Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs)

Christopher J. Caunt, Andrew M. Kidger, Stephen M. Keyse

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

LanguageEnglish
Title of host publicationProtein Tyrosine Phosphatases
Subtitle of host publicationMethods and Protocols
EditorsR. Pulido
PublisherSpringer
Pages197-215
Number of pages19
ISBN (Print)9781493937448
DOIs
StatusPublished - 12 Aug 2016

Publication series

NameMethods in Molecular Biology
Volume1447

Fingerprint

Phosphoprotein Phosphatases
Mitogens
Mitogen-Activated Protein Kinase Phosphatases
MAP Kinase Signaling System
Phosphoric Monoester Hydrolases
Microscopy
Fibroblasts
Population

Cite this

Caunt, C. J., Kidger, A. M., & Keyse, S. M. (2016). Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs). In R. Pulido (Ed.), Protein Tyrosine Phosphatases: Methods and Protocols (pp. 197-215). (Methods in Molecular Biology; Vol. 1447). Springer. DOI: 10.1007/978-1-4939-3746-2_12

Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs). / Caunt, Christopher J.; Kidger, Andrew M.; Keyse, Stephen M.

Protein Tyrosine Phosphatases: Methods and Protocols. ed. / R. Pulido. Springer, 2016. p. 197-215 (Methods in Molecular Biology; Vol. 1447).

Research output: Chapter in Book/Report/Conference proceedingChapter

Caunt, CJ, Kidger, AM & Keyse, SM 2016, Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs). in R Pulido (ed.), Protein Tyrosine Phosphatases: Methods and Protocols. Methods in Molecular Biology, vol. 1447, Springer, pp. 197-215. DOI: 10.1007/978-1-4939-3746-2_12
Caunt CJ, Kidger AM, Keyse SM. Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs). In Pulido R, editor, Protein Tyrosine Phosphatases: Methods and Protocols. Springer. 2016. p. 197-215. (Methods in Molecular Biology). Available from, DOI: 10.1007/978-1-4939-3746-2_12
Caunt, Christopher J. ; Kidger, Andrew M. ; Keyse, Stephen M./ Visualizing and quantitating the spatiotemporal regulation of Ras/ERK signaling by dual-specificity mitogen-activated protein phosphatases (MKPs). Protein Tyrosine Phosphatases: Methods and Protocols. editor / R. Pulido. Springer, 2016. pp. 197-215 (Methods in Molecular Biology).
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