Lentiviral Vectors to Study the Differential Function of ERK1 and ERK2 MAP Kinases
Accumulating evidence indicates that p44ERK1 and p42ERK2 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 sign
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1. Introduction One of the major dilemmas in signal transduction is related to the “redundancy problem”: why does a cell need multiple isoforms of a given signal transducer to perform its physiological function? In recent years, the idea that slightly different isoforms may perform a set of nonoverlapping functions has become a central concept to be tested experimentally, to reach a deeper understanding of the complexity of cell signaling mechanisms. Certainly, the availability of genetically modified mice and RNA interference (RNAi) techniques has shed new light on this complexity as it is Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_12, © Springer Science+Business Media, LLC 2010
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now clear that ablation of different MAP kinases may lead to quantitative and qualitative differences in phenotype. This is particularly evident for the two main ERK isoforms, p44ERK1 and p42ERK2. A number of groups have independently generated genetargeted mice for both isoforms and the general consensus is clear: ERK2 ablation has a large impact on animal survival, while ERK1 loss has not. In fact, Erk2 gene deletion causes an early phenotype, which is incompatible with the completion of the embryonic development (1–6). On the contrary, erk1 gene ablation is fully compatible with the adult life (7–9). In 2006, we published a seminal paper in which we demonstrated for the first time that ERK1 and ERK2 MAP kinases play a differential role in the control of cell proliferation (10). That publication was the culmination of an intense research activity performed by our laboratory in the pursue of a molecular explanation for a crucial observation made already by Pagès et al. in 1999 and later confirmed by us in 2002: ERK1-deficient cells, from embryonic fibroblasts to neurons, show an enhanced phosphorylation of the remaining ERK2 isoform, without significant changes in the ERK2 protein level (7, 9). At first glance, this observation could have been interpreted as a compensatory action put in place by the cell signaling machinery to overcome ERK1 loss. However, using a combination of ERK1 knockout mouse embryonic fibroblasts (MEF), ERK1 and ERK2 knockdown cells obtained via RNAi, and cells overexpressing either epitope tagged ERK1 or ERK2 proteins, we suggested a different scenario. In our view, ERK1 could act, in physiological settings, i.e., without major alterations of the cell signaling machinery and without an extreme, ERK-independent deregulation of cell growth, as a fine regulator of ERK2, which is, in the large majority of tissues, the most abundant of the two MAP kinases. We essentially found, both in MEF and NIH 3T3 cells, that a complete (>90%) ablation of ERK1 protein leads to a significant growth advantage, while ablation of ERK2 is essentially incompatible with cell viability. However, we also found that in NIH 3T3, overexpression of both WT ERK1 and a kinase defective form of ERK1 caused a significant reduction i
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