The NH2-terminal extension of high molecular weight forms of basic fibroblast growth factor (bFGF) is not essential for the binding of bFGF to nuclear chromatin in transfected NIH 3T3 cells.
Immunolocalization of basic fibroblast growth factor (bFGF) was investigated in NIH 3T3 cells transfected with a cDNA encoding for the 18 kD form of human bFGF (18 kD-bFGF) or with a bFGF cDNA encoding for both 18 kD-bFGF and NH2-terminal extended high molecular weight forms of bFGF (HMW-bFGFs). Nuclear and cytoplasmic bFGF-immunoreactivity was observed in both transfectants. Nuclear bFGF immunoreactivity was evenly distributed during interfase and associated with condensed chromosomes throughout the mitotic cycle. Cell fractionation, followed by Western blot analysis, confirmed the presence of 18 kD-bFGF and of HMW-bFGFs in the nucleus of transfected cells. Also, both 18-kD bFGF and HMW-bFGFs copurified with nuclear chromatin. After trypsin digestion, chromatin-bound bFGFs showed a rapid degradation of the nuclear-targeting NH2-terminal extension of HMW-bFGFs which were converted to the 18 kD form. On the contrary, 18 kD-bFGF appeared to be trypsin-resistant when bound to nuclear chromatin or to isolated eukaryote DNA. Thus, our data indicate that: i) both 18 kD-bFGF and HMW-bFGFs localize into the nucleus of transfected NIH 3T3 cells and bind to nuclear chromatin; ii) the interaction of all bFGF isoforms with nuclear chromatin is mediated by one or more sequences present within the 18 kD form; iii) the chromatin-binding domain of HMW-bFGFs is distinct from their nuclear-targeting domain.
Gualandris
A
A
Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Italy.
Coltrini
D
D
Bergonzoni
L
L
Isacchi
A
A
Tenca
S
S
Ginelli
B
B
Presta
M
M
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D002843:Chromatin; D010446:Peptide Fragments; D016222:Fibroblast Growth Factor 2; D004247:DNA; D014357:Trypsin