13,625 results on '"Kinase activity"'
Search Results
2. Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.
- Author
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Póti ÁL, Dénes L, Papp K, Bató C, Bánóczi Z, Reményi A, and Alexa A
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- Phosphorylation, Protein Binding, Ribosomal Protein S6 Kinases, 90-kDa metabolism, Mitogen-Activated Protein Kinases metabolism, Protein Kinases metabolism
- Abstract
Protein kinases are key regulators of cell signaling and have been important therapeutic targets for three decades. ATP-competitive drugs directly inhibit the activity of kinases but these enzymes work as part of complex protein networks in which protein-protein interactions (often referred to as kinase docking) may govern a more complex activation pattern. Kinase docking is indispensable for many signaling disease-relevant Ser/Thr kinases and it is mediated by a dedicated surface groove on the kinase domain which is distinct from the substrate-binding pocket. Thus, interfering with kinase docking provides an alternative strategy to control kinases. We describe activity sensors developed for p90 ribosomal S6 kinase (RSK) and mitogen-activated protein kinases (MAPKs: ERK, p38, and JNK) whose substrate phosphorylation is known to depend on kinase-docking-groove-mediated protein-protein binding. The in vitro assays were based on fragment complementation of the NanoBit luciferase, which is facilitated upon substrate motif phosphorylation. The new phosphorylation-assisted luciferase complementation (PhALC) sensors are highly selective and the PhALC assay is a useful tool for the quantitative analysis of kinase activity or kinase docking, and even for high-throughput screening of academic compound collections.
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- 2023
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3. Subcellular partitioning of protein kinase activity revealed by functional kinome profiling.
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Wegman-Points L, Alganem K, Imami AS, Mathis V, Creeden JF, McCullumsmith R, and Yuan LL
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- Animals, Phosphorylation, Rats, Serine metabolism, Subcellular Fractions metabolism, Threonine metabolism, Peptides metabolism, Protein Kinases metabolism
- Abstract
Protein kinases and their substrates form signaling networks partitioned across subcellular compartments to facilitate critical biological processes. While the subcellular roles of many individual kinases have been elucidated, a comprehensive assessment of the synaptic subkinome is lacking. Further, most studies of kinases focus on transcript, protein, and/or phospho-protein expression levels, providing an indirect measure of protein kinase activity. Prior work suggests that gene expression levels are not a good predictor of protein function. Thus, we assessed global serine/threonine protein kinase activity profiles in synaptosomal, nuclear, and cytosolic fractions from rat frontal cortex homogenate using peptide arrays. Comparisons made between fractions demonstrated differences in overall protein kinase activity. Upstream kinase analysis revealed a list of cognate kinases that were enriched in the synaptosomal fraction compared to the nuclear fraction. We identified many kinases in the synaptic fraction previously implicated in this compartment, while also identifying other kinases with little or no evidence for synaptic localization. Our results show the feasibility of assessing subcellular fractions with peptide activity arrays, as well as suggesting compartment specific activity profiles associated with established and novel kinases., (© 2022. The Author(s).)
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- 2022
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4. Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis.
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Pavinato L, Villamor-Payà M, Sanchiz-Calvo M, Andreoli C, Gay M, Vilaseca M, Arauz-Garofalo G, Ciolfi A, Bruselles A, Pippucci T, Prota V, Carli D, Giorgio E, Radio FC, Antona V, Giuffrè M, Ranguin K, Colson C, De Rubeis S, Dimartino P, Buxbaum JD, Ferrero GB, Tartaglia M, Martinelli S, Stracker TH, and Brusco A
- Subjects
- Adolescent, Adult, Child, Child, Preschool, Cohort Studies, DNA Mutational Analysis, Female, Humans, Male, Metabolome, Middle Aged, Mutation, Mutation, Missense, Neurodevelopmental Disorders enzymology, Pedigree, Protein Interaction Mapping, Protein Kinases metabolism, Exome Sequencing, Young Adult, Chromatin metabolism, Neurodevelopmental Disorders genetics, Protein Kinases genetics
- Abstract
Introduction: The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with 'Mental Retardation Autosomal Dominant 57' (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies., Methods: We re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity., Results: We identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2 , and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage., Conclusion: Our study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2022
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5. The Protein Kinase Activity of NME7 Activates Wnt/β-Catenin Signaling to Promote One-Carbon Metabolism in Hepatocellular Carcinoma.
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Ren X, Rong Z, Liu X, Gao J, Xu X, Zi Y, Mu Y, Guan Y, Cao Z, Zhang Y, Zeng Z, Fan Q, Wang X, Pei Q, Wang X, Xin H, Li Z, Nie Y, Qiu Z, Li N, Sun L, and Deng Y
- Subjects
- Carcinoma, Hepatocellular pathology, Cell Line, Tumor, Cell Proliferation, Humans, Liver Neoplasms pathology, Carbon metabolism, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics, Nucleoside-Diphosphate Kinase metabolism, Protein Kinases metabolism, Wnt Signaling Pathway genetics, beta Catenin metabolism
- Abstract
Metabolic reprogramming by oncogenic signaling is a hallmark of cancer. Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms inducing hyperactivation of Wnt/β-catenin signaling and strategies for targeting this pathway are incompletely understood. In this study, we find nucleoside diphosphate kinase 7 (NME7) to be a positive regulator of Wnt/β-catenin signaling. Upregulation of NME7 positively correlated with the clinical features of HCC. Knockdown of NME7 inhibited HCC growth in vitro and in vivo , whereas overexpression of NME7 cooperated with c-Myc to drive tumorigenesis in a mouse model and to promote the growth of tumor-derived organoids. Mechanistically, NME7 bound and phosphorylated serine 9 of GSK3β to promote β-catenin activation. Furthermore, MTHFD2, the key enzyme in one-carbon metabolism, was a target gene of β-catenin and mediated the effects of NME7. Tumor-derived organoids with NME7 overexpression exhibited increased sensitivity to MTHFD2 inhibition. In addition, expression levels of NME7, β-catenin, and MTHFD2 correlated with each other and with poor prognosis in patients with HCC. Collectively, this study emphasizes the crucial roles of NME7 protein kinase activity in promoting Wnt/β-catenin signaling and one-carbon metabolism, suggesting NME7 and MTHFD2 as potential therapeutic targets for HCC. SIGNIFICANCE: The identification of NME7 as an activator of Wnt/β-catenin signaling and MTHFD2 expression in HCC reveals a mechanism regulating one-carbon metabolism and potential therapeutic strategies for treating this disease., (©2021 American Association for Cancer Research.)
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- 2022
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6. Kinase activity is required for the receptor kinase DROOPY LEAF1 to control leaf droopiness.
- Author
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Wang C, Tang S, Zhang Q, Shang Z, Liu X, Diao X, and Zhao M
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- Crops, Agricultural genetics, Crops, Agricultural growth & development, Crops, Agricultural metabolism, Gene Expression Regulation, Plant, Genes, Plant, Genetic Variation, Genotype, Plant Leaves genetics, Protein Kinases genetics, Brassinosteroids metabolism, Plant Leaves growth & development, Plant Leaves metabolism, Protein Kinases metabolism, Setaria Plant genetics, Setaria Plant growth & development, Setaria Plant metabolism
- Abstract
Plants have evolved many leucine-rich repeat receptor-like kinases (LRR-RLKs) that control all aspects of plant life in a kinase-dependent or -independent manner. DROOPY LEAF1 (DPY1), which is a subfamily II LRR-RLK authentic kinase, controls leaf droopiness by negatively regulating early brassinosteroid (BR) signaling in foxtail millet. In this study, we proved that overexpressing kinase-inactive DPY1 does not rescue the droopy leaf phenotype of dpy1 plants because the mutated DPY1 cannot repress BR signaling, suggesting that kinase activity is required for DPY1 to control BR signaling. Moreover, seven DPY1 sites potentially transphosphorylated by SiBAK1 were identified as crucial for DPY1 activation. These findings highlight the importance of kinase activity for the functionality of DPY1.
