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.
Keywords: Adenoviral expression; DUSP5; High-content microscopy; Ras/ERK signaling; Spatiotemporal regulation.