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.