24 results on '"Kinase activity"'
Search Results
2. Ubiquitin negatively regulates ABA responses by inhibiting SnRK2.2 and SnRK2.3 kinase activity in Arabidopsis.
- Author
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Shao, Zhengyu, Yang, Shuhua, Gu, Yinghui, Guo, Yan, Zhou, Huapeng, and Yang, Yongqing
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ABSCISIC acid , *PROTEIN kinases , *KINASES , *MASS spectrometry , *ARABIDOPSIS , *COMPLEX variables , *UBIQUITIN , *UBIQUITIN ligases - Abstract
Abscisic acid (ABA) is an essential phytohormone for plant responses to complex and variable environmental conditions. The molecular basis of the ABA signaling pathway has been well elucidated. SnRK2.2 and SnRK2.3 are key protein kinases participating in ABA responses, and the regulation of their activity plays an important role in signaling. Previous mass spectroscopy analysis of SnRK2.3 suggested that ubiquitin and homologous proteins may bind directly to the kinase. Ubiquitin typically recruits E3 ubiquitin ligase complexes to target proteins, marking them for degradation by the 26S proteasome. Here, we show that SnRK2.2 and SnRK2.3 interact with ubiquitin but are not covalently attached to the protein, resulting in the suppression of their kinase activity. The binding between SnRK2.2, SnRK2.3, and ubiquitin is weakened under prolonged ABA treatment. Overexpression of ubiquitin positively regulated the growth of seedlings exposed to ABA. Our results thus demonstrate a novel function for ubiquitin, which negatively regulates ABA responses by directly inhibiting SnRK2.2 and SnRK2.3 kinase activity. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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3. Essential role of the CD docking motif of MPK4 in plant immunity, growth, and development.
- Author
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Siodmak, Anna, Shahul Hameed, Umar F., Rayapuram, Naganand, Völz, Ronny, Boudsocq, Marie, Alharbi, Siba, Alhoraibi, Hannah, Lee, Yong‐Hwan, Blilou, Ikram, Arold, Stefan T., and Hirt, Heribert
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DISEASE resistance of plants , *REACTIVE oxygen species , *PROTEIN kinases - Abstract
Summary: MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators.We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand‐bound MPK4 crystal structure.We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild‐type (WT) MPK4‐C181, nonsulfenylatable MPK4‐C181S, and potentially sulfenylation mimicking MPK4‐C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4‐C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4‐C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4.Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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4. Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.
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Schütz, Martin, Müller, Regina, Socher, Eileen, Wangen, Christina, Full, Florian, Wyler, Emanuel, Wong, Diana, Scherer, Myriam, Stamminger, Thomas, Chou, Sunwen, Rawlinson, William D., Hamilton, Stuart T., Sticht, Heinrich, and Marschall, Manfred
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CYTOMEGALOVIRUSES , *AMINO acids , *HUMAN cytomegalovirus , *PROTEIN kinases , *NUCLEOTIDE sequencing , *CYCLIN-dependent kinases , *VIRAL nonstructural proteins - Abstract
The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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5. AMPKβ isoform expression patterns in various adipocyte models and in relation to body mass index.
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Kopietz, Franziska, Degerman, Eva, and Göransson, Olga
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BODY mass index ,AMP-activated protein kinases ,FAT cells ,PROTEIN kinases ,ADIPOSE tissues - Abstract
AMP-activated protein kinase (AMPK) activation is considered a useful strategy for the treatment of type 2 diabetes (T2D). It is unclear whether the expression and/or activity of AMPK in adipocytes is dysregulated in obesity. Also, the expression/activity pattern of AMPKβ isoforms, which are targets for AMPK activators, in adipocytes remains elusive. In this study we show that the two AMPKβ isoforms make roughly equal contributions to AMPK activity in primary human and mouse adipocytes, whereas in cultured 3T3-L1 adipocytes of mouse origin and in primary rat adipocytes, β1-associated activity clearly dominates. Additionally, we found that obesity is not associated with changes in AMPK subunit expression or kinase activity in adipocytes isolated fromsubcutaneous adipose tissue from individuals with various BMI. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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6. Shining Light on Protein Kinase Biomarkers with Fluorescent Peptide Biosensors.
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Morris, May C.
