The Experts below are selected from a list of 60750 Experts worldwide ranked by ideXlab platform
Nobuo Nomura - One of the best experts on this subject based on the ideXlab platform.
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High-throughput Kinase Assay based on surface plasmon resonance.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Hiroyuki Takeda, Naoki Goshima, Nobuo NomuraAbstract:We have designed a novel high-throughput (HTP) Kinase Assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the Kinase Assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute to improved data reliability, and optimization of the procedure for an array-based apparatus achieved multiple sample processing (1,000 samples/day).
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High-throughput Kinase Assay based on surface plasmon resonance suitable for native protein substrates.
Analytical biochemistry, 2006Co-Authors: Hiroyuki Takeda, Naoki Goshima, Akiko Fukumoto, Aya Miura, Nobuo NomuraAbstract:We report a novel in vitro high-throughput (HTP) Kinase Assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope Assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine Kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as Kinase activity screening, Kinase substrate profiling, and Kinase HTP screening of Kinase inhibitors.
Hiroyuki Takeda - One of the best experts on this subject based on the ideXlab platform.
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High-throughput Kinase Assay based on surface plasmon resonance.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Hiroyuki Takeda, Naoki Goshima, Nobuo NomuraAbstract:We have designed a novel high-throughput (HTP) Kinase Assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the Kinase Assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute to improved data reliability, and optimization of the procedure for an array-based apparatus achieved multiple sample processing (1,000 samples/day).
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High-throughput Kinase Assay based on surface plasmon resonance suitable for native protein substrates.
Analytical biochemistry, 2006Co-Authors: Hiroyuki Takeda, Naoki Goshima, Akiko Fukumoto, Aya Miura, Nobuo NomuraAbstract:We report a novel in vitro high-throughput (HTP) Kinase Assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope Assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine Kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as Kinase activity screening, Kinase substrate profiling, and Kinase HTP screening of Kinase inhibitors.
W. Andy Tao - One of the best experts on this subject based on the ideXlab platform.
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Identification of the Direct Substrates of the ABL Kinase via Kinase Assay Linked Phosphoproteomics with Multiple Drug Treatments.
Journal of proteome research, 2019Co-Authors: Justine V. Arrington, Liang Xue, Wen-horng Wang, Robert L. Geahlen, W. Andy TaoAbstract:Ableson tyrosine Kinase (ABL) plays essential roles in cell differentiation, division, adhesion, and stress response. However, fusion of the breakpoint cluster region (BCR) to ABL produces constitutive Kinase activity that causes chronic myelogenous leukemia (CML). Small molecule tyrosine Kinase inhibitors (TKIs) such as imatinib revolutionized the treatment of CML and other cancers, but acquired resistance to these inhibitors is rising. Thus, careful dissection of ABL signaling pathways is needed to find novel drug targets. Here we present a refined proteomic approach for elucidation of direct Kinase substrates called Kinase Assay linked phosphoproteomics (KALIP). Our strategy integrates in vitro Kinase Assays at both the peptide and protein levels with quantitative tyrosine phosphoproteomics in response to treatment by multiple TKIs. Utilizing multiple TKIs permits elimination of off-target effects of these drugs, and overlapping the in vivo and in vitro data sets allows us to define a list of the most ...
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Identification of Plant Kinase Substrates Based on Kinase Assay-Linked Phosphoproteomics.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Chuan-chih Hsu, Justine V. Arrington, Liang Xue, W. Andy TaoAbstract:Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein Kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of Kinases is important for dissecting Kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. Despite the fact that thousands of Kinase-substrate networks have been identified in mammalian cells, the downstream substrates of important plant Kinases are still elusive. Moreover, it remains challenging to identify bona fide Kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant Kinase-substrate cascades. Here, we describe a proteomic strategy termed Kinase Assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant Kinases with high sensitivity and a low false-positive rate.
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Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Liang Xue, Justine V. Arrington, W. Andy TaoAbstract:Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein Kinases have become potential drug targets for treating a wide variety of diseases. Identification of Kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide Kinase substrates has remained challenging, necessitating the development of new methods and techniques. The Kinase Assay linked phosphoproteomics (KALIP) approach integrates in vitro Kinase Assays with global phosphoproteomics experiments to identify the direct substrates of protein Kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for Kinase substrate screening.
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Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics
Molecular & cellular proteomics : MCP, 2013Co-Authors: Liang Xue, Robert L. Geahlen, W. Andy TaoAbstract:Protein Kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of Kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide Kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying Kinase specificity and direct substrates in high throughput. This approach includes an in vitro Kinase Assay-based substrate screening and an endogenous Kinase dependent phosphorylation profiling. In the in vitro Kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the Kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the Kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the Kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein Kinase Assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine Kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine Kinases.
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Sensitive Kinase Assay linked with phosphoproteomics for identifying direct Kinase substrates
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Liang Xue, Wen-horng Wang, Anton Iliuk, Jacob A. Galan, Michael Hans, Robert L. Geahlen, W. Andy TaoAbstract:Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein Kinases. Here we describe an integrated proteomic strategy, termed Kinase Assay linked with phosphoproteomics, which combines a sensitive Kinase reaction with endogenous Kinase-dependent phosphoproteomics to identify direct substrates of protein Kinases. The unique in vitro Kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular Kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the Kinase is either active or inhibited reveals new candidate protein substrates. The Kinase Assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine Kinase (Syk), a protein-tyrosine Kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal Kinase activity.
