Mutagenesis of the nuclear localization sequence in EGF-1 alters protein stability but not mitogenic activity.
Fibroblast growth factor (FGF)-1 is able to translocate to the nucleus as an exogenous protein and a deletion of the nuclear localization sequence near the NH2-terminus of FGF-1 (FGF-1(28-154)) yields a recombinant polypeptide with impaired mitogenic activity. To study the significance within this region (NYKKPK), five FGF-1 point mutations were constructed and their recombinant protein forms analyzed. Interestingly, none of the mutant protein products showed a significant reduction in mitogenic activity when compared to wild-type protein. Because these data suggested possible structural instability within the FGF-1(28-154) deletion mutant, protein structure was examined by fluorescence spectroscopy. Indeed, the FGF-1 point mutations which had similar mitogenic activity to wild-type FGF-1 displayed similar thermal fluorescence spectroscopy patterns as wild-type protein, but FGF-1(28-154) exhibited altered fluorometric profiles. However, the mitogenic activity and structural stability of FGF-1(28-154) was dependent upon the method used to purify the recombinant, whereas purification methods did not effect FGF-1(21-154) mitogenic activity. These studies suggest that the reduced mitogenic activity observed in preparations of the FGF-1(28-154) deletion mutant may be the result of structural instability and fluorescence spectroscopy cannot be used to predict FGF-1 stability.
Friedman
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S
Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, MD 20855.
Zhan
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X
Maciag
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T
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003062:Codon / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D016220:Fibroblast Growth Factor 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013936:Thymidine / Q000378:metabolism
D003062:Codon; D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D004247:DNA; D013936:Thymidine