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PEPTIDES , *PROTEIN kinases , *BIOSENSORS , *BIOMARKERS , *CELLULAR signal transduction , *BIOMOLECULES - Abstract
Protein kinases (PKs) are established gameplayers in biological signalling pathways, and a large body of evidence points to their dysregulation in diseases, in particular cancer, where rewiring of PK networks occurs frequently. Fluorescent biosensors constitute attractive tools for probing biomolecules and monitoring dynamic processes in complex samples. A wide variety of genetically encoded and synthetic biosensors have been tailored to report on PK activities over the last decade, enabling interrogation of their function and insight into their behaviour in physiopathological settings. These optical tools can further be used to highlight enzymatic alterations associated with the disease, thereby providing precious functional information which cannot be obtained through conventional genetic, transcriptomic or proteomic approaches. This review focuses on fluorescent peptide biosensors, recent developments and strategies that make them attractive tools to profile PK activities for biomedical and diagnostic purposes, as well as insights into the challenges and opportunities brought by this unique toolbox of chemical probes. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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7. How can a traffic light properly work if it is always green? The paradox of CK2 signaling.
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Borgo, Christian, D'Amore, Claudio, Cesaro, Luca, Sarno, Stefania, Pinna, Lorenzo A., Ruzzene, Maria, and Salvi, Mauro
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PROTEIN kinase CK2 , *PROTEIN kinases , *ENZYME regulation , *PHOSPHORYLATION , *VIRUS diseases - Abstract
CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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8. PKZ, a Fish-Unique eIF2α Kinase Involved in Innate Immune Response.
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Wu, Chuxin, Zhang, Yibing, and Hu, Chengyu
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IMMUNE response ,PROTEIN kinases ,CATALYTIC domains - Abstract
PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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9. Phosphate-guanidine interaction based fluorometric strategy for protein kinase activity sensing.
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Wang, Mengke, Liu, Yang, Su, Dandan, Chen, Junyang, and Su, Xingguang
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PROTEIN kinases , *FLUORESCENCE resonance energy transfer , *GOLD nanoparticles , *DNA methyltransferases , *FLUORESCENCE quenching , *ADENOSINE triphosphate - Abstract
A fluorometric strategy for protein kinase A (PKA) activity detection was proposed based on the recognition of phosphate and guanidine groups by applying gold nanoclusters (AuNCs) functionalized by peptide as the fluorescent probe. • A fluorometric strategy for PKA activity sensing was constructed based on specific phosphate-guanidine interaction. • Specially designed peptides were applied to modify AuNPs and AuNCs, respectively. • The recognition of phosphate and guanidine groups triggered the FRET process. • The inhibition effect of H-89 on PKA activity was also investigated. • The analysis of PKA activity in complicated biologic sample was demonstrated in HepG2 cell lysates. In this paper, a fluorometric strategy for protein kinase A (PKA) activity detection was proposed based on the recognition of phosphate and guanidine groups. Two artificial substrate peptides (T1 and T2) were specially designed, T1, containing PKA specific recognition domain, was self-assembled on gold nanoclusters (T1-AuNCs). T2, possessing guanidine groups at arginine residues for the interaction with phosphorylation site, was used for modifying gold nanoparticles (T2-AuNPs). In the presence of adenosine triphosphate (ATP) and PKA, T1 was phosphorylated and then the phosphorylated T1 specially combined with T2 via phosphate-guanidine recognition. So, T1-AuNCs and T2-AuNPs were brought in close distance and fluorescence resonance energy transfer (FRET) process from T1-AuNCs to T2-AuNPs was triggered to result in the fluorescence quenching of T1-AuNCs. Accordingly, PKA activity was efficiently determined depending on the fluorescent signal changes within a good linearity from 0.04 to 8.0 U mL−1 with the detection limit down to 0.013 U mL−1. Moreover, this sensing platform was used to investigate the inhibition effect of PKA inhibitor (H-89) with good performance. Also, the practical application of this strategy for analysis of PKA activity in complicated biologic sample was demonstrated in HepG2 cell lysates. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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10. Sensitive electrogenerated chemiluminescence biosensors for protein kinase activity analysis based on bimetallic catalysis signal amplification and recognition of Au and Pt loaded metal-organic frameworks nanocomposites.