- Published
- 2021
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7. The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains.
- Author
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Weissman JD, Singh AK, Devaiah BN, Schuck P, LaRue RC, and Singer DS
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- Amino Acid Motifs, Animals, Mice, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Domains, Protein Kinases genetics, Protein Kinases metabolism, Protein Structure, Quaternary, RNA Polymerase II chemistry, RNA Polymerase II genetics, RNA Polymerase II metabolism, TATA-Binding Protein Associated Factors chemistry, TATA-Binding Protein Associated Factors genetics, TATA-Binding Protein Associated Factors metabolism, Transcription Factor TFIID chemistry, Transcription Factor TFIID genetics, Transcription Factor TFIID metabolism, Transcription Factors genetics, Transcription Factors metabolism, Nuclear Proteins chemistry, Protein Kinases chemistry, Protein Multimerization, Transcription Factors chemistry
- Abstract
Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4's biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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8. Open stomata 1 exhibits dual serine/threonine and tyrosine kinase activity in regulating abscisic acid signaling.
- Author
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Shang Y, Yang D, Ha Y, Lee JY, Kim JY, Oh MH, and Nam KH
- Subjects
- Phosphorylation, Protein-Tyrosine Kinases, Serine, Threonine, Abscisic Acid, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Protein Kinases genetics, Protein Kinases metabolism
- Abstract
Open Stomata 1 (OST1)/SnRK2.6 is a critical component connecting abscisic acid (ABA) receptor complexes and downstream components, including anion channels and transcription factors. Because OST1 is a serine/threonine kinase, several autophosphorylation sites have been identified, and S175 is known to be critical for its kinase activity. We previously reported that BAK1 interacts with and phosphorylates OST1 to regulate ABA signaling. Here, we mapped additional phosphosites of OST1 generated by autophosphorylation and BAK1-mediated transphosphorylation in Arabidopsis. Many phosphosites serve as both auto- and transphosphorylation sites, especially those clustered in the activation loop region. Phospho-mimetic transgenic plants containing quadruple changes in Y163, S164, S166, and S167 rescued ost1 mutant phenotypes, activating ABA signaling outputs. Moreover, we found that OST1 is an active tyrosine kinase, autophosphorylating the Y182 site. ABA induced tyrosine phosphorylation of Y182 in OST1; this event is catalytically important for OST1 activity in plants. ABA-Insensitive 1 (ABI1) and its homologs ABI2 and HAB1, PP2C serine/threonine phosphatases that are known to dephosphorylate OST1 at S175, function as tyrosine phosphatases acting on the phosphorylated Y182 site. Our results indicate that phosphorylation cycles between OST1 and ABI1, which have dual specificity for tyrosine and serine/threonine, coordinately control ABA signaling in Arabidopsis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2021
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9. Granulovirus PK-1 kinase activity relies on a side-to-side dimerization mode centered on the regulatory αC helix.
- Author
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Oliver MR, Horne CR, Shrestha S, Keown JR, Liang LY, Young SN, Sandow JJ, Webb AI, Goldstone DC, Lucet IS, Kannan N, Metcalf P, and Murphy JM
- Subjects
- Baculoviridae metabolism, Crystallography, X-Ray, Granulovirus genetics, Molecular Dynamics Simulation, Phosphorylation, Protein Conformation, Protein Kinases genetics, Protein Subunits metabolism, Viral Proteins metabolism, Dimerization, Granulovirus enzymology, Protein Kinases chemistry, Protein Kinases metabolism
- Abstract
The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.
- Published
- 2021
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10. Oxidative Stress, Kinase Activity and Inflammatory Implications in Right Ventricular Hypertrophy and Heart Failure under Hypobaric Hypoxia.
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Pena E, Brito J, El Alam S, and Siques P
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- Animals, Heart Failure immunology, Heart Failure metabolism, Humans, Hypertrophy, Right Ventricular immunology, Hypertrophy, Right Ventricular metabolism, Inflammation immunology, Inflammation pathology, Heart Failure pathology, Hypertrophy, Right Ventricular pathology, Hypoxia physiopathology, Inflammation complications, Oxidative Stress, Protein Kinases metabolism
- Abstract
High altitude (hypobaric hypoxia) triggers several mechanisms to compensate for the decrease in oxygen bioavailability. One of them is pulmonary artery vasoconstriction and its subsequent pulmonary arterial remodeling. These changes can lead to pulmonary hypertension and the development of right ventricular hypertrophy (RVH), right heart failure (RHF) and, ultimately to death. The aim of this review is to describe the most recent molecular pathways involved in the above conditions under this type of hypobaric hypoxia, including oxidative stress, inflammation, protein kinases activation and fibrosis, and the current therapeutic approaches for these conditions. This review also includes the current knowledge of long-term chronic intermittent hypobaric hypoxia. Furthermore, this review highlights the signaling pathways related to oxidative stress (Nox-derived O
2 .- and H2 ), protein kinase (ERK5, p38α and PKCα) activation, inflammatory molecules (IL-1β, IL-6, TNF-α and NF-kB) and hypoxia condition (HIF-1α). On the other hand, recent therapeutic approaches have focused on abolishing hypoxia-induced RVH and RHF via attenuation of oxidative stress and inflammatory (IL-1β, MCP-1, SDF-1 and CXCR-4) pathways through phytotherapy and pharmacological trials. Nevertheless, further studies are necessary.2 ), protein kinase (ERK5, p38α and PKCα) activation, inflammatory molecules (IL-1β, IL-6, TNF-α and NF-kB) and hypoxia condition (HIF-1α). On the other hand, recent therapeutic approaches have focused on abolishing hypoxia-induced RVH and RHF via attenuation of oxidative stress and inflammatory (IL-1β, MCP-1, SDF-1 and CXCR-4) pathways through phytotherapy and pharmacological trials. Nevertheless, further studies are necessary.- Published
- 2020
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11. Kinase activity-tagged western blotting assay.
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Eto M, Katsuki S, Tanaka Y, and Takeya K
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- Animals, Cells, Cultured, Mice, Muscles cytology, Muscles metabolism, Neurons metabolism, Proteins analysis, Proteins chemistry, Proteins metabolism, Blotting, Western methods, Phosphorylation physiology, Protein Kinases chemistry, Protein Kinases metabolism, Proteomics methods
- Abstract
Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a nonradioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP.
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- 2020
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12. Modulation of BIN2 kinase activity by HY5 controls hypocotyl elongation in the light.
