The Net Repressor Is Regulated by Nuclear Export in Response to Anisomycin, UV, and Heat Shock

Abstract

The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein kinase signalling pathways. They integrate the transcriptional response at the level of serum response elements in early-response genes, such as the c-fosproto-oncogene. An important aim is to understand the individual roles played by the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a strong repressor of transcription. We now show that Net is regulated by nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net is mainly nuclear under both normal and basal serum conditions. Net contains two nuclear localization signals (NLSs); one is located in the Ets domain, and the other corresponds to the D box. Net also has a nuclear export signal (NES) in the conserved Ets DNA binding domain. Net is apparently unique among Ets proteins in that a particular leucine in helix 1, a structural element, generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion of Net through a pathway that involves c-Jun N-terminal kinase kinase and is inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression by Net. The specific induction of nuclear exclusion of Net by particular signalling pathways shows that nuclear-cytoplasmic transport of transcription factors can add to the specificity of the response to signalling cascades.

ACKNOWLEDGMENTS

We thank the following: for generous gifts of reagents and cells, B. Wolff and Novartis (leptomycin B), A. Bradford and A. Gutierrez-Hartmann (UAS-TK-Luc), R. Davis [expression vectors for MKK3(Glu), MKK6(Glu), MKK3 (Ala), and MKK6(Ala)], M. Karin [expression vectors for MEKK1, JNKK, MEKK1(KM), and JNKK (KR)]; for confocal microscopy, Jean-Luc Vonesch; and for invaluable help, the IGBMC facilities staff.

For fellowships, we thank the MRT (C.D.) and the MRT, the Ligue, and BioAvenir (S.-M.M.). For financial assistance, we thank BioAvenir, the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, Fondation pour la Recherche Médicale, Ligue Nationale Française contre le Cancer, Ligue Régionale (Haut-Rhin) contre le Cancer, and Ligue Régionale (Bas-Rhin) contre le Cancer (the Legs Meyer).