FIG.1.
Disruption of both basic amino acid parts but not individual amino acids in the bipartite NLS abolishes p53 nuclear import. (A) Diagram of p53 with positions and amino acid sequences of the bipartite NLS and C-terminal NES indicated. Amino acid substitutions used in the study are indicated in bold face. (B) Combining an NES mutation revealed a nuclear import activity in the p53KKK mutant. U2-OS cells were transfected with the indicated plasmids, and pictures were taken 24 h after transfection with living cells. (C) Degradation of p53KKK mutant by MDM2. U2-OS cells were transiently transfected with indicated p53 and MDM2 plasmid DNAs along with a GFP plasmid. Twenty-four hours after transfection, total cell lysate was prepared from each transfected cell population, electrophoretically separated by SDS-PAGE, and immunoblotted with antibodies specific to MDM2 (SMP14), p53 (DO1), and GFP (Ab2). (D) Various contributions of the basic amino acids in the bipartite NLS to the nuclear import of p53. The conserved basic amino acids in the bipartite NLS of p53 were replaced with alanine residues individually or in combination as indicated in panel A. The plasmid DNA was transfected into U2-OS cells, and the pictures were taken of living cells 24 h after transfection. (E) Quantification of GFP-positive cells. The GFP fluorescence pattern of each transfected cell population expressing the indicated p53 constructs was scored for at least 200 cells. The graph shows the percentage of cells with the indicated GFP patterns.