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.

ACKNOWLEDGMENTS

We appreciate the invaluable help of Danièle Roux with pSUPER-ERK2 cloning and of Christine Bourcier with mouse ERK1 cloning. We are grateful to Gilles Pagès and to Sylvain Méloche for sharing the results of mouse crosses. We are grateful to Elisabeth Goldsmith for helpful discussion concerning ERK2 versus ERK1 binding to the D-motif peptide and to Melanie Cobb for personal communication concerning the lack of ERK1 in the chicken genome. We thank Michiaki Kohno and Sir Philip Cohen for kindly providing us PD184352. We thank Krittalak Chakrabandhu for performing the RabVa immunoblotting, Richard Christen for elaborating genomic sequence analysis, and Robert Hipskind for kindly providing us the vector GST-Elk1^307-428. We are grateful to Aurélie Rossin, Rosana Kral, and Sébastien Huault for helping with fluorescence-activated cell sorter analysis. We thank Emmanuel Chamorey for helping with statistical analysis and M. Christiane Brahimi-Horn for critical reading of the manuscript.

Financial support was provided by the Centre National de la Recherche Scientifique (CNRS), Centre A. Lacassagne, Ministère de l'Education de la Recherche et de la Technologie, Ligue Nationale Contre le Cancer (Equipe Labelisée), and Association pour la Recherche sur le Cancer (ARC contract 3338).