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Journal of Cell Science, Vol 104, Issue 1 77-87, Copyright © 1993 by Company of Biologists
JOURNAL ARTICLES |
Y Cao, M Ekstrom and RF Pettersson
Ludwig Institute for Cancer Research, Stockholm Branch, Sweden.
The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4 degrees C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37 degrees C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.
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