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Molecular and Cellular Biology, April 2005, p. 3087-3108, Vol. 25, No. 8
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.8.3087-3108.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Identification of Nuclear Import and Export Signals within Fli-1: Roles of the Nuclear Import Signals in Fli-1-Dependent Activation of Megakaryocyte-Specific Promoters

Wei Hu, Alana S. Philips, Juliana C. Kwok, Michael Eisbacher, and Beng H. Chong^*

Centre for Thrombosis and Vascular Research, Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales, Australia

Received 22 April 2004/ Returned for modification 1 June 2004/ Accepted 19 November 2004

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMP) and IMPß, with NLS1 and NLS2 being predominantly recognized by IMP and IMPß, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

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* Corresponding author. Mailing address: Department of Medicine, WR Pitney Building, St. George Hospital, Gray St., Kogarah, Sydney, New South Wales 2217, Australia. Phone: 612 93502010. Fax: 612 93503998. E-mail: Beng.chong{at}unsw.edu.au.

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Molecular and Cellular Biology, April 2005, p. 3087-3108, Vol. 25, No. 8
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.8.3087-3108.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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