Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis.
We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1). The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1. In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells. Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus. Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants. These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.
Cell Science and Technology Division, Fermentation Research Institute, Ibaraki, Japan.
T
T
Imamura
Y
Y
Tokita
Y
Y
Mitsui
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002872:Chromosome Deletion; D015180:Electrophoresis, Gel, Two-Dimensional; D016220:Fibroblast Growth Factor 1 / Q000032:analysis* / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D016220:Fibroblast Growth Factor 1