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Li J, Terzaghi W, Gong Y, Li C, Ling JJ, Fan Y, Qin N, Gong X, Zhu D, and Deng XW
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- Arabidopsis genetics, Arabidopsis Proteins genetics, Basic-Leucine Zipper Transcription Factors genetics, Brassinosteroids metabolism, DNA-Binding Proteins metabolism, Gene Expression Regulation, Plant, Glycogen Synthase Kinase 3, Phosphorylation, Protein Kinases genetics, Transcription Factors metabolism, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Basic-Leucine Zipper Transcription Factors metabolism, Hypocotyl metabolism, Light, Protein Kinases metabolism
- Abstract
ELONGATED HYPOCOTYL 5 (HY5), a basic domain/leucine zipper (bZIP) transcription factor, acts as a master regulator of transcription to promote photomorphogenesis. At present, it's unclear whether HY5 uses additional mechanisms to inhibit hypocotyl elongation. Here, we demonstrate that HY5 enhances the activity of GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), a key repressor of brassinosteroid signaling, to repress hypocotyl elongation. We show that HY5 physically interacts with and genetically acts through BIN2 to inhibit hypocotyl elongation. The interaction of HY5 with BIN2 enhances its kinase activity possibly by the promotion of BIN2 Tyr
200 autophosphorylation, and subsequently represses the accumulation of the transcription factor BRASSINAZOLE-RESISTANT 1 (BZR1). Leu137 of HY5 is found to be important for the HY5-BIN2 interaction and HY5-mediated regulation of BIN2 activity, without affecting the transcriptional activity of HY5. HY5 levels increase with light intensity, which gradually enhances BIN2 activity. Thus, our work reveals an additional way in which HY5 promotes photomorphogenesis, and provides an insight into the regulation of GSK3 activity.- Published
- 2020
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13. OsCIPK7 point-mutation leads to conformation and kinase-activity change for sensing cold response.
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Zhang D, Guo X, Xu Y, Li H, Ma L, Yao X, Weng Y, Guo Y, Liu CM, and Chong K
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- Adaptation, Physiological, Amino Acid Sequence, Base Sequence, Plant Proteins metabolism, Protein Conformation, Spectroscopy, Fourier Transform Infrared, Cold Temperature, Oryza enzymology, Oryza physiology, Plant Proteins chemistry, Plant Proteins genetics, Point Mutation genetics, Protein Kinases metabolism
- Abstract
Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular structure conformation level. Here, an OsCIPK7 mutant via TILLING procedure with a point mutation in the kinase domain showed increased chilling tolerance, which could be potentially used in the molecular breeding. We found that this point mutation of OsCIPK7 led to a conformational change in the activation loop of the kinase domain, subsequently with an increase of protein kinase activity, thus conferred an increased tolerance to chilling stress., (© 2019 Institute of Botany, Chinese Academy of Sciences.)
- Published
- 2019
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14. Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro .
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Wong DD, van Zuylen WJ, Hamilton ST, Steingruber M, Sonntag E, Marschall M, and Rawlinson WD
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- Cell Line, Cell Line, Tumor, Cytomegalovirus genetics, Drug Resistance, Viral genetics, Humans, Mutation genetics, Open Reading Frames genetics, Phosphorylation, Protein Kinases genetics, Virus Replication drug effects, Virus Replication genetics, Antiviral Agents pharmacology, Cytomegalovirus drug effects, Cytomegalovirus enzymology, Ganciclovir pharmacology, Protein Kinases metabolism
- Abstract
Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro ., (Copyright © 2019 American Society for Microbiology.)
- Published
- 2019
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15. An Integrative Analysis of Tumor Proteomic and Phosphoproteomic Profiles to Examine the Relationships Between Kinase Activity and Phosphorylation.
- Author
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Arshad OA, Danna V, Petyuk VA, Piehowski PD, Liu T, Rodland KD, and McDermott JE
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- Humans, Neoplasms metabolism, Phosphorylation, Proteomics, Phosphoproteins metabolism, Protein Kinases metabolism
- Abstract
Phosphorylation of proteins is a key way cells regulate function, both at the individual protein level and at the level of signaling pathways. Kinases are responsible for phosphorylation of substrates, generally on serine, threonine, or tyrosine residues. Though particular sequence patterns can be identified that dictate whether a residue will be phosphorylated by a specific kinase, these patterns are not highly predictive of phosphorylation. The availability of large scale proteomic and phosphoproteomic data sets generated using mass-spectrometry-based approaches provides an opportunity to study the important relationship between kinase activity, substrate specificity, and phosphorylation. In this study, we analyze relationships between protein abundance and phosphopeptide abundance across more than 150 tumor samples and show that phosphorylation at specific phosphosites is not well correlated with overall kinase abundance. However, individual kinases show a clear and statistically significant difference in correlation among known phosphosite targets for that kinase and randomly selected phosphosites. We further investigate relationships between phosphorylation of known activating or inhibitory sites on kinases and phosphorylation of their target phosphosites. Combined with motif-based analysis, this approach can predict novel kinase targets and show which subsets of a kinase's target repertoire are specifically active in one condition versus another., (© 2019 Arshad et al.)
- Published
- 2019
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16. LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.
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Bonello F, Hassoun SM, Mouton-Liger F, Shin YS, Muscat A, Tesson C, Lesage S, Beart PM, Brice A, Krupp J, Corvol JC, and Corti O
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- Adult, Aged, Benzodiazepinones pharmacology, Carbonyl Cyanide m-Chlorophenyl Hydrazone analogs & derivatives, Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology, Female, Fibroblasts drug effects, Fibroblasts pathology, Fluorescence Resonance Energy Transfer, Humans, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 antagonists & inhibitors, Male, Middle Aged, Mitochondria genetics, Mitochondria pathology, Mitophagy drug effects, Mutation, Parkinson Disease pathology, Phosphorylation, Primary Cell Culture, Pyrimidines pharmacology, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 genetics, Parkinson Disease genetics, Protein Kinases genetics, Ubiquitin-Protein Ligases genetics
- Abstract
Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2019
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17. Recent Advances in Protein Kinase Activity Analysis Based on Nanomaterials.
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Yan Z, Deng P, and Liu Y
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- Animals, Colorimetry, Electrochemical Techniques, Enzyme Activation, Humans, Phosphates chemistry, Phosphates metabolism, Phosphoproteins chemistry, Phosphoproteins metabolism, Phosphorylation, Photochemistry methods, Protein Binding, Protein Kinases metabolism, Signal Transduction, Biosensing Techniques, Nanostructures chemistry, Nanotechnology, Protein Kinases chemistry
- Abstract
Protein phosphorylation regulated by protein kinases, as well as their dephosphorylation, is one of the most common post-translational modifications, and plays important roles in physiological activities, such as intracellular signal communications, gene transcription, cell proliferation and apoptosis. Over-expression of protein kinases is closely associated with various diseases. Consequently, accurate detection of protein kinases activities and their relevant inhibitors screening is critically important, not only to the biochemical research, but also to the clinical diagnosis and therapy. Nanomaterials, taking advantage of large surface areas, as well as excellent electrical, catalytic, magnetic and optical properties, have been utilized as target concentrators, recognition components, signal transducer or amplification elements in protein kinase related assays. This review summarizes the recent representative works to highlight the applications of nanomaterials in different biosensor technologies for protein kinases activities detection and their inhibitors screening. First, different nanomaterials developed for phosphoprotein/phosphopeptide enrichment and phosphate recognition are introduced. Next, representative works are selected that mainly focus on the utilization of nanomaterials as signal transducer or amplification elements in various protein kinases sensing platforms, such as electrochemical, colorimetric, fluorescent, and mass spectroscopy-based approaches. Finally, the major challenges and perspectives of nanomaterials being applied in protein kinases related assays are discussed.
- Published
- 2019
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18. Kinase activity of SOBIR1 and BAK1 is required for immune signalling.
