A point mutant of p53 containing arginine instead of proline at amino acid 71 (P71R) fails to interact with Sin3 in vivo or to be stabilized by exogenous Sin3. This mutant shows transactivation potential, however, that is indistinguishable from wt p53. (A) Transfection of H1299 cells with wt p53 or point mutants containing leucine at amino acid 80 (P80L) or arginine at amino acid 71 (P71R) followed by IP with Sin3 antiserum (AK-11; Santa Cruz Biotechnology) and immunoblotting with p53 antiserum (Ab-6; Calbiochem). The data indicate that the P71R mutant shows impaired interaction with Sin3 in transfected cells. (B) Transfection of H1299 cells with 50 ng of p53 or the P71R mutant, along with increasing concentrations of Sin3 expression plasmid (0.5 and 2.5 μg, lanes 2, 3, 5, and 6). Immunoblot analysis of p53 levels indicates that p53 is stabilized by Sin3 in a dose-dependent manner but that neither dose of Sin3 is capable of stabilizing the P71R mutant, which shows a defective interaction with Sin3. A β-actin control is included for protein loading. (C) Luciferase activity of the p53-inducible luciferase vector SpVII (a minimal promoter containing the E1B TATA box and a single p53 consensus element) transiently transfected into H1299 cells in the presence of parental vector (pRc/CMV) or increasing concentrations of wt p53 (25 and 100 ng) or the P71R point mutant of p53. The data depicted represent the fold increase in luciferase activity obtained after transfection, averaged from five independent experiments. Error bars depict the standard error of the mean for the five experiments. (D) Western analysis depicting comparable p53 levels expressed in the transfected cells analyzed in the results shown in panel C. A total of 1.25 μg of the SpVII luciferase reporter construct was cotransfected with either vector alone or increasing concentrations of wt p53 (25 and 100 ng), P71R (25 and 100 ng), and 100 ng of pEGFP (transfection control) in six-well plates and analyzed for p53 levels by Western analysis. Western analysis of the GFP levels between samples is used to show relatively equal transfection efficiencies, and equivalent protein loading is depicted by Western analysis for β-actin.
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