Single and Combined Silencing of ERK1 and ERK2 Reveals Their Positive Contribution to Growth Signaling Depending on Their Expression Levels

Abstract

The proteins ERK1 and ERK2 are highly similar, are ubiquitously expressed, and share activators and substrates; however, erk2 gene invalidation is lethal in mice, while erk1 inactivation is not. We ablated ERK1 and/or ERK2 by RNA interference and explored their relative roles in cell proliferation and immediate-early gene (IEG) expression. Reducing expression of either ERK1 or ERK2 lowered IEG induction by serum; however, silencing of only ERK2 slowed down cell proliferation. When both isoforms were silenced simultaneously, compensating activation of the residual pool of ERK1/2 masked a more deleterious effect on cell proliferation. It was only when ERK2 activation was clamped at a limiting level that we demonstrated the positive contribution of ERK1 to cell proliferation. We then established that ERK isoforms are activated indiscriminately and that their expression ratio correlated exactly with their activation ratio. Furthermore, we determined for the first time that ERK1 and ERK2 kinase activities are indistinguishable in vitro and that erk gene dosage is essential for survival of mice. We propose that the expression levels of ERK1 and ERK2 drive their apparent biological differences. Indeed, ERK1 is dispensable in some vertebrates, since it is absent from chicken and frog genomes despite being present in all mammals and fishes sequenced so far.Numerous cell surface agonists activate the signaling cascade Ras/Raf/MEK/ERK. In contrast to the upstream activation steps of the cascade, which have few known substrates, ERK phosphorylates hundreds of substrates on serine and threonine residues (52). ERK substrates are localized in all cell compartments: at the membrane, (e.g., epidermal growth factor receptor), in the cytosol (e.g., DUSP6-MKP-3), on the cytoskeleton (e.g., cortactin), and in the nucleus (e.g., Elk-1). The serine/threonine protein kinase ERKs play key roles in cell proliferation, cell differentiation, and cell death. The spatio-temporal regulation of ERK activation dictates the biological outcome (34). Therefore, for all these reasons, ERK activation is a key signaling step for the regulation of many biological responses.Two isoforms convey ERK activity in many vertebrates; these are ERK1 and ERK2, which are 84% identical at the amino acid level (8). Among the mitogen-activated protein kinases, ERK5 (55) is the closest kinase to ERK1/2, with all three kinases being activated on the threonine and tyrosine residues of the TEY sequence. However, ERK5 cannot be considered an ERK1/2 isoform, since ERK5 and ERK1/2 are activated by different kinase modules. ERK5 is activated by the kinase MEK5 (55), unlike ERK1 and -2, which are activated by MEK1/2 (23).Many observations indicate that ERK1 and ERK2 are very similar. ERK1 and ERK2 are ubiquitously expressed; however, their relative distributions across tissues differ (8). ERK1 and -2 display the same subcellular localization; both isoforms translocate from the cytosol to the nucleus upon stimulation of resting cells (25). ERK1 and -2 are serine/threonine pr...

RNAi-mediated ERK2 knockdown inhibits growth of tumor cells in vitro and in vivo

“…Using B-cell-specifictargeted mice, Sanjo et al show the absolute normal proliferation of B cells lacking ERK2, and propose that ERK1 compensate for the loss of ERK2. Lefloch et al (2008) showed that ERK1 and 2 kinase activities are indistinguishable in NIH3T3 cells and proposed that erk gene dosage is essential and drive their apparent biological differences.…”

Total ERK1/2 activity regulates cell proliferation

“…59 As shown in Figure 1A, Xenopus MEK is orthologous to human MEK1 and Xenopus ERK is ortologous to human ERK2 indicating that MEK2 and ERK1 have been lost since they are present in fish genomes. Similarly to Xenopus, the pre-vertebrates Drosophila and C. elegans possess only one MEK and one ERK protein.…”

“…66 Loosing one allele of ERK1 in mice is harmless when both alleles of ERK2 are present, however in the heterozygous ERK2 +/-animal we have shown that the loss of one allele of ERK1 was highly deleterious (3% animals born instead of 50% expected). 59 This re-enforces the notion that reducing the generally least expressed isoform is harmful when the other is limiting (or when the pathway is stressed markedly).…”

“…58 Both ERK1 and ERK2 phosphorylate substrates on the consensus PxS/TP sites with similar specific activities in vitro (demonstrated with bacterially expressed ERK1 and ERK2, 58 and we verified it by immuno-precipitating epitope-tagged ERK1 and epitope-tagged ERK2.) 59 Substrates bind to ERK mainly via two docking sites, the CD (Common-Docking) and the ED (ERK Docking site), which interact with complementary docking sites on the substrates, respectively the D-domain (also called DEJL) and the DEF (docking site for ERK, FXF). Co-crystallisation of the CD domain of ERK with a D-domain peptide substrate indicated that among the 11 amino acids implicated, ERK1 and ERK2 diverge only for a leucine instead of an isoleucine.…”

“…The lack of MEK2 and ERK1 is Xenopus tropicalis genome and in Xenopus laevis EST databases was demonstrated by search strategies described previously. 59 Other sequences for phylograms have the following accession numbers in Ensembl: Drosophila. melanogaster Ras (FBtr0082122), Raf (FBgn0003079), MEK FBgn0010269, ERK (FBgn0003256); C.elegans Ras (ZK792.6), Raf (Y73B6A.5a.3), MEK (Y54E10BL.6), ERK (F43C1.2).…”