ArticleMolecular & cellular proteomics : MCP2013
A high-throughput assay for phosphoprotein-specific phosphatase activity in cellular extracts.
Article in Molecular & cellular proteomics : MCP, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 25 citations in OpenAlex.
- Cells of the Maternal-Fetal Interface May Contribute to Epidural-Related Maternal Fever After Administration of Ropivacaine: The Role of Phosphatases DUSP9 and PHLPP1.International journal of molecular sciences · 2025Article
- MAP kinases differentially bind and phosphorylate NOS3 via two unique NOS3 sites.FEBS open bio · 2022Article
- LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome.Cell · 2021Article
- The Convergence of Cell-Based Surface Plasmon Resonance and Biomaterials: The Future of Quantifying Bio-molecular Interactions-A Review.Annals of biomedical engineering · 2020Review
- Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways.Histochemistry and cell biology · 2020Article
- An ultrasensitive fiveplex activity assay for cellular kinases.Scientific reports · 2019Article
- Association of Intestinal Alkaline Phosphatase With Necrotizing Enterocolitis Among Premature Infants.JAMA network open · 2019Article
- Optical control of protein phosphatase function.Nature communications · 2019Article
- Article
- Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities.Methods in enzymology · 2018Article
- ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker.Journal of Cancer · 2018Article
- VEGFR1 promotes cell migration and proliferation through PLCγ and PI3K pathways.NPJ systems biology and applications · 2018Article
- Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells.Scientific reports · 2017Article
- Profiling Subcellular Protein Phosphatase Responses to Coxsackievirus B3 Infection of Cardiomyocytes.Molecular & cellular proteomics : MCP · 2017Article
- Studying Cellular Signal Transduction with OMIC Technologies.Journal of molecular biology · 2015Review
- An analysis of critical factors for quantitative immunoblotting.Science signaling · 2015Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.