Lentiviral vectors to study the differential function of ERK1 and ERK2 MAP kinases.
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.
Indrigo
Marzia
M
Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Foundation and University, Milano, Italy.
Papale
Alessandro
A
Orellana
Daniel
D
Brambilla
Riccardo
R
D000818:Animals; D002130:Calcium Phosphates / Q000378:metabolism; D049109:Cell Proliferation; D057075:Enzyme Assays / Q000379:methods*; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D002130:Calcium Phosphates; D035683:MicroRNAs; D034741:RNA, Small Interfering; C020243:calcium phosphate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3