissue origin by blockade of nuclear FOXO4 degradation and induction of caspase-dependent apoptosis. Our findings challenge the current paradigm that nuclear export regulates the proteolysis of FOXO3A/4 tumour suppressors in the context of cancer and illustrates how oncogenic RAS-mediated degradation of FOXOs, via post-translational mechanisms, blocks these important tumour suppressors.


Assuntos
Caseína Quinase Ialfa/genética , Núcleo Celular/patologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Caseína Quinase Ialfa/antagonistas & inibidores , Caseína Quinase Ialfa/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína Forkhead Box O3/metabolismo , Humanos , Imidazóis/farmacologia , Mutação , Neoplasias/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética
2.
Oncogene ; 35(33): 4302-11, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-26751776

RESUMO

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.


Assuntos
Transição Epitelial-Mesenquimal , Metástase Neoplásica , Animais , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia
3.
Oncogene ; 35(17): 2197-207, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-26257057

RESUMO

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Proteínas de Membrana/genética , Proteínas Wnt/genética , Aciltransferases , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Processamento de Proteína Pós-Traducional , Células-Tronco/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Oncogene ; 34(4): 474-84, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24469046

RESUMO

Sprouty2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase signaling, and is implicated as a tumor suppressor. SPRY2 inhibits FGF-RAS-ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2-SOS (son of sevenless) complex that transduces signals from FGFR to RAS. SPRY2 binding to GRB2 is modulated by phosphorylation but the key regulatory kinase(s) are not known. Prior studies identified the frequent presence of CK1 phosphorylation motifs on SPRY2. We therefore tested if CK1 has a role in SPRY2 phosphorylation and function. Loss of CK1 binding and inhibition of CK1 activity by two structurally distinct small molecules abrogated SPRY2 inhibition of FGF-ERK signaling, leading to decreased SPRY2 interaction with GRB2. Moreover, CK1 activity and binding are necessary for SPRY2 inhibition of FGF-stimulated neurite outgrowth in PC12 cells. Consistent with its proposed role as an inhibitor of FGF signaling, we find that CSNK1E transcript abundance negatively correlates with FGF1/FGF7 message in human gastric cancer samples. Modulation of CK1 activity may be therapeutically useful in the treatment of FGF/SPRY2-related diseases.


Assuntos
Caseína Quinase I/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Proteína Adaptadora GRB2/fisiologia , Humanos , Neuritos/fisiologia , Fosforilação , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Neoplasias Gástricas/metabolismo
5.
Oncogene ; 31(2): 213-25, 2012 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-21666721

RESUMO

Using a screen for Wnt/ß-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific ß-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on ß-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse ß-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.


Assuntos
Proliferação de Células , Compostos Ferrosos/metabolismo , Engenharia Genética , Quelantes de Ferro/farmacologia , Neoplasias/tratamento farmacológico , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Modelos Animais de Doenças , Quelantes de Ferro/uso terapêutico , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia
6.
Oncogene ; 30(22): 2558-69, 2011 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-21258417

RESUMO

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/ß-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/ß-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/ß-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.


Assuntos
Apoptose/efeitos dos fármacos , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Iépsilon/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Indóis/farmacologia , Floroglucinol/análogos & derivados , Fuso Acromático/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/metabolismo , Linhagem Celular Tumoral , Proteínas Desgrenhadas , Humanos , Neoplasias/metabolismo , Floroglucinol/farmacologia , Fosfoproteínas/metabolismo , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-18419299

