ERF Nuclear Shuttling, a Continuous Monitor of Erk Activity That Links It to Cell Cycle Progression

Abstract

The ets domain transcriptional repressor ERF is an effector of the receptor tyrosine kinase/Ras/Erk pathway, which, it has been suggested, is regulated by subcellular localization as a result of Erk-dependent phosphorylation and is capable of suppressing cell proliferation and ras-induced tumorigenicity. Here, we analyze the effect of ERF phosphorylation on nuclear import and export, the timing of its phosphorylation and dephosphorylation in relation to its subcellular location, Erk activity, and the requirements for ERF-induced cell cycle arrest. Our findings indicate that ERF continuously shuttles between the nucleus and the cytoplasm and that both phosphorylation and dephosphorylation of ERF occur within the nucleus. While nuclear import is not affected by phosphorylation, ERF nuclear export and cytoplasmic release require multisite phosphorylation and dephosphorylation. ERF export is CRM1 dependent, although ERF does not have a detectable nuclear export signal. ERF phosphorylation and export correlate with the levels of nuclear Erk activity. The cell cycle arrest induced by nonphosphorylated ERF requires the wild-type retinoblastoma protein and can be suppressed by overexpression of cyclin. These data suggest that ERF may be a very sensitive and constant sensor of Erk activity that can affect cell cycle progression through G₁, providing another link between the Ras/Erk pathway and cellular proliferation.

We thank Valérie Lefevbre for help with the localization experiments, Kaliopi Borboudaki for technical assistance, Katerina Mpilitou for help with the antibody work, Meropi Athanasiou (BRP-SAIC, Frederick, Md.) for immunohistochemistry reagents, Jane Leitch (Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, Dundee, Scotland) for the production of the anti-phospho-ERF antibodies, Thierry Dubois (University of Edinburgh) and others in the Aitken laboratory for the 14-3-3 reagents, and Pantelis Hatzis for help with the manuscript.

This work was supported by the EU grants ERBFMRXCT 96-0041 and BIO4CT975133 (to G.M.) and by the UK Medical Research Council and The Royal Society (to P.C.).