- Author
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van der Burgh AM, Postma J, Robatzek S, and Joosten MHAJ
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- Gene Expression Regulation, Plant, Phosphorylation genetics, Phosphorylation physiology, Plant Immunity genetics, Plants, Genetically Modified, Signal Transduction genetics, Signal Transduction physiology, Arabidopsis Proteins metabolism, Plant Immunity physiology, Protein Kinases metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Leucine-rich repeat-receptor-like proteins (LRR-RLPs) and LRR-receptor-like kinases (LRR-RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR-RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR-RLK Suppressor of BIR1-1/EVERSHED (SOBIR1/EVR) to form signalling-competent receptor complexes. Ligand perception by LRR-RLPs initiates recruitment of the co-receptor BRI1-Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR-RLP/SOBIR1 complex, thereby activating LRR-RLP-mediated immunity. We employed phosphorylation analysis of in planta-produced proteins, live cell imaging, gene silencing and co-immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses when overexpressed in planta, is highly phosphorylated. Moreover, in addition to the kinase activity of SOBIR1 itself, kinase-active BAK1 is essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered by the tomato LRR-RLP Cf-4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase-active BAK1. We argue that, in addition to the trans-autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling-competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP-mediated immunity., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)
- Published
- 2019
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19. EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis .
- Author
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Ding Y, Lv J, Shi Y, Gao J, Hua J, Song C, Gong Z, and Yang S
- Subjects
- Cell Membrane metabolism, Cold-Shock Response genetics, Cold-Shock Response physiology, Enzyme Activation genetics, Fatty Acids, Monounsaturated metabolism, Freezing, Gene Expression Regulation, Plant, Phosphorylation, Plants, Genetically Modified, Protein Processing, Post-Translational genetics, Signal Transduction, Acclimatization genetics, Arabidopsis enzymology, Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Arabidopsis Proteins physiology, Cold Temperature adverse effects, Protein Kinases metabolism, Protein Phosphatase 2C physiology
- Abstract
OST1 (open stomata 1) protein kinase plays a central role in regulating freezing tolerance in Arabidopsis ; however, the mechanism underlying cold activation of OST1 remains unknown. Here, we report that a plasma membrane-localized clade-E growth-regulating 2 (EGR2) phosphatase interacts with OST1 and inhibits OST1 activity under normal conditions. EGR2 is N-myristoylated by N-myristoyltransferase NMT1 at 22°C, which is important for its interaction with OST1. Moreover, myristoylation of EGR2 is required for its function in plant freezing tolerance. Under cold stress, the interaction of EGR2 and NMT1 is attenuated, leading to the suppression of EGR2 myristoylation in plants. Plant newly synthesized unmyristoylated EGR2 has decreased binding ability to OST1 and also interferes with the EGR2-OST1 interaction under cold stress. Consequently, the EGR2-mediated inhibition of OST1 activity is released. Consistently, mutations of EGRs cause plant tolerance to freezing, whereas overexpression of EGR2 exhibits decreased freezing tolerance. This study thus unravels a molecular mechanism underlying cold activation of OST1 by membrane-localized EGR2 and suggests that a myristoyl switch on EGR2 helps plants to adapt to cold stress., (© 2018 The Authors.)
- Published
- 2019
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20. A Temperature-Sensitive Misfolded bri1-301 Receptor Requires Its Kinase Activity to Promote Growth.
- Author
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Zhang X, Zhou L, Qin Y, Chen Y, Liu X, Wang M, Mao J, Zhang J, He Z, Liu L, and Li J
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Cell Membrane metabolism, Endoplasmic Reticulum metabolism, Mutation, Phenotype, Plants, Genetically Modified, Protein Folding, Protein Kinases genetics, Protein Stability, Temperature, Arabidopsis growth & development, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism, Protein Kinases chemistry, Protein Kinases metabolism
- Abstract
BRASSINOSTEROID-INSENSITIVE1 (BRI1) is a leucine-rich-repeat receptor-like kinase that functions as the cell surface receptor for brassinosteroids (BRs). Previous studies showed that BRI1 requires its kinase activity to transduce the extracellular BR signal into the nucleus. Among the many reported mutant bri1 alleles, bri1-301 is unique, as its glycine-989-to-isoleucine mutation completely inhibits its kinase activity in vitro but only gives rise to a weak dwarf phenotype compared with strong or null bri1 alleles, raising the question of whether kinase activity is essential for the biological function of BRI1. Here, we show that the Arabidopsis ( Arabidopsis thaliana ) bri1-301 mutant receptor exhibits weak BR-triggered phosphorylation in vivo and absolutely requires its kinase activity for the limited growth that occurs in the bri1-301 mutant. We also show that bri1-301 is a temperature-sensitive misfolded protein that is rapidly degraded in the endoplasmic reticulum and at the plasma membrane by yet unknown mechanisms. A temperature increase from 22°C to 29°C reduced the protein stability and biochemical activity of bri1-301, likely due to temperature-enhanced protein misfolding. The bri1-301 protein could be used as a model to study the degradation machinery for misfolded membrane proteins with cytosolic structural lesions and the plasma membrane-associated protein quality-control mechanism., (© 2018 American Society of Plant Biologists. All rights reserved.)
- Published
- 2018
- Full Text
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21. Aptamer based electrochemical assay for protein kinase activity by coupling hybridization chain reaction.
- Author
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Jia LP, Zhao RN, Wang LJ, Ma RN, Zhang W, Shang L, and Wang HS
- Subjects
- Aptamers, Nucleotide, Electrodes, Enzyme Activation drug effects, Enzyme Assays, Gold chemistry, Isoquinolines pharmacology, Limit of Detection, Nucleic Acid Hybridization, Sulfonamides pharmacology, Biosensing Techniques instrumentation, Biosensing Techniques methods, Electrochemical Techniques, Protein Kinases metabolism
- Abstract
The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au-S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 - 4 ×10
5 U L-1 ) and a low detection limit (1.5 U L-1 ) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
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22. Neurodegeneration in SCA14 is associated with increased PKCγ kinase activity, mislocalization and aggregation.
- Author
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Wong MMK, Hoekstra SD, Vowles J, Watson LM, Fuller G, Németh AH, Cowley SA, Ansorge O, Talbot K, and Becker EBE
- Subjects
- Adult, Aged, Autopsy, Catalytic Domain drug effects, Cerebellum pathology, Female, Humans, Induced Pluripotent Stem Cells pathology, Male, Middle Aged, Models, Biological, Mutation genetics, Protein Aggregation, Pathological genetics, Protein Kinase C genetics, Protein Kinase C metabolism, Nerve Degeneration enzymology, Nerve Degeneration etiology, Protein Aggregation, Pathological etiology, Protein Kinases metabolism, Protein Transport genetics, Spinocerebellar Ataxias complications, Spinocerebellar Ataxias genetics, Spinocerebellar Ataxias pathology
- Abstract
Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.
- Published
- 2018
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23. Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity.
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Hu Q, Wang Q, Jiang C, Zhang J, Kong J, and Zhang X
- Subjects
- Cyclic AMP-Dependent Protein Kinases analysis, Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors, Cyclic AMP-Dependent Protein Kinases metabolism, Drug Evaluation, Preclinical instrumentation, Drug Evaluation, Preclinical methods, Electrochemical Techniques instrumentation, Enzyme Assays instrumentation, Equipment Design, Hep G2 Cells, Humans, Limit of Detection, Peptides chemistry, Peptides metabolism, Phosphorylation, Polymerization, Protein Kinase Inhibitors pharmacology, Protein Kinases metabolism, Electrochemical Techniques methods, Enzyme Assays methods, Polymers chemistry, Protein Kinases analysis
- Abstract
Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr
4+ -phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL-1 . Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
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24. Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.
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Maryu G, Miura H, Uda Y, Komatsubara AT, Matsuda M, and Aoki K
- Subjects
- Biosensing Techniques, Fluorescence Resonance Energy Transfer, Humans, Luminescent Proteins genetics, Luminescent Proteins metabolism, Protein Kinases genetics, Genes, Reporter, Microscopy, Fluorescence, Protein Kinases metabolism
- Abstract
Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.
- Published
- 2018
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25. Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG.
