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Methods Mol Biol. 2010;661:205-20. doi: 10.1007/978-1-60761-795-2_12.

Lentiviral vectors to study the differential function of ERK1 and ERK2 MAP kinases.

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1
Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Foundation and University, Milano, Italy.

Abstract

Accumulating evidence indicates that p44(ERK1) and p42(ERK2) mitogen-activated protein kinases (MAPKs) have distinct quantitative roles in cell signaling. In our recently proposed model of regulation of ERK1 and ERK2, p42 plays a major role in delivering signals from the cell membrane to the nucleus, while p44 acts as a partial agonist of ERK2 toward effectors and downstream activators, thus providing a fine tuning system of the global signaling output. Here, we describe systems to modulate MAPK signaling in vitro and in vivo via lentiviral vector (LV)-mediated gene transfer, using three systems: RNAi with small hairpin RNAs, microRNA-mediated gene knockdown, and expression of signaling-interfering mutants of MEK1. We show, by using proliferation assays in mouse embryo fibroblasts (MEF) and NIH 3T3 cells, that gene knockdown of ERK1 promotes cell proliferation in a manner indistinguishable from a constitutively active MEK1 construct, while ERK2 RNAi causes a significant growth arrest, similar to that observed with the ectopic expression of a dominant negative MEK1 mutant.

PMID:
20811985
DOI:
10.1007/978-1-60761-795-2_12
[Indexed for MEDLINE]
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