MULTISITE PHOSPHORYLATION AND THE NUCLEAR-LOCALIZATION OF PHOSPHATASEINHIBITOR 2-GREEN FLUORESCENT PROTEIN FUSION PROTEIN DURING S-PHASE OF THE CELL-GROWTH CYCLE

Human phosphatase inhibitor 2 (Inh2) is a phosphoprotein that complexe s with type 1 protein phosphatase, and its expression peaks during S p hase and mitosis during the cell cycle. Localization of Inh2 was visua lized in HS68 human fibroblasts by fusing Inh2 to green fluorescent pr otein (GFP). During G(1) phase, Inh2-GFP was localized in the cytoplas m, and as cells progressed into S phase Inh2-GFP accumulated in the nu cleus, Known phosphorylation sites of Inh2 at Thr-72, Ser-86, and Ser1 20/121 were each replaced with alanine. None of the mutated Inh2-GFP p roteins accumulated in the nucleus during S phase, indicating that all of these phosphorylation sites were required, Mutation of two lysine residues in a putative nuclear localization sequence in Inh2 also prev ented the Inh2-GFP fusion protein from accumulating in the nucleus dur ing S phase. Recombinant Inh2 was phosphorylated by kinases in cytosol s prepared from G(1) and S phase cells, The amount of Inh2 kinase attr ibuted to casein kinase 2, based on inhibition by heparin, increased 2 .6-fold from G(1) to S phase, In addition, kinases in G(1) versus S ph ase cytosols produced distinct Inh2 phosphopeptides. The results indic ate that changes in phosphorylation of Inh2 are involved in intracellu lar redistribution of the protein during the cell cycle.