- Author
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Ito T, Ishida S, and Takahashi Y
- Subjects
- Catalytic Domain genetics, Enzyme Activation genetics, Homeostasis, Phosphorylation, Plants, Genetically Modified, Protein Kinases genetics, Protein Processing, Post-Translational genetics, Serine genetics, Nicotiana genetics, Basic-Leucine Zipper Transcription Factors metabolism, Plant Proteins metabolism, Protein Kinases chemistry, Protein Kinases metabolism, Repressor Proteins metabolism, Serine metabolism, Nicotiana enzymology
- Abstract
Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.
- Published
- 2018
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26. Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity.
- Author
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Gongol B, Marin TL, Jeppson JD, Mayagoitia K, Shin S, Sanchez N, Kirsch WM, Vinters HV, Wilson CG, Ghribi O, and Soriano S
- Subjects
- Alzheimer Disease metabolism, Alzheimer Disease pathology, Animals, Cell Line, Tumor, Forkhead Box Protein O1 metabolism, Gene Expression Regulation drug effects, Humans, Intracellular Signaling Peptides and Proteins metabolism, Intracellular Space metabolism, Male, Mice, Phosphorylation drug effects, Rats, Amyloid beta-Protein Precursor genetics, Hormesis, Hydroxycholesterols pharmacology, Intracellular Space drug effects, Microtubule-Associated Proteins metabolism, Protein Kinases metabolism, Protein Serine-Threonine Kinases metabolism, Transcription, Genetic drug effects
- Abstract
The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.
- Published
- 2017
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27. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.
- Author
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Wilkes EH, Casado P, Rajeeve V, and Cutillas PR
- Subjects
- Algorithms, Cell Line, Tumor, Cell Survival drug effects, Epidermal Growth Factor pharmacology, Humans, Insulin-Like Growth Factor I pharmacology, Models, Biological, Reproducibility of Results, Protein Kinases metabolism, Proteomics methods
- Abstract
Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
- Full Text
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28. The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.
- Author
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Ando M, Fiesel FC, Hudec R, Caulfield TR, Ogaki K, Górka-Skoczylas P, Koziorowski D, Friedman A, Chen L, Dawson VL, Dawson TM, Bu G, Ross OA, Wszolek ZK, and Springer W
- Subjects
- Fibroblasts metabolism, HeLa Cells, Humans, Mitochondria metabolism, Protein Stability, Ubiquitin metabolism, Ubiquitin-Protein Ligases genetics, Ubiquitination genetics, Mutation genetics, Parkinson Disease genetics, Protein Kinases genetics, Ubiquitin genetics
- Abstract
Background: Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future., Methods: Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays., Results: Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity., Conclusions: Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients' fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.
- Published
- 2017
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29. Quantitative analysis of protein orientation in membrane environments by kinase activity.
- Author
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Xiong W, Quan C, Zhang X, Wang L, Liu B, Jin L, and Fan S
- Subjects
- Bacterial Proteins analysis, Membrane Proteins analysis, Phosphorylation, Protein Kinases analysis, Proteolipids metabolism, Quorum Sensing, Sulfhydryl Compounds chemistry, Time Factors, Bacterial Proteins metabolism, Biocatalysis, Enzyme Assays, Membrane Proteins metabolism, Protein Kinases metabolism, Staphylococcus aureus enzymology
- Abstract
AgrC is an integral membrane receptor protein with histidine kinase activity in the accessory gene regulator (agr) quorum-sensing system of Staphylococcus aureus. In this study, proteoliposomes were used as a model to investigate AgrC orientation. Many approaches have been described to determine membrane protein orientation, but they are often complicated and time consuming. In this study, AgrC orientation in liposomes was determined by thiol-reactive reagent labeling and a kinase activity assay. Our results suggest use of a kinase activity assay could get an accurate percentage of functional protein orientation and only cost nearly one-sixth of the time compared with the method based on thiol-reactive reagent labeling. We present an effective and rapid method for determining the orientation of membrane protein kinases like AgrC., (Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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30. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.
- Author
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Janeček M, Rossmann M, Sharma P, Emery A, Huggins DJ, Stockwell SR, Stokes JE, Tan YS, Almeida EG, Hardwick B, Narvaez AJ, Hyvönen M, Spring DR, McKenzie GJ, and Venkitaraman AR
- Subjects
- Cell Line, Tumor, HeLa Cells, Humans, Mitosis drug effects, Neoplasm Proteins metabolism, Phosphoproteins metabolism, Protein Binding drug effects, Spindle Apparatus drug effects, Aurora Kinase A metabolism, Cell Cycle Proteins metabolism, Microtubule-Associated Proteins metabolism, Nuclear Proteins metabolism, Protein Interaction Maps drug effects, Protein Kinases metabolism, Small Molecule Libraries pharmacology
- Abstract
The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.
- Published
- 2016
- Full Text
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31. A signal "on" photoelectrochemical biosensor for assay of protein kinase activity and its inhibitor based on graphite-like carbon nitride, Phos-tag and alkaline phosphatase.
- Author
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Yin H, Sun B, Dong L, Li B, Zhou Y, and Ai S
- Subjects
- Electrodes, Enzyme Activation, Equipment Design, Equipment Failure Analysis, Graphite chemistry, Photometry instrumentation, Protein Kinase Inhibitors analysis, Protein Kinases analysis, Alkaline Phosphatase chemistry, Biosensing Techniques instrumentation, Conductometry instrumentation, Nitriles chemistry, Protein Kinase Inhibitors chemistry, Protein Kinases chemistry, Pyridines chemistry
- Abstract
A highly sensitive and selective photoelectrochemical (PEC) biosensor is fabricated for the detection of protein kinase activity based on visible-light active graphite-like carbon nitride (g-C3N4) and the specific recognition utility of Phos-tag for protein kinase A (PKA)-induced phosphopeptides. For assembling the substrate peptides, g-C3N4 and gold nanoparticles (g-C3N4-AuNPs) complex is synthesized and characterized. When the immobilized peptides on g-C3N4-AuNPs modified ITO electrode are phosphorylated under PKA catalysis, they can be specifically identified and binded with biotin functionalized Phos-tag (Phos-tag-biotin) in the presence of Zn(2+). Then, through the specific interaction between biotin and avidin, avidin functionalized alkaline phosphatase (avidin-ALP) is further assembled to catalyze its substrate of l-ascorbic acid-2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting an increased photocurrent compared with the absence of phosphorylation event. Based on the specific identification effect of Phos-tag, the fabricated biosensor presents excellent selectivity for capturing the phosphorylated serine residues in the substrate peptides. With the good photoactivity of g-C3N4 and ALP-catalyzed signal amplification, the fabricated biosensor presents high sensitivity and low detection limit (0.015 unit/mL, S/N = 3) for PKA. The applicability of this PEC biosensor is further testified by the evaluation of PKA inhibition by HA-1077 with the IC50 value of 1.18μM. This new strategy is also successfully applied to detect the change of PKA activity in cancer cell lysate with and without drug stimulation. Therefore, the developed PEC method has great potential in screening of kinase inhibitors and highly sensitive detection of kinase activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
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32. High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes.
