Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed mutagenesis of a putative nuclear translocation sequence.
Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.
Presta
M
M
Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Italy.
Gualandris
A
A
Urbinati
C
C
Rusnati
M
M
Coltrini
D
D
Isacchi
A
A
Caccia
P
P
Bergonzoni
L
L
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2