RESUMO

Protein phosphorylation regulates the period of the circadian clock within mammalian cells. Circadian rhythms are an approximately 24-hour cycle that regulates key biological processes. Daily fluctuations of wakefulness, stress hormones, lipid metabolism, immune function, and the cell division cycle are controlled by the molecular clocks that function throughout our bodies. Mutations in regulatory components of the clock can shorten or lengthen the timing of the rhythms and have significant physiological consequences. The clock is formed by a negative feedback loop of transcription, translation, and inhibition of transcription. The precision of clock timing is controlled by protein kinases and phosphatases. Casein kinase Iepsilon is a protein kinase that regulates the circadian clock by periodic phosphorylation of the proteins PER1 and PER2, controlling their stability and localization. The role of phosphorylation in regulating PER function in the clock has been explored in detail. Quantitative modeling has proven to be very useful in making important predictions about how changes in phosphorylation alter the clock's behavior. Quantitative data from biological studies can be used to refine the quantitative model and make additional testable predictions. A detailed understanding of how reversible protein phosphorylation regulates circadian rhythms and a detailed quantitative model that makes clear, testable, and accurate predictions about the clock and how we may manipulate it can have important benefits for human health. Pharmacological manipulation of rhythms could mitigate stress from jet lag, shift work, and perhaps even seasonal affective disorder.


Assuntos
Ritmo Circadiano/fisiologia , Proteínas/metabolismo , Animais , Caseína Quinase Iépsilon/antagonistas & inibidores , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ritmo Circadiano/genética , Retroalimentação Fisiológica , Mamíferos , Camundongos , Modelos Biológicos , Mutação , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo
8.
J Biol Chem ; 276(49): 45921-7, 2001 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-11591712

RESUMO

The timing of mammalian circadian rhythm is determined by interlocking negative and positive transcriptional feedback loops that govern the cyclic expression of both clock regulators and output genes. In mammals, nuclear localization of the circadian regulators PER1-3 is controlled by multiple mechanisms, including multimerization with PER and CRY proteins. In addition, nuclear entry of mammalian PER1 (mPER1) can be regulated by a phosphorylation-dependent masking of its nuclear localization signal. Here we present evidence suggesting that nuclear localization of PER proteins is a dynamic process determined by both nuclear import and previously unrecognized nuclear export pathways. Examination of the subcellular localization of a series of truncated mPER1 proteins demonstrated that cytoplasmic localization is mediated by an 11-amino acid region with homology to leucine-rich nuclear export signals (NESs). Similar sequences were identified in mPER2 and mPER3 as well as in several insect PER proteins. The putative NESs from mPER1 and mPER2 were able to direct cytoplasmic accumulation when fused to a heterologous protein. Mutations in conserved NES residues and the nuclear export inhibitor leptomycin B each blocked the function of the NES. Full-length mPER1 was also exported from microinjected Xenopus laevis oocyte nuclei in an NES-dependent manner. The presence of a functional NES in mPER1 and mPER2 as well as related sequences in a variety of other PER proteins suggests that nuclear export may be a conserved and important feature of circadian regulators.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Citoplasma/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Oócitos/metabolismo , Proteínas Circadianas Period , Transporte Proteico , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Proteínas de Xenopus , Xenopus laevis
9.
EMBO J ; 20(15): 4122-31, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11483515

RESUMO

Wnt signaling increases beta-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, beta-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3beta and axin but upstream of beta-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the beta-catenin degradation complex. PP2A appears to be essential for beta-catenin degradation, since beta-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transdução de Sinais , Transativadores , Proteínas de Peixe-Zebra , Animais , Antígenos Virais de Tumores/metabolismo , Proteína Axina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Domínio Catalítico , Extratos Celulares , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/farmacologia , Epistasia Genética , Quinase 3 da Glicogênio Sintase , Humanos , Microcistinas , Ácido Okadáico/farmacologia , Óvulo , Peptídeos Cíclicos/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Testes de Precipitina , Proteína Fosfatase 2 , Proteínas/metabolismo , Proteínas/farmacologia , Proteínas Proto-Oncogênicas/genética , Ratos , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis/embriologia , beta Catenina
10.
IUBMB Life ; 51(2): 73-8, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11463166

RESUMO

The casein kinase I (CKI) family of protein kinases is a group of highly related, ubiquitously expressed serine/threonine kinases found in all eukaryotic organisms from protozoa to man. Recent advances in diverse fields, including developmental biology and chronobiology, have elucidated roles for CKI in regulating critical processes such as Wnt signaling, circadian rhythm, nuclear import, and Alzheimer's disease progression.


