Cell cycle-dependent nuclear localization of exogenously added fibroblast growth factor-1 in BALB/c 3T3 and human vascular endothelial cells.
Previous studies have shown that the presence of a functional nuclear targeting sequence in the primary structure of fibroblast growth factor (FGF)-1 correlates with its activity as a mitogen, but not with its potential for inducing receptor tyrosine phosphorylation, suggesting the presence of a yet undefined function of FGF-1 as a nuclear protein. In the present study we have investigated the cytosolic and nuclear localization of exogenously added FGF-1. FGF-1-specific monoclonal antibodies were raised. By an extensive screening, highly specific antibody clones were isolated. For both BALB/c 3T3 and human umbilical vein endothelial (HUVE) cells, immunofluorescence studies performed with those clones delineated that during G1 stage of cell cycle, FGF-1 transits from cytosol to nucleus. This was followed by a shift to the perinuclear and juxtanuclear region just prior to the onset of S-phase in BALB/c 3T3 cells. Confocal microscopical examinations confirmed that the nuclear staining resides throughout the nuclear matrix with some enrichment at the envelope boundary and in the nucleoli. Immunoblot analysis of the fractionated BALB/c 3T3 cells that had been induced to proliferate by serum and pulsed with exogenous FGF-1 at various timings revealed that the incorporation of exogenous FGF-1 into cytosol took place constantly, whereas the nuclear translocation significantly increased after 5 h following stimulation of the quiescent cells. The cytosolic form of FGF-1 is indicated to be present in soluble cytosolic fraction rather than membrane-enveloped compartments, endosomes, by the microinjection of anti FGF-1 antibody to HUVE cells cultured in the presence of FGF-1. The data demonstrate that the exogenously added FGF-1 is constantly endocytosed and fractioned into the cytosol soluble compartment, whereas its nuclear localization is regulated at the nuclear translocation level and takes place preferably at late G1 phase of the cell cycle.
Imamura
T
T
Cell Biology Laboratory, National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Oka
S
S
Tanahashi
T
T
Okita
Y
Y
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D002453:Cell Cycle; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice
D000911:Antibodies, Monoclonal; D005346:Fibroblast Growth Factors