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.
Highlights
In cell culture acidic fibroblast growth factor1 stimulates DNA synthesis, cell migration, and cell differentiation
To investigate if this were the case with Acidic fibroblast growth factor (aFGF)(K132E)-DT-A, Vero cells expressing diphtheria toxin receptor, but resistant to the toxin due to inability of their elongation factor 2 to be modified by the toxin [38, 39], were first kept on ice with radiolabeled fusion protein reconstituted with diphtheria toxin B-fragment (DT-B), exposed to low pH in order to translocate the fusion protein, subsequently treated with Pronase, washed, and further incubated for 0, 24, or 48 h
The main findings in this paper are that aFGF(K132E) appears to enter cells in a similar manner as wild-type growth factor and that after artificial translocation of the mutant as a fusion protein with DT, it is not able to stimulate DNA synthesis
Summary
Rabbit reticulocyte lysate and rRNasin were from Promega, Madison, WI; T3 RNA-polymerase from Life Technologies, Inc., Gaithersburg, MD; [35S]methionine from NEN Research Products, Wilmington, DE; protein A-Sepharose and heparin-Sepharose from Pharmacia, Up-. Fragments from paFGF(K132E) and vector from pHBGF-DT1 were ligated, creating a plasmid containing the sequence encoding aFGF(K132E) in front of DTA. Recombinant proteins were expressed and purified as described previously [12] except that for aFGF(K132E) and aFGF(K132E)-CAAX the clarified bacterial lysates were applied to heparin-Sepharose in a buffer containing 0.1 M NaCl instead of 0.5 M and were further purified using an Econo-Pac Q cartridge (Bio-Rad). The plasmids were linearized, transcribed in vitro, and translated in a rabbit reticulocyte lysate system in the presence of unlabeled methionine or [35S]methionine as described [4]. The lysates were dialyzed against dialysis buffer (20 mM HEPES, pH 7.0, 140 mM NaCl, 2 mM CaCl2) to remove free [35S]methionine and reducing agent, allowing disulfide bridges to be formed [11]
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