NDLI: A high-throughput assay for phosphoprotein-specific phosphatase activity in cellular extracts.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Bose, Anjun K. Janes, Kevin A.
Description	Country affiliation: United States Author Affiliation: Bose AK ( Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.)
Abstract	Protein phosphatases undo the post-translational modifications of kinase-signaling networks, but phosphatase activation in cells is difficult to measure and interpret. Here, we report the design of a quantitative and high-throughput assay platform for monitoring cellular phosphatase activity toward specific phosphoprotein targets. Protein substrates of interest are purified recombinantly, phosphorylated in vitro using the upstream kinase, and adsorbed to 96-well plates. Total phosphatase extracts from cells are then added to trigger a solid-phase dephosphorylation reaction. After stopping the reaction, phosphoprotein levels are quantified by ELISA with a phospho-specific antibody, and the loss of phospho-specific immunoreactivity is used as the readout of phosphatase activity. We illustrate the generality of the method by developing specific phosphatase-activity assays for the three canonical mitogen-activated protein phospho-kinases: ERK, JNK, and p38. The assays capture changes in activity with a dynamic range of 25-100-fold and are sensitive to a limit of detection below 25,000 cells. When applied to cytokine-induced signaling, the assays revealed complex and dynamic regulation of phosphatases suggesting cross-communication and a means for cellular memory. Our assay platform should be beneficial for phosphoproteomic surveys and computational-systems models of signaling, where phosphatases are known to be important but their activities are rarely measured.
File Format	HTM / HTML
ISSN	15359476
e-ISSN	15359484
DOI	10.1074/mcp.O112.024059
Journal	Molecular & Cellular Proteomics
Issue Number	3
Volume Number	12
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2013-03-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Proteomics Cell Extracts Enzyme Assays Mitogen-activated Protein Kinases Metabolism Phosphoprotein Phosphatases Phosphoproteins Animals Blotting, Western Dual-specificity Phosphatases Genetics Enzyme Activation Drug Effects Instrumentation Enzyme-linked Immunosorbent Assay Epidermal Growth Factor Pharmacology Ht29 Cells Interferon-gamma Mice Mitogen-activated Protein Kinase Phosphatases Phosphorylation Proteomics Rna Interference Reproducibility Of Results Tumor Necrosis Factor-alpha Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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