Exercise and CaMK activation both increase the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo

2007 ◽  
Vol 292 (2) ◽  
pp. E413-E420 ◽  
Author(s):  
James A. H. Smith ◽  
Malcolm Collins ◽  
Liesl A. Grobler ◽  
Carrie J. Magee ◽  
Edward O. Ojuka
Keyword(s):  
Dominant Negative ◽  
Binding Assays ◽  
Cis Element ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Autonomous Activity ◽  
Exercise Induced ◽  
Camk Iv

In vitro binding assays have indicated that the exercise-induced increase in muscle GLUT4 is preceded by increased binding of myocyte enhancer factor 2A (MEF2A) to its cis-element on the Glut4 promoter. Because in vivo binding conditions are often not adequately recreated in vitro, we measured the amount of MEF2A that was bound to the Glut4 promoter in rat triceps after an acute swimming exercise in vivo, using chromatin immunoprecipitation (ChIP) assays. Bound MEF2A was undetectable in nonexercised controls or at 24 h postexercise but was significantly elevated ∼6 h postexercise. Interestingly, the increase in bound MEF2A was preceded by an increase in autonomous activity of calcium/calmodulin-dependent protein kinase (CaMK) II in the same muscle. To determine if CaMK signaling mediates MEF2A/DNA associations in vivo, we performed ChIP assays on C2C12 myotubes expressing constitutively active (CA) or dominant negative (DN) CaMK IV proteins. We found that ∼75% more MEF2A was bound to the Glut4 promoter in CA compared with DN CaMK IV-expressing cells. GLUT4 protein increased ∼70% 24 h after exercise but was unchanged by overexpression of CA CaMK IV in myotubes. These results confirm that exercise increases the binding of MEF2A to the Glut4 promoter in vivo and provides evidence that CaMK signaling is involved in this interaction.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Echinococcus Granulosus ◽  
Calcium Content ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. Methods The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. Results In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. Conclusions Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

scholarly journals Reconstitution of Calcium-Regulated Parathyroid Hormone Secretion from Streptolysin-O-Permeabilized Parathyroid Cells by Guanosine 5′-O-(Thio)Triphosphate*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1170-1179 ◽  
Author(s):  
Lisa M. Matovcik ◽  
Steven S. Rhee ◽  
Jean F. Schaefer ◽  
Barbara K. Kinder
Keyword(s):  
Hormone Secretion ◽  
Dependent Manner ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Calmodulin Dependent ◽  
Streptolysin O ◽  
Dependent Protein ◽  
Vitro Model

Abstract Intracellular Ca2+ levels determine the amount of PTH secretion from parathyroid cells. Dissociated calf parathyroid cells were permeabilized with streptolysin-O (SLO) to provide an in vitro model system to examine Ca2+-dependent regulation of hormone secretion. PTH release from these cells was energy dependent and increased by cytosolic cofactors. Guanosine 5′-O-(thio)triphosphate (GTPγS) increased PTH secretion from SLO-permeabilized cells in a dose-dependent manner from 0.1–100 μm. In the absence of GTPγS there was no relationship between the ambient Ca2+ concentration and the rate of PTH secretion. However, in the presence of GTPγS, intracellular Ca2+ inhibited PTH secretion with an EC50 of approximately 0.1 μm, corresponding to physiological intracellular Ca2+ levels. Thus, the addition of GTPγS to SLO-permeabilized parathyroid cells reconstituted the inverse relationship between extracellular Ca2+ and PTH secretion that is observed in vivo and in intact cells. The data indicate that this effect is mediated at least in part by heterotrimeric guanosine triphosphatases. In addition, calcium/calmodulin-dependent protein kinase II appears to mediate low Ca2+-dependent PTH secretion from these cells.

scholarly journals Yeast Pak1 Kinase Associates with and Activates Snf1

2003 ◽  
Vol 23 (11) ◽  
pp. 3909-3917 ◽  
Author(s):  
Nandita Nath ◽  
Rhonda R. McCartney ◽  
Martin C. Schmidt
Keyword(s):  
Protein Kinase ◽  
Specific Activity ◽  
Nutrient Stress ◽  
Snf1 Kinase ◽  
Calcium Calmodulin Dependent ◽  
Upstream Kinase ◽  
Dependent Protein ◽  
Additional Protein

