RESUMO
Glucotoxicity, impaired insulin secretion, suppression of insulin gene expression, and apoptosis, in pancreatic ß-cells caused by chronic hyperglycemia is a key component of the pathogenesis of type 2 diabetes. Recently, it has been reported that rare sugar d-allulose has antihyperglycemic and antihyperlipidemic effects in diabetic rats. However, the direct effects of rare sugars including d-allulose on pancreatic ß-cell function are unclear. In this study, we investigated whether chronic exposure to ketohexoses causes glucotoxicity, suppression of insulin gene expression, and apoptosis, in INS-1 rat pancreatic insulinoma cells. d-Fructose, d-tagatose, l-allulose, and l-sorbose treatment for 1-week reduced insulin gene expression, whereas d-allulose, d-sorbose, l-fructose, and l-tagatose did not. All ketohexoses were transported into INS-1 cells, but were not metabolized. In addition, the ketohexoses did not induce apoptosis and did not affect glucose metabolism. These results suggest that long-term administration of d-allulose, d-sorbose, l-fructose, and l-tagatose does not affect pancreatic ß-cell function.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Insulinoma , Neoplasias Pancreáticas , Ratos , Animais , Sorbose , Frutose , Insulina/metabolismo , Açúcares , Glucose/metabolismoRESUMO
HHUA endometrial adenocarcinoma cells aggregated into spheroids when cultured on collagen type I gels. 12-O-Tetradecanoylphorbol 13-acetate, a PKC activator, disassembled the spheroids through epithelial-mesenchymal transition and increased their proliferation rate, while inducing cell death under monolayer culture conditions. These unusual behaviors of endometrial epithelial cells with collagen fibers could be a target for the treatment of some endometrial diseases.
Assuntos
Transição Epitelial-Mesenquimal , Doenças Uterinas , Acetatos/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Feminino , Géis/metabolismo , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.
Assuntos
Relógios Biológicos/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ritmo Circadiano/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Animal , Relógios Biológicos/efeitos dos fármacos , Proteínas CLOCK/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Ritmo Circadiano/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células NIH 3T3 , Neurônios/enzimologia , Fosforilação , Ratos , Transdução de SinaisRESUMO
Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease-modifying effects of anti-diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti-diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription-polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.
Assuntos
Encéfalo/metabolismo , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Pessoa de Meia-IdadeRESUMO
Chronic hyperglycemia causes pancreatic ß-cell dysfunction, impaired insulin secretion and suppression of insulin gene expression, referred to as glucotoxicity. Insulin gene expression is regulated by several protein kinases and protein phosphatases. However, the molecular mechanisms of the suppressed insulin gene expression in glucotoxicity are not fully understood. In this study, we employed rat insulinoma INS-1â¯cells as a model of pancreatic glucotoxicity. In INS-1â¯cells, insulin gene expression is up-regulated by incubation with 11.2â¯mM glucose for 7 days and down-regulated by incubation with 22.4â¯mM glucose for the same period. To identify the protein kinases and protein phosphatases involved in the suppression of insulin gene expression, we analyzed gene expression in INS-1â¯cells cultured with 11.2â¯mM or 22.4â¯mM glucose for 7 days using microarray analysis and real-time PCR. The expression levels of nine protein kinases were affected by glucotoxic conditions. In particular, CPG16 expression level was increased in INS-1â¯cells under these conditions. Transfection of CPG16 decreased insulin promoter activity, whereas kinase-dead mutant of CPG16 did not affect this. These results suggest that CPG16 plays a role in the suppression of insulin gene expression in pancreatic ß-cells under glucotoxic conditions.
Assuntos
Regulação para Baixo , Hiperglicemia/genética , Insulina/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Linhagem Celular Tumoral , Quinases Semelhantes a Duplacortina , Hiperglicemia/complicações , Células Secretoras de Insulina/metabolismo , Insulinoma/complicações , Insulinoma/genética , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/genética , Ratos , Regulação para CimaRESUMO
Alterations in microRNAs (miRNAs) levels deeply correlate with tumorigenesis. However, the molecular mechanism for the regulation of the miRNA production in tumors is not fully understood. We previously reported that downregulation of miR-7, which is an anti-oncogenic miRNA, was caused by overexpression of the nuclear factor 90 (NF90)-nuclear factor 45 (NF45) complex through the binding of double-stranded (ds) RNA-binding proteins to primary miR-7, resulting in promotion of tumorigenesis (Higuchi et al 2016). During this study, we found that the level of NF90 protein was dramatically decreased by overexpression of miR-7. Interestingly, the miR-7-mediated reduction in NF90 family proteins was only observed in NF90 protein, but not in NF110 protein, which is a longer form of the NF90 gene. Luciferase reporter analysis indicated that the overexpression of miR-7 significantly repressed the luciferase activity in the coding region of NF90 mRNA harboring a predicted target sequence of miR-7. The luciferase activity of the reporter vector, which has a mutated miR-7 target site in the coding region, was the same in the control and miR-7 overexpressed cells. Furthermore, the translation of TARGET-tagged NF90 mRNA without the 3'UTR of the NF90 mRNA was inhibited by the overexpression of miR-7. These results imply that miR-7 suppresses NF90 at the protein level through the binding of miR-7 to the complementary site of the seed sequence in the coding region of the NF90 mRNA. We further confirmed increased endogenous NF90 protein levels in SK-N-SH cells transfected with antisense oligonucleotides targeting miR-7, indicating that miR-7-mediated translational repression of NF90 is a physiological event. Taken together with our previous findings (Higuchi et al 2016), it suggests that the level of NF90 is increased by a negative feedback loop between NF90 and miR-7 in tumor tissues under physiological conditions.
Assuntos
Retroalimentação Fisiológica , MicroRNAs/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Proteínas do Fator Nuclear 90/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.
Assuntos
Bacteriófago lambda/enzimologia , Caseína Quinase I , Escherichia coli , Expressão Gênica , Fosfoproteínas Fosfatases/biossíntese , Proteínas Virais/biossíntese , Bacteriófago lambda/genética , Caseína Quinase I/biossíntese , Caseína Quinase I/química , Caseína Quinase I/genética , Caseína Quinase I/isolamento & purificação , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Fosfoproteínas Fosfatases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/genéticaRESUMO
MicroRNA-7 (miR-7)has been characterized as an anti-oncogenic microRNA (miRNA) in several cancers, including hepatocellular carcinoma (HCC). However, the mechanism for the regulation of miR-7 production in tumors remains unclear. Here, we identified nuclear factor 90 (NF90) and NF45 complex (NF90-NF45) as negative regulators of miR-7 processing in HCC. Expression of NF90 and NF45 was significantly elevated in primary HCC tissues compared with adjacent non-tumor tissues. To examine which miRNAs are controlled by NF90-NF45, we performed an miRNA microarray and quantitative RT-PCR analyses of HCC cell lines. Depletion of NF90 resulted in elevated levels of mature miR-7, whereas the expression of primary miR-7-1 (pri-miR-7-1) was decreased in cells following knockdown of NF90. Conversely, the levels of mature miR-7 were reduced in cells overexpressing NF90 and NF45, although pri-miR-7-1 was accumulated in the same cells. Furthermore, NF90-NF45 was found to bind pri-miR-7-1 in vitro These results suggest that NF90-NF45 inhibits the pri-miR-7-1 processing step through the binding of NF90-NF45 to pri-miR-7-1. We also found that levels of the EGF receptor, an oncogenic factor that is a direct target of miR-7, and phosphorylation of AKT were significantly decreased in HCC cell lines depleted of NF90 or NF45. Of note, knockdown of NF90 or NF45 caused a reduction in the proliferation rate of HCC cells. Taken together, NF90-NF45 stimulates an elevation of EGF receptor levels via the suppression of miR-7 biogenesis, resulting in the promotion of cell proliferation in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína do Fator Nuclear 45/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , RNA Neoplásico/metabolismo , Adulto , Carcinoma Hepatocelular/genética , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Proteína do Fator Nuclear 45/genética , Proteínas do Fator Nuclear 90/genética , RNA Neoplásico/genéticaRESUMO
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization.
Assuntos
Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Frações Subcelulares/enzimologia , Animais , Linhagem Celular , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/fisiologia , Camundongos , Neurônios/ultraestrutura , Fosforilação , Quinases DyrkRESUMO
To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.
Assuntos
Fosfoproteínas Fosfatases/química , Proteínas Quinases/química , Animais , Humanos , Camundongos , Fosforilação , Especificidade por SubstratoRESUMO
We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.