- Author
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Anderson JC, Willey CD, Mehta A, Welaya K, Chen D, Duarte CW, Ghatalia P, Arafat W, Madan A, Sudarshan S, Naik G, Grizzle WE, Choueiri TK, and Sonpavde G
- Subjects
- Carcinoma, Renal Cell pathology, Cluster Analysis, Disease Progression, Female, Gene Expression Regulation, Neoplastic, Humans, Kidney Neoplasms pathology, Male, Middle Aged, Protein Kinases genetics, Treatment Outcome, Carcinoma, Renal Cell genetics, Gene Expression Profiling, Kidney Neoplasms genetics, Protein Kinases metabolism
- Abstract
Despite the widespread use of kinase-targeted agents in clear cell renal cell carcinoma (CC-RCC), comprehensive kinase activity evaluation (kinomic profiling) of these tumors is lacking. Thus, kinomic profiling of CC-RCC may assist in devising a classification system associated with clinical outcomes, and help identify potential therapeutic targets. Fresh frozen CC-RCC tumor lysates from 41 clinically annotated patients who had localized disease at diagnosis were kinomically profiled using the PamStation®12 high-content phospho-peptide substrate microarray system (PamGene International). Twelve of these patients also had matched normal kidneys available that were also profiled. Unsupervised hierarchical clustering and supervised comparisons based on tumor vs. normal kidney and clinical outcome (tumor recurrence) were performed and coupled with advanced network modeling and upstream kinase prediction methods. Unsupervised clustering analysis of localized CC-RCC tumors identified 3 major kinomic groups associated with inflammation (A), translation initiation (B), and immune response and cell adhesions (C) processes. Potential driver kinases implicated include PFTAIRE (PFTK1), PKG1, and SRC, which were identified in groups A, B, and C, respectively. Of the 9 patients who had tumor recurrence, only one was found in Group B. Supervised analysis showed decreased kinase activity of CDK1 and RSK1-4 substrates in those which progressed compared to others. Twelve tumors with matching normal renal tissue implicated increased PIM's and MAPKAPK's in tumors compared to adjacent normal renal tissue. As such, comprehensive kinase profiling of CC-RCC tumors could provide a functional classification strategy for patients with localized disease and identify potential therapeutic targets.
- Published
- 2015
- Full Text
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33. Lack of Evidence for PKM2 Protein Kinase Activity.
- Author
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Hosios AM, Fiske BP, Gui DY, and Vander Heiden MG
- Subjects
- Adenosine Triphosphate metabolism, Animals, Carrier Proteins genetics, Cell Line, Tumor, Cell Proliferation physiology, Cells, Cultured, Gene Deletion, Glycolysis, Humans, Membrane Proteins deficiency, Membrane Proteins genetics, Mice, Phosphoenolpyruvate metabolism, Phosphorylation, Protein Kinases deficiency, Protein Kinases genetics, Pyruvate Kinase deficiency, Pyruvate Kinase genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Thyroid Hormones deficiency, Thyroid Hormones genetics, Thyroid Hormone-Binding Proteins, Carrier Proteins metabolism, Membrane Proteins metabolism, Protein Kinases metabolism, Pyruvate Kinase metabolism, Thyroid Hormones metabolism
- Abstract
The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [(32)P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [(32)P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
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34. Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.
- Author
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Lim B, Park JI, Lee KJ, Lee JW, Kim TW, and Kim YP
- Subjects
- Adenosine Triphosphate pharmacology, Enzyme Assays, Magnesium pharmacology, Peptides metabolism, Phosphorylation drug effects, Substrate Specificity drug effects, Time Factors, Biosensing Techniques methods, Fluorescence Resonance Energy Transfer methods, Nanoparticles chemistry, Protein Kinases metabolism, Quantum Dots chemistry, Zinc chemistry
- Abstract
We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.
- Published
- 2015
- Full Text
- View/download PDF
35. Multiple signal amplification electrogenerated chemiluminescence biosensors for sensitive protein kinase activity analysis and inhibition.
- Author
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Wang Z, Yan Z, Sun N, and Liu Y
- Subjects
- DNA chemistry, Gold, Humans, Hydrogen Peroxide chemistry, Hypoxanthine chemistry, Limit of Detection, Luminol chemistry, Protein Kinase Inhibitors chemistry, Protein Kinases chemistry, Xanthine Oxidase chemistry, Biosensing Techniques, Metal Nanoparticles chemistry, Protein Kinases isolation & purification
- Abstract
A novel electrogenerated chemiluminescence (ECL) biosensor was built for the detection of kinase activity based on multiple signal amplification nanoprobes. In this strategy, the Xanthine oxidase (XOD) and 5'-phosphate group end DNA conjugated AuNPs was integrated with the phosphorylated peptide by Zr(4+). The XOD on gold nanoparticles can catalyze dissolved oxygen to produce H2O2 in the presence of hypoxanthine (HA) which acts as a coreactor for luminol ECL reaction. In addition, due to the excellent catalytic activity of gold nanoparticle toward luminol ECL reaction and its large surface area that can accommodate large number of XOD and DNA on the surface, the ECL signal of luminol was significantly amplified, affording a highly sensitive ECL analysis of kinase activity. The as-proposed biosensor presents a low detection limit of 0.09 U mL(-1) for protein kinase A (PKA) activity, wide linear range (from 0.1 to 10 U mL(-1)) and excellent stability even in serum samples. This biosensor can also be applied for quantitative kinase inhibitor evaluation. The robust ECL biosensor provides a valuable tool for the high throughput assay in the applications of clinic diagnostic and therapeutic., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
36. The extracytoplasmic linker peptide of the sensor protein SaeS tunes the kinase activity required for staphylococcal virulence in response to host signals.
- Author
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Liu Q, Cho H, Yeo WS, and Bae T
- Subjects
- Amino Acid Sequence, Animals, Bacterial Proteins, Blotting, Western, Flow Cytometry, Gene Expression Regulation, Bacterial physiology, Humans, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Neutrophils microbiology, Polymerase Chain Reaction, Protein Kinases genetics, Staphylococcal Infections genetics, Staphylococcus aureus genetics, Virulence physiology, Protein Kinases metabolism, Staphylococcal Infections metabolism, Staphylococcus aureus pathogenicity
- Abstract
Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS's basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases.
- Published
- 2015
- Full Text
- View/download PDF
37. TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions.
- Author
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Brandao K, Deason-Towne F, Zhao X, Perraud AL, and Schmitz C
- Subjects
- Animals, B-Lymphocytes metabolism, Cell Line, Cell Membrane metabolism, HEK293 Cells, Homeostasis, Humans, Immunoblotting, Microscopy, Confocal, Models, Molecular, Mutation, Phosphorylation, Protein Kinases chemistry, Protein Kinases genetics, Protein Multimerization, Protein Serine-Threonine Kinases, Protein Structure, Quaternary, Protein Transport, Serine genetics, Serine metabolism, TRPM Cation Channels chemistry, TRPM Cation Channels genetics, Cell Proliferation, Magnesium metabolism, Protein Kinases metabolism, TRPM Cation Channels metabolism
- Abstract
The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg(2+) homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other's cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg(2+)-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg(2+), but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect-a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg(2+)-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg(2+)-uptake is thus important knowledge.
- Published
- 2014
- Full Text
- View/download PDF
38. Microfluidic bead-based sensing platform for monitoring kinase activity.
- Author
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Lee SH, Rhee HW, van Noort D, Lee HJ, Park HH, Shin IS, Hong JI, and Park TH
- Subjects
- Enzyme Assays instrumentation, Equipment Design, HeLa Cells, Humans, Peptides metabolism, Phosphorylation, Biosensing Techniques instrumentation, Microfluidic Analytical Techniques instrumentation, Protein Kinases metabolism
- Abstract
Protein kinases control cellular functions by regulating protein phosphorylation. Monitoring protein kinase activity is essential for medical diagnosis and drug screening. Here, we present a novel microfluidic device for performing simple and versatile protein kinase assays, which utilizes a microbead-based chemosensor. An automatic mix-and-measure technique was achieved using integrated pneumatic valves. After mixing each reagent for the kinase assay, the mixture was transferred to the sensing chamber. Then, phosphorylated and fluorescence-labeled peptides were captured and detected by the chemosensor. A fluorescence signal was observed depending on the presence of the kinase. Furthermore, activities of various kinases in the cell lysate and the inhibitory effect of specific chemicals on the kinases were monitored. These results indicate that chemosensor-based microfluidic chips can be developed as a versatile kinase assay system., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
39. A gold nanoparticles colorimetric assay for label-free detection of protein kinase activity based on phosphorylation protection against exopeptidase cleavage.
