FIGURE 1.

Identification of nuclear localization signal of I₂^PP2A. A, the structural similarities between human, chimpanzee, pig, bovine, rat, mouse, and Drosophila were analyzed to determine the conservation and homology of potential I₂^PP2A NLS among various species. B, schematic diagram of the constructs of I₂^PP2A and its NLS mutants employed to study their intracellular localization. C, photomicrographs of COS7 cells transiently transfected with vector (vec), HA-tagged human I₂^PP2A (I₂^PP2AWT), and its NLS mutants (I₂^PP2AAA, I₂^PP2AAAA, and I₂^PP2AAA-AAA) for the identification of critical regions of I₂^PP2A required for its translocation from the cell nucleus to the cytoplasm. Following 48 h of transfection with HA-tagged I₂^PP2A (WT and NLS mutants), COS7 cells were subjected to immunocytochemical staining with anti-HA, and TOPRO-3 was used for nuclear staining. Mutations at amino acid residues 178–181 markedly increased the cytoplasmic retention of I₂^PP2A. D, COS7 cells transiently transfected with R179A-I₂^PP2A and K180A-I₂^PP2A, followed by immunostaining with anti-HA (I₂^PP2A). Immunofluorescence images indicate that lysine 180 is required for I₂^PP2A nuclear localization (i.e. NLS). Scale bar, 50 μm.