ABSTRACT Members of the Snf1/AMP-activated protein kinase family are activated under conditions of nutrient stress by a distinct upstream kinase. Here we present evidence that the yeast Pak1 kinase functions as a Snf1-activating kinase. Pak1 associates with the Snf1 kinase in vivo, and the association is greatly enhanced under glucose-limiting conditions when Snf1 is active. Snf1 kinase complexes isolated from pak1Δ mutant strains show reduced specific activity in vitro, and affinity-purified Pak1 kinase is able to activate the Snf1-dependent phosphorylation of Mig1 in vitro. Purified Pak1 kinase promotes the phosphorylation of the Snf1 polypeptide on threonine 210 within the activation loop in vitro, and an increased dosage of the PAK1 gene causes increased Snf1 threonine 210 phosphorylation in vivo. Deletion of the PAK1 gene does not produce a Snf phenotype, suggesting that one or more additional protein kinases is able to activate Snf1 in vivo. However, deletion of the PAK1 gene suppresses many of the phenotypes associated with the deletion of the REG1 gene, providing genetic evidence that Pak1 activates Snf1 in vivo. The closest mammalian homologue of yeast Pak1 kinase, calcium-calmodulin-dependent protein kinase kinase beta, may play a similar role in mammalian nutrient stress signaling.

scholarly journals Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling

2016 ◽  
Vol 113 (26) ◽  
pp. E3773-E3781 ◽  
Author(s):  
Yaling Yin ◽  
Di Gao ◽  
Yali Wang ◽  
Zhi-Hao Wang ◽  
Xin Wang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Camp Response Element Binding ◽  
Memory Deficit ◽  
Nuclear Fraction ◽  
Wild Type ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Intracellular accumulation of wild-type tau is a hallmark of sporadic Alzheimer’s disease (AD), but the molecular mechanisms underlying tau-induced synapse impairment and memory deficit are poorly understood. Here we found that overexpression of human wild-type full-length tau (termed hTau) induced memory deficits with impairments of synaptic plasticity. Both in vivo and in vitro data demonstrated that hTau accumulation caused remarkable dephosphorylation of cAMP response element binding protein (CREB) in the nuclear fraction. Simultaneously, the calcium-dependent protein phosphatase calcineurin (CaN) was up-regulated, whereas the calcium/calmodulin-dependent protein kinase IV (CaMKIV) was suppressed. Further studies revealed that CaN activation could dephosphorylate CREB and CaMKIV, and the effect of CaN on CREB dephosphorylation was independent of CaMKIV inhibition. Finally, inhibition of CaN attenuated the hTau-induced CREB dephosphorylation with improved synapse and memory functions. Together, these data indicate that the hTau accumulation impairs synapse and memory by CaN-mediated suppression of nuclear CaMKIV/CREB signaling. Our findings not only reveal new mechanisms underlying the hTau-induced synaptic toxicity, but also provide potential targets for rescuing tauopathies.

scholarly journals Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase Ⅱ response in vitro and in a murine infection model

2021 ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
Calcium Content ◽  
Mouse Serum ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background: Echinococcosis caused by the larvae of cestodes of the genus Echinococcus is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and the assocaiated side effects after long-time use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of verapamil on Echinococcus with a particular focus on the regulatory effect of verapamil on calcium/calmodulin-dependent protein kinase Ⅱ (Ca2+/CaM-CamKⅡ) in infected mice.Methods: The anti-echinococcal effects of verapamil on E. granulosus protoscoleces (PSC) in vitro and E. multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by verapamil were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and Alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CamKⅡ in infected mice, and in the mRNA levels of CamKⅡ in E. granulosus PSC were evaluated after treatment with verapamil by immunohistochemistry and/or real-time quantitative polymerase chain reaction.Results: In vitro, E. granulosus PSC could be killed by verapamil at a concentration of 0.5 μg/mL or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied obvious calcium loss and downregulation of CamKⅡ mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg verapamil, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CamKⅡ protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with verapamil.Conclusions: Verapamil exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CamKⅡ, which were over-activated by parasitic infection. The results suggested that Ca2+/CaM-CamKⅡ may be a novel drug-target, and verapamil was shown to be a potential anti-echinococcal drug for controlling echinococcosis in the future.

