Inability of the Acidic Fibroblast Growth Factor Mutant K132E to Stimulate DNA Synthesis after Translocation into Cells*
- From the Department of Biochemistry, The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
Abstract
Acidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated.
Footnotes
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↵* This work was supported by The Norwegian Cancer Society, Novo Nordisk Foundation, The Norwegian Research Council for Science and Humanities, Blix Legat, 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. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Fellow of The Norwegian Cancer Society.,
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↵§ Fellow of The Norwegian Cancer Society. On leave of absence from The Institute of Immunology and Experimental Therapy of The Polish Academy of Sciences, Wroclaw, Poland.
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↵¶ Supported by a Personal Investigator Fellowship from the Spanish Ministry of Education and Science and from the European Union.
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↵‖ To whom correspondence should be addressed. Tel.: 4722935640; Fax: 4722508692; E-mail: olsnes{at}radium.uio.no.
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↵1 The abbreviations used are: aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; DT-A, diphtheria toxin A-fragment; DT-B, diphtheria toxin B-fragment; BrdUrd, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium.
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- Received August 22, 1997.
- Revision received January 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