- Author
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Yan, Zhiyong, Wang, Feng, Deng, Pingye, Wang, Yu, Cai, Kai, Chen, Yanhui, Wang, Zonghua, and Liu, Yang
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CHEMILUMINESCENCE , *BIOSENSORS , *PROTEIN kinases , *METAL-organic frameworks , *PLATINUM nanoparticles - Abstract
In this work, a novel and sensitive electrogenerated chemiluminescence (ECL) biosensor for protein kinase A (PKA) activity analysis and relevant inhibitor screening was proposed based on bimetallic catalysis signal amplification and recognition of Au and Pt nanoparticles loaded metal-organic frameworks (Au&Pt@UiO-66) nanocomposite. After being phosphorylated by PKA in the presence of ATP, Au&Pt@UiO-66 probes were specifically chelated to the modified electrode by forming Zr-O-P bonds between the surface defects of UiO-66 and the phosphorylated kemptide. Due to the high synergistic catalysis of Au&Pt@UiO-66 nanocomposites to the luminol-H 2 O 2 reaction, the ECL signal of luminol was greatly enhanced. Moreover, UiO-66 afford numerous Zr defect sites for high efficient phosphate group recognition, and can also prevent the nanoparticles from aggregating during catalytic reactions. Thus, the excellent performance of the ECL biosensor with high sensitivity and superior stability was obtained. Under the optimized conditions, the detection limit for PKA activity was 0.009 UmL −1 (S/N = 3). Meanwhile, the ECL biosensor was successfully applied in inhibitor screening and cell lysates PKA activity analysis, showing great promise in kinase related research. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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11. Drug design targeting the AMPK signalling pathway with herbal medicines for atherosclerosis therapy.
- Author
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Hanheng Zuo, Yinping Li, Peng Hao, and Jing-Hua Liu
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ATHEROSCLEROSIS treatment ,CELLULAR signal transduction ,COMPUTER simulation ,HERBAL medicine ,MOLECULAR structure ,PHOSPHOTRANSFERASES ,PROTEIN kinases ,DRUG development ,IN vitro studies - Abstract
Background: Atherosclerosis (AS) is a vascular disease which causes cardiovascular diseases such as stroke, heart attack and acute coronary syndrome. It is a major cause of death worldwide, especially in developed countries. In AS, the artery walls become thickened due to the accumulation and invasion of white blood cells (foam cells) and form a fibro-fatty plaque. Hence, these investigations aim to identify the potential applications of herbal compounds targeting the AMPK signalling pathway for AS therapy. Methods: In this investigation, in vitro kinase activity was determined for a set of herbal compounds present in herbal plants and herbal medicines against 5' AMP-activated protein kinase (AMPK). This enzyme plays a vital role in AS, having an effect on the invasion and accumulation of white blood cells, leading to the thickening of artery walls and vascular remodelling. In addition, a molecular docking simulation study was carried out for the AMPK enzyme (PDB ID: 3AQV) against the herbal compounds. Results: The in vitro kinase activity showed that 10 of the herbal compounds possessed IC
50 values lower than 10 µM, which showed the potent inhibitory effect of the AMPK enzyme. In addition, a molecular docking simulation carried out on the AMPK enzyme (PDB ID: 3AQV) against the herbal compounds observed a positive correlation with IC50 values and docking scores of the docked compounds with an ICµ <10 µM. The subsequent protein-ligand interaction analyses for the best docking hits showed favourable interactions. Also, the molecular dynamics simulation enabled the generation of a trajectory file for the RMSD backbone, illustrating the stability of the docked protein-ligand complex without any structural fluctuations. Conclusions: The study concludes that baicalin, curcumin, campesterol, emodin and gingerol provide a promising lead for AS and can therefore be prescribed for the treatment of AS. [ABSTRACT FROM AUTHOR]- Published
- 2017
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12. An atlas of human kinase regulation.
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Ochoa, David, Jonikas, Mindaugas, Lawrence, Robert T, El Debs, Bachir, Selkrig, Joel, Typas, Athanasios, Villén, Judit, Santos, Silvia DM, and Beltrao, Pedro
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PROTEIN kinases , *CELL determination , *PHOSPHOPROTEINS , *SALMONELLA diseases , *PROTEOLYSIS - Abstract
The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinasedriven decision-making. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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13. Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies.
