Abstract
Mitogen 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. Identification of tyrosine and dual specificity 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 specific 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 specific anti-phospho MAPK antibodies and flow cytometry-based determination of fluorescence produced by GFP whose expression is driven by MAPK-regulated promoters are the selected techniques for monitoring these readouts.
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Acknowledgements
We acknowledge the Centro de Citometría y Microscopía de Fluorescencia (UCM, Madrid, Spain) for flow cytometry analysis. This work was made possible thanks to grant BIO2013-44112-P from Ministerio de Economía y Competitividad, (Spain) and S2011/BMD-2414 from Comunidad Autónoma de Madrid (Spain). A.S-R is a recipient of a FPU fellowship from Ministerio de Educación y Ciencia (Spain).
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Sacristán-Reviriego, A., Molina, M., Martín, H. (2016). Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs. In: Pulido, R. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 1447. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3746-2_21
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DOI: https://doi.org/10.1007/978-1-4939-3746-2_21
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