E2F4 Is Exported from the Nucleus in a CRM1-Dependent Manner

Abstract

E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G₁. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16^INK4a, an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G₁ in association with a suitable pocket protein.

ACKNOWLEDGMENTS

We thank Stefanie Hauser, Ulrike Kutay, Fabio Martelli, Pamala Silver, and our laboratory and divisional colleagues for many helpful conversations. We thank Sara Nakielny and G. Dreyfus for sending us the nucleoplasmin expression plasmid and for important advice on the use of the heterokaryon fusion assay. We also thank G. Grosveld (St. Jude Children's Hospital) for the polyclonal CRM1 antiserum and CRM1 expression plasmid and B. Wolff (Novartis, Vienna, Austria) for her gift of leptomycin B.

This work was supported by grants from the NIH-NCI to D.M.L., by fellowships from the European Molecular Biology Organization and the Leukemia and Lymphoma Society to S.G., and by a fellowship from the National Health and Medical Research Council of Australia to G.J.L.