Assuntos
Proteínas Quinases/fisiologia , Proteínas de Peixe-Zebra , Doença de Alzheimer/patologia , Animais , Caseína Quinases , Ritmo Circadiano/fisiologia , Progressão da Doença , Humanos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt
11.
Chronobiol Int ; 18(3): 389-98, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11475410

RESUMO

Multiple components of the circadian central clock are phosphoproteins, and it has become increasingly clear that posttranslational modification is an important regulator of circadian rhythm in diverse organisms, from dinoflagellates to humans. Genetic studies in Drosophila have identified double-time (dbt), a serine/threonine protein kinase that is highly homologous to human casein kinase I epsilon (CKIepsilon), as the first kinase linked to behavioral rhythms. Identification of a missense mutation in CKIepsilon as the tau mutation in the Syrian hamster places CKIepsilon within the core clock machinery in mammals. Most recently, identification of a phosphorylation site mutant of hPER2 in a family with an inherited circadian rhythm abnormality strongly suggests that PER2 is a physiologically relevant substrate of CKI. Phosphorylation may regulate multiple properties of clock proteins, including stability and intracellular localization.


Assuntos
Caseína Quinase Iépsilon , Ritmo Circadiano , Proteínas de Drosophila , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caseína Quinases , Cricetinae , Drosophila , Mesocricetus , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Fosforilação , Proteínas Quinases/genética , Homologia de Sequência de Aminoácidos
12.
Science ; 291(5506): 1040-3, 2001 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-11232563

RESUMO

Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.


Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas/metabolismo , Transtornos do Sono do Ritmo Circadiano/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Caseína Quinases , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Éxons , Feminino , Ligação Genética , Glicina , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas Nucleares/química , Linhagem , Proteínas Circadianas Period , Fosforilação , Polimorfismo Conformacional de Fita Simples , Proteínas Quinases/metabolismo , Proteínas/química , Serina , Transtornos do Sono do Ritmo Circadiano/fisiopatologia , Fatores de Transcrição
13.
FEBS Lett ; 489(2-3): 159-65, 2001 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-11165242

RESUMO

Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Animais , Caseína Quinases , Proteínas de Ciclo Celular , Linhagem Celular , DNA Recombinante , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas Nucleares/genética , Proteínas Circadianas Period , Fosforilação , Ligação Proteica , Proteínas Quinases/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Núcleo Supraquiasmático/enzimologia , Fatores de Transcrição , Transfecção
14.
Mol Cell Biol ; 20(13): 4888-99, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10848614

RESUMO

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I epsilon (CKIepsilon) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Caseína Quinases , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Ritmo Circadiano/fisiologia , Citoplasma/metabolismo , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Circadianas Period , Fosforilação , Proteínas Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Curr Opin Cell Biol ; 12(2): 180-5, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10712915

RESUMO

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Transativadores , Proteínas de Peixe-Zebra , Animais , Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Replicação do DNA , Quinase 3 da Glicogênio Sintase , Humanos , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2 , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Wnt , beta Catenina
16.
Biochem Biophys Res Commun ; 268(2): 562-6, 2000 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-10679243

RESUMO

Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates.


Assuntos
Proteínas Quinases/metabolismo , Reticulócitos/enzimologia , Caseína Quinases , Domínio Catalítico , Clonagem Molecular , Biblioteca Gênica , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Reticulócitos/metabolismo , Especificidade por Substrato
17.
J Biol Chem ; 274(45): 32063-70, 1999 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-10542239

RESUMO

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.


Assuntos
Proteínas Quinases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Caseína Quinases , Linhagem Celular , Ritmo Circadiano , Sequência Consenso , Reparo do DNA , Replicação do DNA , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Fosforilação , Proteínas Quinases/genética , Homologia de Sequência de Aminoácidos
19.
Science ; 283(5410): 2089-91, 1999 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-10092233

RESUMO

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores , Proteínas de Peixe-Zebra , Proteína da Polipose Adenomatosa do Colo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Genes Reporter , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Humanos , Leupeptinas/farmacologia , Complexos Multienzimáticos/metabolismo , Mutação , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma , Proteína Fosfatase 2 , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteínas Wnt , Xenopus , Proteínas de Xenopus , beta Catenina
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