title: Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs.
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.
authors: Departamento de Microbiología II, Facultad de Farmacia, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040, Madrid, Spain.
authors: Almudena
authors: A
authors: Sacristán-Reviriego
authors: Departamento de Microbiología II, Facultad de Farmacia, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040, Madrid, Spain.
authors: María
authors: M
authors: Molina
authors: Departamento de Microbiología II, Facultad de Farmacia, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040, Madrid, Spain. humberto@ucm.es.
authors: Humberto
authors: H
authors: Martín
mesh_terms: D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases
keywords: Anti-phospho MAPK antibody; DSP; Dual specificity phosphatase; Flow cytometry; GFP; MAPK; MAPK phosphatase; PTP; Protein tyrosine phosphatase; Yeast