A monopartite sequence is essential for p45 NF-E2 nuclear translocation, transcriptional activity and platelet production.
BACKGROUND
p45 NF-E2 is a bZIP transcription factor crucial for thrombopoiesis, as indicated by the fact that loss of p45 NF-E2 function results in dramatic embryonic lethal thrombopoietic defects and its overexpression boosts platelet release.


OBJECTIVE
In the present study, we set out to identify the sequences responsible for p45 NF-E2 nuclear import, evaluate its transport mechanism and ascertain its functional significance.


METHODS
A series of p45 NF-E2 deletion constructs fused to green fluorescent protein (GFP) was created and their cellular localization examined in mammalian cells, with the factor responsible for nuclear import identified using an in vitro transport assay. A p45 NF-E2 derivative mutated in the nuclear targeting sequence (NLS) was generated and its biological activity compared with wild type (wt) in luciferase assays, and proplatelet and platelet production measured in murine megakaryocytes transduced with a retroviral vector.


RESULTS
Here we show that residues 271-273 are essential for nuclear import of p45 NF-E2 in COS-7 and in primary bone marrow cells. The p45 NF-E2 NLS facilitates nuclear import specifically via importin (IMP) 7. Although within the DNA-binding domain of p45 NF-E2, the NLS is not essential for DNA-binding, but is crucial for transcriptional activation and biological activity; where, in contrast to wt, a mutant derivative with a mutated NLS failed to promote proplatelet and platelet production in murine megakaryocytes.


CONCLUSIONS
The NLS is critical for p45 NF-E2 function, with the present study being the first to demonstrate the importance of NLS-dependent nuclear import of p45 NF-E2 for platelet development.
Perdomo
J
J
Centre for Vascular Research, University of New South Wales, Sydney, New South Wales, Australia.
Fock
E-L
EL
Kaur
G
G
Yan
F
F
Khachigian
L M
LM
Jans
D A
DA
Chong
B H
BH
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000378:metabolism*; D019913:Nuclear Localization Signals; D011485:Protein Binding; D034061:Thrombopoiesis; D015533:Transcriptional Activation
D050984:NF-E2 Transcription Factor, p45 Subunit; D019913:Nuclear Localization Signals; D004247:DNA; D008156:Luciferases