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Naz, Huma, Shahbaaz, Mohd., Bisetty, Krishna, Islam, Asimul, Ahmad, Faizan, and Hassan, Md. Imtaiyaz
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PH effect , *PHYSIOLOGICAL effects of hydrogen-ion concentration , *PHYSIOLOGICAL effects of calcium , *PROTEIN kinases , *PHOSPHOTRANSFERASES - Abstract
Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca2+, which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca2+ concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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14. Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators.
- Author
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Rajamohan, Francis, Reyes, Allan R., Frisbie, Richard K., Hoth, Lise R., Sahasrabudhe, Parag, Magyar, Rachelle, Landro, James A., Withka, Jane M., Caspers, Nicole L., Calabrese, Matthew F., Ward, Jessica, and Kurumbail, Ravi G.
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ENZYME kinetics , *ALLOSTERIC regulation , *ALLOSTERIC enzymes , *PROTEIN kinases , *PHYSIOLOGICAL effects of adenylic acid - Abstract
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ 1, α1β2γ 1, α1β2γ 3, α2β1γ 1, α2β2γ1 and α2β2γ 3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lowerKm values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ~25- fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1- complexes. Compared with β1-containing heterotrimers, β2- containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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15. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.
- Author
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Steingruber, Mirjam, Socher, Eileen, Hutterer, Corina, Webel, Rike, Bergbrede, Tim, Lenac, Tihana, Sticht, Heinrich, and Marschall, Manfred
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CYCLIN-dependent kinases , *CYTOMEGALOVIRUSES , *PROTEIN kinases , *HOST-virus relationships , *CELL communication , *PROTEIN-protein interactions , *GENE expression - Abstract
Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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16. Atg11-mediated activation of Atg1 kinase in fission yeast.
- Author
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Pan, Zhao-Qian and Du, Li-Lin
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AUTOPHAGY ,PROTEIN kinases ,SCHIZOSACCHAROMYCES pombe ,AMINO acids ,AUTOPHOSPHORYLATION - Abstract
The protein kinase Atg1 is a key player in macroautophagy/autophagy, but how its activity is regulated in various organisms is inadequately understood. Our recent study showed that in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity depends on Atg11, but not Atg13, Atg17, or Atg101. Notably, a 62 amino acid region of S. pombe Atg11 is sufficient for activating Atg1. This region is composed of two parts: an Atg1-binding domain and a homodimerization domain. Atg11 uses this region to dimerize Atg1. Dimerized Atg1 is activated through cis-autophosphorylation. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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17. Lord Kelvin's method of images in semigroup theory.
- Author
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Bobrowski, Adam
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SEMIGROUPS (Algebra) , *SEMIGROUPS of operators , *DIFFERENTIAL equations , *MATHEMATICAL models , *PROTEIN kinases - Abstract
We show that Lord Kelvin's method of images is a way to prove generation theorems for semigroups of operators. To this end we exhibit three examples: a more direct semigroup-theoretic treatment of abstract delay differential equations, a new derivation of the form of the McKendrick semigroup, and a generation theorem for a semigroup describing kinase activity in the recent model of Kaźmierczak and Lipniacki (J. Theor. Biol. 259:291-296, ). [ABSTRACT FROM AUTHOR]
- Published
- 2010
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18. Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases
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Sullivan, Stuart, Thomson, Catriona E., Kaiserli, Eirini, and Christie, John M.
