title: Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs).
abstract: The spatiotemporal regulation of the Ras/ERK pathway is critical in determining the physiological and pathophysiological outcome of signaling. Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (DUSPs or MKPs) are key regulators of pathway activity and may also localize ERK to distinct subcellular locations. Here we present methods largely based on the use of high content microscopy to both visualize and quantitate the subcellular distribution of activated (p-ERK) and total ERK in populations of mouse embryonic fibroblasts derived from mice lacking DUSP5, a nuclear ERK-specific MKP. Such methods in combination with rescue experiments using adenoviral vectors encoding wild-type and mutant forms of DUSP5 have allowed us to visualize specific defects in ERK regulation in these cells thus confirming the role of this phosphatase as both a nuclear regulator of ERK activity and localization.
authors: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
authors: Christopher J
authors: CJ
authors: Caunt
authors: Cancer Research UK Stress Response Laboratory, Division of Cancer Research, Jacqui Wood Cancer Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY, UK.
authors: Andrew M
authors: AM
authors: Kidger
authors: Cancer Research UK Stress Response Laboratory, Division of Cancer Research, Jacqui Wood Cancer Centre, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY, UK. s.m.keyse@dundee.ac.uk.
authors: Stephen M
authors: SM
authors: Keyse
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D054637:Dual-Specificity Phosphatases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique / Q000379:methods; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D020935:MAP Kinase Signaling System; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015398:Signal Transduction; D018631:ras Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases; D054637:Dual-Specificity Phosphatases; C546197:Dusp5 protein, mouse; D018631:ras Proteins
keywords: Adenoviral expression; DUSP5; High-content microscopy; Ras/ERK signaling; Spatiotemporal regulation