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 ge nerated during oogenesis, This maternal nucleolin is distributed throu ghout the cytoplasm of the egg during oocyte maturation and after fert ilization it is gradually reaccumulated in the nuclei of the embryo, C ytoplasmic localization of nucleolin coincides with massive phosphoryl ation by p34(cdc2) kinase, and nuclear translocation is accompanied by net dephosphorylation. Multiple phosphorylation consensus sites for t he cell cycle-dependent p34(cdc2) kinase and for protein kinase CK2 ar e clustered in the N-terminal domain of nucleolin, To assess the effic iency of the bipartite nuclear localization signal, we have constructe d fusion proteins consisting of maltose binding protein (MBP) and the nuclear localization signal of nucleolin, In addition, either an acidi c domain of nucleolin without phosphorylation sites, or an acidic doma in containing 4 CK2 sites, or a cluster of 5 cdc2 sites was fused to t he MBP-nuclear localization signal (MBP-NLS), Nuclear translocation of these constructs was tested in an in vitro system consisting of Xenop us egg extract and sperm nuclei, Nuclear targetting of MBP by the bipa rtite nuclear localization signal of nucleolin became significantly mo re efficient after addition of either CK2 sites or cdc2 sites to the M BP-NLS construct, Yet the cdc2 sites play a dual role, They enhance nu clear translocation exclusively in their dephosphorylated state and pr omote cytoplasmic localization when phosphorylated, thereby providing a powerful cell cycle-dependent regulatory element of the nuclear loca lization signal.