Multisite phosphorylation and the nuclear localization of phosphatase inhibitor 2-green fluorescent protein fusion protein during S phase of the cell growth cycle

Human phosphatase inhibitor 2 (Inh2) is a phosphoprotein that complexes with type 1 protein phosphatase, and its expression peaks during S phase and mitosis during the cell cycle. Localization of Inh2 was visualized in HS68 human fibroblasts by fusing Inh2 to green fluorescent protein (GFP). During G1 phase, Inh2-GFP was localized in the cytoplasm, and as cells progressed into S phase Inh2-GFP accumulated in the nucleus. Known phosphorylation sites of Inh2 at Thr-72, Ser-86, and Ser-120/121 were each replaced with alanine. None of the mutated Inh2-GFP proteins 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 prevented the Inh2-GFP fusion protein from accumulating in the nucleus during S phase. Recombinant Inh2 was phosphorylated by kinases in cytosols prepared from G1 and S phase cells. The amount of Inh2 kinase attributed to casein kinase 2, based on inhibition by heparin, increased 2.6-fold from G1 to S phase. In addition, kinases in G1 versus S phase cytosols produced distinct Inh2 phosphopeptides. The results indicate that changes in phosphorylation of Inh2 are involved in intracellular redistribution of the protein during the cell cycle.