Assuntos
Biotina/química , Fosfoproteínas/análise , Proteômica/métodos , Piridinas/química , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Western Blotting , Bovinos , Linhagem Celular , Peroxidase do Rábano Silvestre/química , Humanos , Indicadores e Reagentes/química , Medições Luminescentes , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfoproteínas/química , Fosforilação , Análise Serial de Proteínas , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32. METHODS: CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined. RESULTS: CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase. CONCLUSIONS: CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses. GENERAL SIGNIFICANCE: In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.
Assuntos
Coprinus/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Protein kinases regulate almost all biological processes including cell proliferation, differentiation, apoptosis, and gene expression. Dysregulation of protein phosphorylation caused by abnormal activity and expression of protein kinases results in the onset of various diseases such as cancer and metabolic syndromes. The activities of a large number of protein kinases are regulated by phosphorylation. Therefore, analysis of the phosphorylation status of protein kinases is important for elucidation of biological phenomena and the pathogenesis of diseases. To investigate protein phosphorylation, phosphate-binding tag molecule "Phos-tag" was developed. In addition, various techniques and tools using Phos-tag such as Phos-tag SDS-PAGE, have been developed for analysis and profiling of protein phosphorylation. Here, we describe the methods and analytical techniques that use Phos-tag for investigation of protein kinase phosphorylation and the applications of phosphorylation analysis. SIGNIFICANCE: Protein kinases play pivotal roles in regulating many biological processes and pathogenesis of diseases. Determination of phosphorylation status of protein kinases can provide the essential information for their activation. This review provides analytical techniques for analysis of phosphorylation status of protein kinases by Phos-tag SDS-PAGE. We believe that this review would help readers to study in kinomics research.
Assuntos
Fosfoproteínas , Proteínas Quinases , Eletroforese em Gel de Poliacrilamida , Fosfoproteínas/análise , Fosforilação , Proteínas Quinases/metabolismo , PiridinasRESUMO
The Nuclear Factor 90 (NF90)-NF45 complex has been known to regulate the progression of transcription, mRNA stability, translational inhibition, RNA export and microRNA biogenesis. However, the physiological functions of the NF90-NF45 complex remain unclear. We newly discovered that the NF90-NF45 complex was expressed in primary ß cells and established cell lines. Therefore, in this study, we focused on the function of the endogenous NF90-NF45 complex in the ß cells. To investigate this issue, we generated ß-cell-specific NF90-NF45 deficient mice. These mice exhibited hyperglycaemia and lower plasma insulin levels under a high fat diet together with decreased islet mass. To uncover this mechanism, we performed a whole-genome expression microarray of the total RNA prepared from ß cell lines treated with siRNAs targeting both NF90 and NF45. In this result, we found an activation of p53 signaling in the NF90-NF45-knockdown cells. This activation was supported by elevation of luciferase activity derived from a reporter plasmid harboring p53 binding sites in the NF90-NF45-knockdown cells. Furthermore, the knockdown of NF90-NF45 resulted in a significant retardation of the ß cell line growth rates. Importantly, a dominant negative form of p53 rescues the growth retardation in BTC6 cells depleted of NF90-NF45, suggesting that NF90-NF45 would be positively involved in ß cell proliferation through suppression of p53 signal pathway. Taken together, NF90-NF45 is essential for ß cell compensation under obesity-inducing metabolic stress via repression of p53 signaling.
Assuntos
Proteína do Fator Nuclear 45 , Proteínas do Fator Nuclear 90 , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína do Fator Nuclear 45/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Obesidade/genética , RNA , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismoRESUMO
Dnmt1 (DNA methyltansferase 1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1-290) was prepared using Dnmt1(1-290)-affinity, DNA-cellulose and gel-filtration columns. When the proteins in this fraction were analysed by LC-MS/MS (liquid chromatography tandem MS), CK1delta/epsilon (casein kinase 1delta/epsilon) was the only protein kinase identified. Recombinant CK1delta/epsilon was found to bind to the N-terminal domain of Dnmt1 and significantly phosphorylated this domain, especially in the presence of DNA. Phosphorylation analyses using various truncation and point mutants of Dnmt1 revealed that the major priming site phosphorylated by CK1delta/epsilon was Ser146, and that subsequent phosphorylation at other sites may occur after phosphorylation of the priming site. When the DNA-binding activity of phosphorylated Dnmt1 was compared with that of the non-phosphorylated form, phosphorylation of Dnmt1 was found to decrease the affinity for DNA. These results suggest that CK1delta/epsilon binds to and phosphorylates the N-terminal domain of Dnmt1 and regulates Dnmt1 function by reducing the DNA-binding activity.