Kurt W. Vogel - One of the best experts on this subject based on the ideXlab platform.
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Multiplexing terbium- and europium-based TR-FRET readouts to increase Kinase Assay capacity.
Journal of biomolecular screening, 2010Co-Authors: Robert A. Horton, Kurt W. VogelAbstract:Identification and characterization of Kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single Kinase, and hits from that screen are then profiled against other Kinases to determine specificity. By developing Kinase Assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop Assays for 2 Kinases that can be performed simultaneously in the same well. This not only increases the information content provided per Assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed Assays for 2 examples systems: EGFR and IKKβ, as well as lipid Kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed Assays to characterize selective Kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single Kinase Assays performed separately. (Journal of Biomolecular Screening 2010:1008-1015) Key w ords: TR-FRET, Kinase Assay, mTOR, homogeneous time-resolved fluorescence (HTRF), time-resolved fluorescence energy transfer (TRET)
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development and applications of a broad coverage tr fret based Kinase binding Assay platform
Journal of Biomolecular Screening, 2009Co-Authors: Connie S Lebakken, Steven M Riddle, Upinder Singh, Jack W Frazee, Hildegard C Eliason, Yi Gao, Laurie Reichling, Bryan D Marks, Kurt W. VogelAbstract:The expansion of Kinase Assay technologies over the past decade has mirrored the growing interest in Kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based met...
Pierre Colas - One of the best experts on this subject based on the ideXlab platform.
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development of a cdk10 cycm in vitro Kinase screening Assay and identification of first small molecule inhibitors
Frontiers in Chemistry, 2020Co-Authors: Thomas Robert, Roxane Guichaoua, Tomer Yaron, Stéphane Bach, Lewis Cantley, Jared L Johnson, Pierre ColasAbstract:Cyclin-dependent Kinases (CDKs) constitute a family of 20 serine/threonine protein Kinases that play pivotal roles in the regulation of numerous important molecular and cellular processes. CDKs have long been considered promising therapeutic targets in a variety of pathologies, and the recent therapeutic success of CDK4/6 inhibitors in breast cancers has renewed interest in their therapeutic potential. Small-molecule inhibitors have been identified for every human CDK, except for CDK10. The only recent discovery of an activating cyclin (CycM) for CDK10 enabled us to identify its first phosphorylation substrates and gain insights into its biological functions. Yet, our knowledge of this Kinase remains incomplete, despite it being the only member of its family that causes severe human developmental syndromes, when mutated either on the cyclin or the CDK moiety. CDK10 small-molecule inhibitors would be useful in exploring the functions of this Kinase and gauging its potential as a therapeutic target for some cancers. Here, we report the identification of an optimized peptide phosphorylation substrate of CDK10/CycM and the development of the first homogeneous, miniaturized CDK10/CycM in vitro Kinase Assay. We reveal the ability of known CDK inhibitors, among which clinically tested SNS-032, riviciclib, flavopiridol, dinaciclib, AZD4573 and AT7519, to potently inhibit CDK10/CycM. We also show that NVP-2, a strong, remarkably selective CDK9 inhibitor is an equally potent CDK10/CycM inhibitor. Finally, we validate this Kinase Assay for applications in high-throughput screening campaigns to discover new, original CDK10 inhibitors.
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Development of a CDK10/CycM in vitro Kinase Screening Assay and Identification of First Small-Molecule Inhibitors
Frontiers in Chemistry, 2020Co-Authors: Thomas Robert, Jared Johnson, Roxane Guichaoua, Tomer Yaron, Stéphane Bach, Lewis Cantley, Pierre ColasAbstract:Cyclin-dependent Kinases (CDKs) constitute a family of 20 serine/threonine protein Kinases that play pivotal roles in the regulation of numerous important molecular and cellular processes. CDKs have long been considered promising therapeutic targets in a variety of pathologies, and the recent therapeutic success of CDK4/6 inhibitors in breast cancers has renewed interest in their therapeutic potential. Small-molecule inhibitors have been identified for every human CDK, except for CDK10. The only recent discovery of an activating cyclin (CycM) for CDK10 enabled us to identify its first phosphorylation substrates and gain insights into its biological functions. Yet, our knowledge of this Kinase remains incomplete, despite it being the only member of its family that causes severe human developmental syndromes, when mutated either on the cyclin or the CDK moiety. CDK10 small-molecule inhibitors would be useful in exploring the functions of this Kinase and gauging its potential as a therapeutic target for some cancers. Here, we report the identification of an optimized peptide phosphorylation substrate of CDK10/CycM and the development of the first homogeneous, miniaturized CDK10/CycM in vitro Kinase Assay. We reveal the ability of known CDK inhibitors, among which clinically tested SNS-032, riviciclib, flavopiridol, dinaciclib, AZD4573 and AT7519, to potently inhibit CDK10/CycM. We also show that NVP-2, a strong, remarkably selective CDK9 inhibitor is an equally potent CDK10/CycM inhibitor. Finally, we validate this Kinase Assay for applications in high-throughput screening campaigns to discover new, original CDK10 inhibitors.
