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• W2149299946 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." @default.
• W2149299946 created "2016-06-24" @default.
• W2149299946 creator A5027602726 @default.
• W2149299946 creator A5028741546 @default.
• W2149299946 date "2013-03-01" @default.
• W2149299946 modified "2023-10-16" @default.
• W2149299946 title "A High-throughput Assay for Phosphoprotein-specific Phosphatase Activity in Cellular Extracts" @default.
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• W2149299946 doi "https://doi.org/10.1074/mcp.o112.024059" @default.
• W2149299946 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3591670" @default.
• W2149299946 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23233447" @default.
• W2149299946 hasPublicationYear "2013" @default.
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