Phosphorylation Dependent Nuclear Transport of Human Dutpase

The nuclear isoform of human dUTPase plays an important role in maintaining genomic integrity. Its expression is strictly cell cycle regulated and is known to be a phosphoprotein in vivo. However, the role of this phosphorylation remained unknown.Here we show regulation of the nuclear transport of human dUTPase via phosphorylation of a serine residue on its nuclear localisation signal. We found that hyperphosphorylation mimicking mutants (glutamic acid) are localized solely in the cytoplasm while hypophosphorylation mimicking mutants (glutamine) localize in the nucleus as the endogenously regulated protein. Our video microscopy studies have also shed light on the nuclear import dynamics of the wild type dUTPase and that of the mutants. These results showed that the phosphorylated wild type form may re-enter the nucleus (after cell division) only after a considerable delay of several hours while mutants that cannot be phosphorylated re-accumulate within the nucleus much faster. The delay observed with the wild type enzyme may indicate that either dephosphorylation or de novo protein synthesis is required. To reveal the mechanism by which cells accumulate sufficient amount of dUTPase in their nucleus after cell division, we are currently conducting protein transfection based experiments.We are also trying to characterize the interaction of the human dUTPase with its possible partner in nuclear trafficking, importin-alpha. Based on Native-PAGE and ThermoFluor experiments, we detected a relatively high affinity complex of dUTPase with importin-alpha. Complex formation was also observed in the case of the hypophosphorylation mimicking mutant (S11Q), but not with the hyperphosporylation mimicking mutant (S11E). We also conduct crystallographic studies of the complex using various dUTPase NLS peptides.