Translocation to Cytosol of Exogenous, CAAX-tagged Acidic Fibroblast Growth Factor

doi:10.1074/jbc.270.51.30680

Translocation to Cytosol of Exogenous, CAAX-tagged Acidic Fibroblast Growth Factor (*)

From the Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

Abstract

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

Footnotes

↵^§ On leave of absence from The Institute of Immunology and Experimental Therapy of The Polish Academy of Sciences, Wroclaw, Poland.
↵^¶ Fellow of The Norwegian Cancer Society.
↵** Fellow of the Spanish Ministry of Education and Science.
↵* This work was supported by The Norwegian Cancer Society, Novo Nordisk Foundation, The Norwegian Research Council, Blix Fund for the Promotion of Medical Research, Rachel and Otto Kr. Bruun's legat, and The Jahre Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

aFGF

acidic fibroblast growth factor

aFGF-CAAX

aFGF modified to contain the C-terminal amino acids Cys-Val-Ile-Met

dtA-CAAX

diphtheria toxin A fragment with the C-terminal amino acids Cys-Val-Ile-Met

FGFR4

fibroblast growth factor receptor 4

PBS

phosphate-buffered saline

DMEM

Dulbecco's minimal essential medium

FCS

fetal calf serum

PAGE

polyacrylamide gel electrophoresis.
- Received April 14, 1995.
- Revision received September 15, 1995.