- Author
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Zhou J, Xu X, Liu X, Li H, Nie Z, Qing M, Huang Y, and Yao S
- Subjects
- Exopeptidases chemistry, Gold chemistry, Humans, Limit of Detection, Metal Nanoparticles chemistry, Phosphorylation, Protein Kinases chemistry, Biosensing Techniques methods, Colorimetry methods, Protein Kinases isolation & purification
- Abstract
Protein kinases are significant regulators in the cell signaling pathways, and it is still greatly desirable to achieve simple and quick kinase detection. Herein, we present a novel colorimetric gold nanoparticles (AuNPs)/peptide platform for probing the activity and inhibition of protein kinases based on phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. This AuNPs/peptide platform can easily monitor the kinase activity by a UV-vis spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the cAMP-dependent protein kinase (PKA) activity with a low detection limit of 0.232 mU/µL and assessment of kinase inhibition by H-89 with an IC50 value of 18.13 nM. The assay was also successfully put into practice for the detection of kinase activity in cell lysate. Because of its label-free, homogenous and colorimetric merits, the proposed assay presents great potential in high-throughput screening for kinase-targeted drug discovery., (© 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
40. E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.
- Author
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Pruneda JN, Smith FD, Daurie A, Swaney DL, Villén J, Scott JD, Stadnyk AW, Le Trong I, Stenkamp RE, Klevit RE, Rohde JR, and Brzovic PS
- Subjects
- Animals, Cell Line, Crystallography, X-Ray, Humans, Mice, Models, Molecular, Protein Conformation, Protein Kinases chemistry, Protein Multimerization, Ubiquitin chemistry, Ubiquitin-Conjugating Enzymes chemistry, Virulence Factors chemistry, Protein Kinases metabolism, Shigella flexneri metabolism, Ubiquitin metabolism, Ubiquitin-Conjugating Enzymes metabolism, Virulence Factors metabolism
- Abstract
Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.
- Published
- 2014
- Full Text
- View/download PDF
41. Light-induced conformational changes of LOV1 (light oxygen voltage-sensing domain 1) and LOV2 relative to the kinase domain and regulation of kinase activity in Chlamydomonas phototropin.
- Author
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Okajima K, Aihara Y, Takayama Y, Nakajima M, Kashojiya S, Hikima T, Oroguchi T, Kobayashi A, Sekiguchi Y, Yamamoto M, Suzuki T, Nagatani A, Nakasako M, and Tokutomi S
- Subjects
- Chlamydomonas reinhardtii genetics, Phototropins genetics, Phototropins metabolism, Protein Kinases genetics, Protein Kinases metabolism, Protein Structure, Tertiary, Scattering, Small Angle, Signal Transduction physiology, X-Ray Diffraction, Chlamydomonas reinhardtii enzymology, Light, Models, Molecular, Phototropins chemistry, Protein Kinases chemistry, Signal Transduction radiation effects
- Abstract
Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.
- Published
- 2014
- Full Text
- View/download PDF
42. Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone.
- Author
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Sharma P, Stagge S, Bekker M, Bettenbrock K, and Hellingwerf KJ
- Subjects
- Escherichia coli, Escherichia coli Proteins genetics, Membrane Proteins genetics, Oxidation-Reduction, Phosphorylation, Protein Kinases genetics, Signal Transduction genetics, Ubiquinone genetics, Vitamin K 2 metabolism, Escherichia coli Proteins metabolism, Gene Expression Regulation, Bacterial, Membrane Proteins metabolism, Protein Kinases metabolism, Ubiquinone metabolism, Vitamin K 2 analogs & derivatives
- Abstract
Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.
- Published
- 2013
- Full Text
- View/download PDF
43. Light-gated integrator for highlighting kinase activity in living cells.
- Author
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Lin, Wei, Phatarphekar, Abhishek, Zhong, Yanghao, Liu, Longwei, Kwon, Hyung-Bae, Gerwick, William H., Wang, Yingxiao, Mehta, Sohum, and Zhang, Jin
- Subjects
ONCOGENIC proteins ,PROTEIN kinases ,HIGH throughput screening (Drug development) ,TRANSIENT analysis ,BIOSENSORS - Abstract
Protein kinases are key signaling nodes that regulate fundamental biological and disease processes. Illuminating kinase signaling from multiple angles can provide deeper insights into disease mechanisms and improve therapeutic targeting. While fluorescent biosensors are powerful tools for visualizing live-cell kinase activity dynamics in real time, new molecular tools are needed that enable recording of transient signaling activities for post hoc analysis and targeted manipulation. Here, we develop a light-gated kinase activity coupled transcriptional integrator (KINACT) that converts dynamic kinase signals into "permanent" fluorescent marks. KINACT enables robust monitoring of kinase activity across scales, accurately recording subcellular PKA activity, highlighting PKA activity distribution in 3D cultures, and identifying PKA activators and inhibitors in high-throughput screens. We further leverage the ability of KINACT to drive signaling effector expression to allow feedback manipulation of the balance of Gα
s R201C -induced PKA and ERK activation and dissect the mechanisms of oncogenic G protein signaling. Real-time biosensors have been developed for visualizing kinase activity dynamics, but they are confronted with many challenges. Here, the authors develop a light-gated KINACT integrator for post hoc analysis of transient kinase activity and signalling manipulation. [ABSTRACT FROM AUTHOR]- Published
- 2024
- Full Text
- View/download PDF
44. Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue.
- Author
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Ziwei Zhao, Brooks, David, Yungui Guo, and Geisbrecht, Erika R.
- Subjects
- *
PROTEIN kinases , *DIGITAL image processing , *GENETIC mutation , *INSECT larvae , *MUSCLES , *ANIMAL experimentation , *CELLULAR signal transduction , *GENE expression , *RESEARCH funding , *DESCRIPTIVE statistics , *INSECTS , *DATA analysis software , *BIOLOGICAL assay , *LIGATURE (Surgery) , *PHOSPHORYLATION - Abstract
Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phosphomimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
45. Differentiating Benign from Malignant Thyroid Tumors by Kinase Activity Profiling and Dabrafenib BRAF V600E Targeting.