scholarly journals Anti-echinococcal effect of verapamil involving regulating calcium/calmodulin-dependent protein kinase Ⅱ response in vitro and in a murine infection model

2020 ◽  
Author(s):  
Hai-Jun Gao ◽  
Xu-Dong Sun ◽  
Yan-Ping Luo ◽  
Hua-Sheng Pang ◽  
Xing-Ming Ma ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Calcium Content ◽  
Morphological Alteration ◽  
Mrna Levels ◽  
Down Regulation ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Abstract Background: Echinococcosis caused by the larval stage of cestode of the genus Echinococcus is a parasitic zoonosis, imposing serious threat on the health of humans and animals globally. Albendazole is the drug of choice for treatment of echinococcosis, but it is difficult to meet the clinical challenge in chemotherapy due to its low curative rate and severe side effects. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. Previous study has showed that verapamil can suppress the growth of Echinococcus granulosus (E. granulosus) larva, but the mechanism remains unclear. The aim of the present study is to gain insight into the anti-echinococcal effect of verapamil on Echinococcus with particular focus on the regulatory role of verapamil to calcium/calmodulin-dependent protein kinase Ⅱ (Ca2+/CaM-CamK Ⅱ) in infected mice.Methods: The anti-echinococcal effect of verapamil on E. granulosus protoscolex (PSCx) in vitro and Echinococcus multilocularis (E. multilocularis) metacestodes in infected mice were assessed. The morphological alteration of Echinococcus spp. induced by verapamil was observed by scanning electron microscope (SEM), and the changes of calcium content in both parasite and mice sera and livers were measured by SEM-energy dispersive spectrometer, inductively coupled plasma mass spectrometry and alizarin red staining accordingly. Additionally, the changes on the protein and mRNA levels of CaM and CamK Ⅱ in infected mice, and on the mRNA levels of CamK Ⅱ in E. granulosus PSCx after treatment of verapamil were evaluated by immunohistochemistry or/and real-time quantitative polymerase chain reaction (RT-qPCR).Results: In vitro, E. granulosus PSCx could be killed by verapamil at 0.5 μg/mL or more within 8 days. Under these conditions, the ultrastructure of PSCx was damaged and accompanied with obvious calcium loss and down-regulation of CamK Ⅱ mRNA. In vivo, the weight and the calcium content of E. multilocularis metacestodes from the mice were reduced after treatment with verapamil at 40 mg/kg, meanwhile, an elevation of calcium content in the serum and liver of infected mice was observed. In addition, down-regulation of both the over-expressed CaM and CamK Ⅱ proteins and mRNAs in the liver of mice infected with E. multilocularis metacestodes were found after treatment of verapamil.Conclusions: Verapamil exerted a parasiticidal activity on Echinococcus both in vitro and in vivo through down-regulation of Ca2+/CaM-CamK Ⅱ that was over-activated by parasitic infection. It has been speculated that the Ca2+/CaM-CamK Ⅱ can be a novel drug-target and verapamil is shown to be a potential anti-echinococcal drug for controlling echinococcosis in the future.

scholarly journals An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Janire Urrutia ◽  
Alejandra Aguado ◽  
Carolina Gomis-Perez ◽  
Arantza Muguruza-Montero ◽  
Oscar R. Ballesteros ◽  
...  
Keyword(s):  
Structural Instability ◽  
Binding Assays ◽  
Iq Motif ◽  
Channel Function ◽  
Human Epilepsy ◽  
Pathogenic Variants ◽  
In Silico Studies ◽  
Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

scholarly journals LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
You-hong Wang ◽  
Zhen Guo ◽  
Liang An ◽  
Yong Zhou ◽  
Heng Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nasopharyngeal Cancer ◽  
Noncoding Rnas ◽  
Dependent Protein Kinase ◽  
Damage Repair ◽  
Dependent Protein ◽  
Cancer Tissues

AbstractRadioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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