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PROTEIN-protein interactions , *PLANT proteins , *ARABIDOPSIS thaliana , *PROTEIN kinases , *PLANT growth , *PHOSPHORYLATION , *PHOTOCHEMISTRY - Abstract
Abstract: Phototropin receptor kinases play an important role in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (λ) isoform from Arabidopsis. 14-3-3λ and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins are specific to phot1 signalling. Structured summary: MINT-7146953: PHOT1 (uniprotkb:O48963) physically interacts (MI:0915) with ARF7 (uniprotkb:Q9LFJ7) by two hybrid (MI:0018) MINT-7147335: PHOT1 (uniprotkb:O48963) physically interacts (MI:0914) with 14-3-3 phi (uniprotkb:P46077) by far Western blotting (MI:0047) MINT-7146854: PHOT1 (uniprotkb:O48963) physically interacts (MI:0915) with RPT2 (uniprotkb:Q682S0) by two hybrid (MI:0018) MINT-7147215: PHOT1 (uniprotkb:O48963) physically interacts (MI:0914) with 14-3-3 lambda (uniprotkb:P48349) by anti tag coimmunoprecipitation (MI:0007) MINT-7147044, MINT-7147185, MINT-7147200, MINT-7147413: PHOT1 (uniprotkb:O48963) physically interacts (MI:0914) with 14-3-3 lambda (uniprotkb:P48349) by far Western blotting (MI:0047) MINT-7146983: PHOT1 (uniprotkb:O48963) physically interacts (MI:0915) with 14-3-3 lambda (uniprotkb:P48349) by two hybrid (MI:0018) MINT-7146871: PHOT1 (uniprotkb:O48963) physically interacts (MI:0915) with NPH3-like (uniprotkb:Q9S9Q9) by two hybrid (MI:0018) MINT-7146905: PHOT1 (uniprotkb:O48963) physically interacts (MI:0915) with ARF2 (uniprotkb:Q9M1P5) by two hybrid (MI:0018) MINT-7147364: PHOT1 (uniprotkb:O48963) physically interacts (MI:0914) with 14-3-3 upsilon (uniprotkb:P42645) by far Western blotting (MI:0047) MINT-7147234: PHOT1 (uniprotkb:O48963) physically interacts (MI:0914) with 14-3-3 kappa (uniprotkb:P48348) by far Western blotting (MI:0047) [Copyright &y& Elsevier]
- Published
- 2009
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19. Cyclin Y, a novel membrane-associated cyclin, interacts with PFTK1
- Author
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Jiang, Mei, Gao, Yankun, Yang, Tao, Zhu, Xueliang, and Chen, Jiangye
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PROTEIN-protein interactions , *CYCLINS , *PROTEIN kinases , *CELL membranes , *ENZYME activation , *FLUORESCENCE microscopy - Abstract
Abstract: A novel cyclin, CCNY, was identified as a PFTK1 interacting protein in a yeast two-hybrid screen. The cyclin box in CCNY and the PFTAIRE motif in PFTK1 are both required for the interaction which was confirmed by in vivo and in vitro assays. Two transcripts (4 and 2kb), of CCNY were detected by Northern blot analysis and CCNY was enriched at the plasma membrane due to an N-terminal myristoylation signal. We propose that binding of CCNY to PFTK1 enhances PFTK1 kinase activity and changes its intracellular location. Structured summary: MINT-7147585, MINT-7147598, MINT-7147614, MINT-7147628, MINT-7147647, MINT-7147665, MINT-7147680: pftk1 (uniprotkb:O94921) physically interacts (MI:0915) with CCNY (uniprotkb:Q8ND76) by two hybrid (MI:0018) MINT-7147725, MINT-7147743: pftk1 (uniprotkb:O94921) physically interacts (MI:0914) with CCNY (uniprotkb:Q8ND76) by anti tag coimmunoprecipitation (MI:0007) MINT-7147758: pftk1 (uniprotkb:O35495) physically interacts (MI:0914) with CCNY (uniprotkb:Q8BGU5) by anti bait coimmunoprecipitation (MI:0006) MINT-7147695, MINT-7147713: pftk1 (uniprotkb:O94921) and CCNY (uniprotkb:Q8ND76) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [Copyright &y& Elsevier]
- Published
- 2009
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20. LSA-50 paper: An alternative to P81 phosphocellulose paper for radiometric protein kinase assays.
