Molina, MaríaMartín, HumbertoSacristán Reviriego, AlmudenaMolina Martín, MaríaMartín Brieva, HumbertoPulido, Rafael2025-01-152025-01-152016Sacristán-Reviriego, Almudena, et al. «Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs». Protein Tyrosine Phosphatases, editado por Rafael Pulido, vol. 1447, Springer New York, 2016, pp. 385-98. DOI.org (Crossref), https://doi.org/10.1007/978-1-4939-3746-2_21.978149393744897814939374621064-37451940-602910.1007/978-1-4939-3746-2_21https://hdl.handle.net/20.500.14352/114428Mitogen activated protein kinases (MAPK) pathways play a key role in orchestrating the eukaryotic cellular response to different stimuli. In this process, phosphorylation of both conserved threonine and tyrosine residues of MAPKs is essential for their activation. Identifi cation of tyrosine and dual specifi city protein phosphatases capable of dephosphorylating these phosphosites is thus critical to gain insight into their regulation. Due to the conservation of pivotal elements in eukaryotic signaling, yeast has turned into a valuable tool to increase the knowledge of MAPK signaling in other cell types. Here we describe an in vivo method to evaluate the capacity of a protein, from yeast or other origin, to act as a MAPK phosphatase. It relies on the ability of the phosphatase to reduce, when overexpressed, both the amount of activated MAPK and the transcription from a specifi c promoter regulated by the corresponding pathway. To this end, the pathway has to be previously activated, preferentially through overexpression of a hyperactive allele of an upstream component within the MAPK module. Additionally, the ability of an overexpressed “trapping” inactive phosphatase version to modify these readouts is also analyzed. Western blotting analysis with specifi c anti-phospho MAPK antibodies and fl ow cytometry-based determination of fl uorescence produced by GFP whose expression is driven by MAPK-regulated promoters are the selected techniques for monitoring these readouts.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/book parthttps://doi.org/10.1007/978-1-4939-3746-2_21restricted access579576.8Protein tyrosine phosphataseDual specifi city phosphatasePTPDSPMAPKMAPK phosphataseAnti-phospho MAPK antibodyYeastGFPFlow cytometryMicrobiología (Farmacia)Parasitología (Farmacia)32 Ciencias Médicas