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.
Kakinoki
Y
Y
Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Somers
J
J
Brautigan
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D016193:G1 Phase; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D000097847:Protein Phosphatase Inhibitory Proteins
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins