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

Abstract

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 (IMPα) and IMPβ, with NLS1 and NLS2 being predominantly recognized by IMPα and IMPβ, 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.

ACKNOWLEDGMENTS

We are indebted to Yoshihiro Yoneda (Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan) for providing IMPα/β expression constructs. We are grateful to Yao Wang (Orthopedic Research Institute, University of New South Wales, Sydney, Australia) for helpful discussions and constructive criticisms of the manuscript.

This work was supported by an NHMRC program grant.