title: A high-throughput assay for phosphoprotein-specific phosphatase activity in cellular extracts.
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.
authors.affiliation: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Anjun K
authors.initials: AK
authors.lastname: Bose
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Janes
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D051379:Mice; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D051381:Rats; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D002457:Cell Extracts; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D004815:Epidermal Growth Factor; D007371:Interferon-gamma; D020928:Mitogen-Activated Protein Kinases; C120407:DUSP10 protein, human; D054639:Mitogen-Activated Protein Kinase Phosphatases; D010749:Phosphoprotein Phosphatases; C439065:DUSP16 protein, human; D054637:Dual-Specificity Phosphatases