Protein phosphorylation sites regulate the function of the bipartite NLS of nucleolin.

Nucleolin is a major component of the nucleolus. In Xenopus laevis, a maternal store of nucleolin is accumulated in the multiple nucleoli generated during oogenesis. This maternal nucleolin is distributed throughout the cytoplasm of the egg during oocyte maturation and after fertilization it is gradually reaccumulated in the nuclei of the embryo. Cytoplasmic localization of nucleolin coincides with massive phosphorylation by p34cdc2 kinase, and nuclear translocation is accompanied by net dephosphorylation. Multiple phosphorylation consensus sites for the cell cycle-dependent p34cdc2 kinase and for protein kinase CK2 are clustered in the N-terminal domain of nucleolin. To assess the efficiency of the bipartite nuclear localization signal, we have constructed fusion proteins consisting of maltose binding protein (MBP) and the nuclear localization signal of nucleolin. In addition, either an acidic domain of nucleolin without phosphorylation sites, or an acidic domain containing 4 CK2 sites, or a cluster of 5 cdc2 sites was fused to the MBP-nuclear localization signal (MBP-NLS). Nuclear translocation of these constructs was tested in an in vitro system consisting of Xenopus egg extract and sperm nuclei. Nuclear targetting of MBP by the bipartite nuclear localization signal of nucleolin became significantly more efficient after addition of either CK2 sites or cdc2 sites to the MBP-NLS construct. Yet the cdc2 sites play a dual role. They enhance nuclear translocation exclusively in their dephosphorylated state and promote cytoplasmic localization when phosphorylated, thereby providing a powerful cell cycle-dependent regulatory element of the nuclear localization signal.