- Author
-
Hilhorst, Riet, van den Berg, Adrienne, Boender, Piet, van Wezel, Tom, Kievits, Tim, de Wijn, Rik, Ruijtenbeek, Rob, Corver, Willem E., and Morreau, Hans
- Subjects
- *
PROTEIN kinases , *DRUG efficacy , *GENETIC mutation , *SEQUENCE analysis , *THYROID gland tumors , *PAPILLARY carcinoma , *PROTEIN kinase inhibitors , *DIFFERENTIAL diagnosis , *ANTINEOPLASTIC agents , *PROTEIN microarrays , *GRAVES' disease , *TRANSFERASES , *SORAFENIB , *DESCRIPTIVE statistics , *EVALUATION - Abstract
Simple Summary: Recurrent non-medullary thyroid cancer (NMTC) is difficult to treat and therapy options are limited. Of the available compounds, serine/threonine kinase (STK) inhibitors are currently widely used. However, this form of targeted therapy is not always effective and additional response-related biological information may improve both accuracy and efficacy. Using in-depth STK activity profiling, in this study, we aimed to determine whether benign and malignant thyroid tumors can be differentiated based on these profiles. In addition, we analyzed the impact of the BRAF V600E-specific inhibitor dabrafenib, as well as the generic RAF inhibitors sorafenib and regorafenib, in a subgroup of BRAF V600E and non-BRAF V600E papillary thyroid carcinomas. We demonstrate that STK activity profiling can differentiate benign from malignant thyroid tumors. Furthermore, the BRAF V600E-specific inhibitor dabrafenib can distinguish BRAF V600E from non-BRAF V600E papillary thyroid carcinomas. We conclude that STK activity profiling is beneficial when the goal is to differentiate benign from malignant thyroid tumors. In addition, this approach aids in the selection of likely effective (novel) kinase inhibitors for treatment of recurrent thyroid and other cancers. Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
46. Ubiquitin negatively regulates ABA responses by inhibiting SnRK2.2 and SnRK2.3 kinase activity in Arabidopsis.
- Author
-
Shao, Zhengyu, Yang, Shuhua, Gu, Yinghui, Guo, Yan, Zhou, Huapeng, and Yang, Yongqing
- Subjects
- *
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
- Full Text
- View/download PDF
47. Bumping up kinase activity with an ATP-derived neo-substrate.
- Author
-
Kleiner RE and Kapoor TM
- Subjects
- Animals, Humans, Mitochondria metabolism, Parkinson Disease pathology, Protein Kinases genetics, Protein Kinases metabolism
- Abstract
Pharmacologic agents capable of increasing kinase function would be useful for treating diseases associated with reduced kinase activity, such as inherited forms of Parkinson's disease. In this issue, Hertz et al. report an innovative approach for activating the Parkinson's-associated kinase PINK1 in cells with an ATP-derived neo-substrate., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
48. CK1δ kinase activity is modulated by Chk1-mediated phosphorylation.
- Author
-
Bischof J, Randoll SJ, Süßner N, Henne-Bruns D, Pinna LA, and Knippschild U
- Subjects
- Amino Acid Sequence, Animals, Casein Kinase Idelta metabolism, Cell Line, Tumor, Checkpoint Kinase 1, Fibroblasts cytology, Humans, Molecular Sequence Data, Mutation, Phosphorylation, Protein Kinases metabolism, Rats, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Serine genetics, Serine metabolism, Signal Transduction, Threonine genetics, Threonine metabolism, Casein Kinase Idelta genetics, Fibroblasts metabolism, Gene Expression Regulation, Protein Kinases genetics
- Abstract
CK1δ, a member of the casein kinase 1 family, is involved in the regulation of various cellular processes and has been associated with the pathophysiology of neurodegenerative diseases and cancer. Therefore recently, interest in generating highly specific inhibitors for personalized therapy has increased enormously. However, the efficacy of newly developed inhibitors is affected by the phosphorylation state of CK1δ. Cellular kinases phosphorylating CK1δ within its C-terminal domain have been identified but still more information regarding the role of site-specific phosphorylation in modulating the activity of CK1δ is required. Here we show that Chk1 phosphorylates rat CK1δ at serine residues 328, 331, 370, and threonine residue 397 as well as the human CK1δ transcription variants 1 and 2. CK1δ mutant proteins bearing one, two or three mutations at these identified phosphorylation sites exhibited significant differences in their kinetic properties compared to wild-type CK1δ. Additionally, CK1δ co-precipitates with Chk1 from HT1080 cell extracts and activation of cellular Chk1 resulted in a significant decrease in cellular CK1δ kinase activity. Taken together, these data point towards a possible regulatory relationship between Chk1 and CK1δ.
- Published
- 2013
- Full Text
- View/download PDF
49. The amplitude and inactivation properties of the delayed potassium currents are regulated by protein kinase activity in hair cells of the frog semicircular canals.
- Author
-
Martini M, Canella R, Fesce R, and Rossi ML
- Subjects
- Animals, Cadmium pharmacology, Calcium metabolism, Carbazoles pharmacology, Cations, Divalent, Hair Cells, Auditory cytology, Hair Cells, Auditory drug effects, Ion Transport drug effects, Isoquinolines pharmacology, Membrane Potentials drug effects, Patch-Clamp Techniques, Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Semicircular Canals cytology, Semicircular Canals drug effects, Sulfonamides pharmacology, Time Factors, Hair Cells, Auditory metabolism, Membrane Potentials physiology, Potassium metabolism, Potassium Channels metabolism, Protein Kinases metabolism, Rana esculenta physiology, Semicircular Canals metabolism
- Abstract
In hair cells dissected from the frog crista ampullaris, the combination of a calcium-dependent (IKCa) and a purely voltage-dependent component (IKV) gives rise to the delayed potassium current complex (IKD). These currents have been recently reported to display slow depolarization-induced inactivation and biphasic inactivation removal by hyperpolarization. The amplitude and inactivation kinetics of both IKCa and IKV are drastically modulated by a previously unrecognized mechanism of protein phosphorylation (sensitive to kinase inhibitors H89 and KT5823), which does not interfere with the transient potassium current (IA) or the calcium current (ICa). IKD amplitude was stable in cells patched with pipettes containing 8 mM ATP or under perforated-patch; under these conditions, a 10 min treatment with 10 µM H89 or 1-10 µM KT5823 reduced IKD amplitude by a mean of 67% at +40 mV. Similarly affected was the isolated IKV component (ICa blocked with Cd(2+)). Thus, a large potassium conductance can be activated by depolarization, but it is made available to the cell to a variable extent that depends on membrane potential and protein kinase activity. The total gKD ranged 4.6-44.0 nS in control cells, according to the level of steady-state inactivation, and was reduced to 1.4-2.7 nS after protein kinase inhibition. When sinusoidal membrane potential changes in the -70/-10 mV range were applied, to mimic receptor response to hair bundle deflection, IKD proved the main current dynamically activated and the only one regulated by PK: H89 decreased the total outward charge during each cycle by 60%. Phosphorylation appears to control both the amount of IKCa and IKV conductance activated by depolarization and the fraction thereof which can be rescued by removal of inactivation. The balance between the depolarizing transduction current and the repolarizing potassium current, and eventually the transmitter release at the cytoneural junction, are therefore modulated by a phosphorylation-mediated process.
- Published
- 2013
- Full Text
- View/download PDF
50. A phytotoxin Solanapyrone-A downregulates calcium-dependent protein kinase activity in potato.
- Author
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Hassan A, Hatsugai N, and Shah MM
- Subjects
- Calcium pharmacology, Solanum lycopersicum drug effects, Magnesium pharmacology, Phosphorylation, Plant Leaves drug effects, Recombinant Fusion Proteins metabolism, Down-Regulation drug effects, Naphthalenes pharmacology, Protein Kinases metabolism, Pyrones pharmacology, Solanum tuberosum drug effects, Solanum tuberosum enzymology
- Abstract
We previously demonstrated that alternaric acid, a host-specific toxin produced by the plant pathogenic fungus Alternaria solani, in the presence of Ca(2+) and Mg(2+), stimulated in vitro phosphorylation of His-tagged calcium-dependent protein kinase 2 from potato cultivar Rishiri (RiCDPK2). Herein, we report that Solanapyrone-A (SpA), a non-host-specific toxin produced by A. solani, inhibited the phosphorylation of RiCDPK2 in the presence of Ca(2+) and Mg(2+). However, SpA stimulated RiCDPK2 phosphorylation in the absence of these cations. Based on the current findings, we suggest that RiCDPK2 may mediate SpA-induced signaling independent of Ca(2+) and Mg(2+), leading to a compatible interaction between potato and A. solani.
- Published
- 2013
- Full Text
- View/download PDF
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