Assuntos
Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Camundongos , Fosforilação , Ligação Proteica , Espectrometria de Massas em TandemRESUMO
Chronic hyperglycemia causes pancreatic ß-cell dysfunction, impaired insulin secretion and the suppression of insulin gene expression. This phenomenon is referred to as glucotoxicity, and is a critical component of the pathogenesis of type 2 diabetes. We previously reported that the expression of candidate plasticity gene 16 (CPG16) was higher in rat pancreatic INS-1 ß-cells under glucotoxic conditions and CPG16 suppressed insulin promoter activity. However, the molecular mechanisms of the CPG16-mediated suppression of insulin gene expression are unclear. In this study, we found that CPG16 directly bound and phosphorylated jun dimerization protein 2 (JDP2), an AP-1 family transcription factor. CPG16 co-localized with JDP2 in the nucleus of INS-1 cells. JDP2 bound to the G1 element of the insulin promoter and up-regulated promoter activity. Finally, CPG16 suppressed the up-regulation of insulin promoter activity by JDP2 in a kinase activity-dependent manner. These results suggest that CPG16 suppresses insulin promoter activity by phosphorylating JDP2.
Assuntos
Regulação da Expressão Gênica , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Animais , Linhagem Celular , Quinases Semelhantes a Duplacortina , Feminino , Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Ratos Wistar , Proteínas Repressoras/genéticaRESUMO
Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.
RESUMO
d-Allose, a C-3 epimer of d-glucose, is a naturally occurring rare monosaccharide that shows anti-proliferative activity against several human cancer cell lines. However, d-allose requires a relatively high concentration for the activity to be observed. Thus, developing more potent derivatives is needed for application. In cells, d-allose is converted to d-allose-6-phosphate (A6P), which is responsible for the anti-proliferative activity of d-allose. In this study, we synthesized A6P derivative 1 with biodegradable protecting groups, which showed higher anti-proliferative activity than A6P against a MOLT-4F human leukemia cell line. Similarly protected derivative of d-glucose-6-phosphate (G6P) (2) and tetraacetyl-A6P (3) showed weaker and less activity compared with 1, respectively, suggesting that both A6P moiety and the protecting group on the phosphate group are responsible for the activity. In addition, significantly weaker induction of thioredoxin-interacting protein (TXNIP) expression by 1 compared with d-allose suggests that 1 exhibited cytotoxicity through the synergetic effect of inducing TXNIP expression and other mechanisms.
Assuntos
Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
D-Allose (D-All), a C-3 epimer of D-glucose (D-Glc), is a naturally rare monosaccharide, which shows anti-proliferative activity against several human cancer cell lines. Unlike conventional anticancer drugs, D-All targets glucose metabolism and is non-toxic to normal cells. Therefore, it has attracted attention as a unique "seed" compound for anticancer agents. However, the anti-proliferative activities of the other rare aldohexoses have not been examined yet. In this study, we evaluated the anti-proliferative activity of rare aldohexoses against human leukemia MOLT-4F and human prostate cancer DU-145 cell lines. We found that D-All and D-idose (D-Ido) at 5 mM inhibited cell proliferation of MOLT-4F cells by 46 % and 60 %, respectively. On the other hand, the rare aldohexoses at 5 mM did not show specific anti-proliferative activity against DU-145 cells. To explore the structure-activity relationship of D-Ido, we evaluated the anti-proliferative activity of D-sorbose (D-Sor), 6-deoxy-D-Ido, and L-xylose (L-Xyl) against MOLT-4F cells and found that D-Sor, 6-deoxy-D-Ido, and L-Xyl showed no inhibitory activity at 5 mM, suggesting that the aldose structure and the C-6 hydroxy group of D-Ido are important for its activity. Cellular glucose uptake assay and western blotting analysis of thioredoxin-interacting protein (TXNIP) expression suggested that the anti-proliferative activity of D-Ido is induced by inhibition of glucose uptake via TXNIP-independent pathway.
RESUMO
Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.