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Appelmans, Olivia, Kashyap, Rudra Shankar, Gilles, Philippe, De Borggraeve, Wim M., Voet, Arnout, and Van Lint, Johan
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PROTEIN kinases , *SCIENTIFIC community , *KINASE inhibitors - Abstract
Radiometric assays have widely been used for measuring protein kinase activity for decades. In addition, several non-radiometric kinase assay formats have been developed over the years, including luciferase-based and fluorescence-based assays. However, radiometric assays are still considered as the "gold standard" for protein kinase assays, because of their direct readout, high sensitivity, reproducibility, reliability, and very low background signals. These radiometric assays rely on P81 phosphocellulose paper to capture the phosphorylated substrate and wash out unreacted [γ-32P] ATP. However, recently the production of P81 was discontinued by the manufacturer, causing major concern within the protein kinase research community. The advantages of radiometric assays over other kinase assay methods call for an urgent alternative to the discontinued P81 paper. In this report, we demonstrate that the LSA-50 paper is a worthy alternative for radiometric protein kinase assays originally using P81 phosphocellulose paper. [Display omitted] • P81 phosphocellulose paper, the gold standard for radiometric kinase assays, was discontinued. • Thus, an alternative paper for radiometric kinase assays is urgently needed. • Efficiency of P81 and LSA-50 paper in radiometric kinase assays was compared. • Kinase activities and inhibitor IC 50 's were very similar for P81 and LSA-50 paper. • LSA-50 paper is a high-quality alternative for P81 paper in radiometric kinase assays. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
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21. Hsp70 associates with Rictor and is required for mTORC2 formation and activity
- Author
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Martin, Jheralyn, Masri, Janine, Bernath, Andrew, Nishimura, Robert N., and Gera, Joseph
- Subjects
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PROTEIN kinases , *GROWTH factors , *RNA , *HEAT shock proteins - Abstract
Abstract: mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
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22. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation
- Author
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Gibson, Candace, Schanen, Brian, Chakrabarti, Debopam, and Chakrabarti, Ratna
- Subjects
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PROTEIN kinases , *CYCLIC adenylic acid , *GIARDIA lamblia , *CLONE cells - Abstract
Abstract: To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3′5′-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation. [Copyright &y& Elsevier]
- Published
- 2006
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23. A kinase-dependent role for EphA2 receptor in promoting tumor growth and metastasis.
- Author
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Wei Bin Fang, Brantley-Sieders, Dana M., Parker, Monica A., Reith, Alastair D., and Jin Chen
- Subjects
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TUMOR growth , *PROTEIN-tyrosine kinases , *METASTASIS , *GUANOSINE triphosphatase , *PROTEIN kinases , *TUMORS - Abstract
Receptor tyrosine kinases of the Eph family are upregulated in several different types of cancer. One family member in particular, the EphA2 receptor, has been linked to breast, prostate, lung and colon cancer, as well as melanoma. However, mechanisms by which EphA2 contributes to tumor progression are far from clear. In certain tumor cell lines, EphA2 receptor is underphosphorylated, raising the question of whether ligand-induced receptor phosphorylation and its kinase activity play a role in oncogenesis. To test directly the role of EphA2 receptor phosphorylation/kinase activity in tumor progression, we generated EphA2 receptor variants that were either lacking the cytoplasmic domain or carrying a point mutation that inhibits its kinase activity. Expression of these EphA2 mutants in breast cancer cells resulted in decreased tumor volume and increased tumor apoptosis in primary tumors. In addition, the numbers of lung metastases were significantly reduced in both experimental and spontaneous metastasis models. Reduced tumor volume and metastasis are not due to defects in tumor angiogenesis, as there is no significant difference in tumor vessel density between wild-type tumors and tumors expressing EphA2-signaling-defective mutants. In contrast, tumor cells expressing the EphA2 mutants are defective in RhoA GTPase activation and cell migration. Taken together, these results suggest that receptor phosphorylation and kinase activity of the EphA2 receptor, at least in part, contribute to tumor malignancy.Oncogene (2005) 24, 7859–7868. doi:10.1038/sj.onc.1208937; published online 15 August 2005 [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
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24. FRAT1 peptide decreases Aβ production in swAPP751 cells
- Author
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Li, Baolin, Ryder, John, Su, Yuan, Zhou, Yan, Liu, Feng, and Ni, Binhui
- Subjects
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AMYLOID , *GLYCOGEN , *PROTEIN kinases , *CHEMICAL inhibitors - Abstract
Recently, LiCl has been shown to inhibit amyloid β peptide secretion in association with diminished glycogen synthase kinase β (GSK3β) activity. However, it remains unclear if direct inhibition of GSK3β activity will result in decreased Aβ production.
requentlyF earranged inr dvanceda -cell lymphomasT (FRAT1) protein is a negative regulator of GSK3α/β kinase activity. To examine whether direct inhibition of GSK3α/β kinase activity can lower Aβ production, a FRAT1 peptide was expressed in swAPP751 cells that produce high levels of Aβ. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP751 cells increases both GSK3α and β phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Aβ and Aβ1-42 was significantly decreased. Thus, we provide evidence that direct regulation of GSK3α/β by FRAT1 peptide significantly decreases Aβ production in swAPP751 cells. [Copyright &y& Elsevier]1 - Published
- 2003
- Full Text
- View/download PDF
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