Nucleophosmin Blocks Mitochondrial Localization of p53 and Apoptosis

Sanjit Kumar Dhar,Daret K St Clair

doi:10.1074/jbc.m109.005736

Abstract

Activation of p53 is an important mechanism in apoptosis. However, whether the presence of p53 in mitochondria plays an important role in p53-mediated apoptosis is unclear. Here, we demonstrate that overexpression of NPM (nucleophosmin) significantly suppresses 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated apoptosis, in part, by blocking the mitochondrial localization of p53. Within 1 h following TPA treatment of skin epithelial (JB6) cells, p53 accumulated in mitochondria. Expression of NPM enhances p53 levels in the nucleus but reduces p53 levels in mitochondria, as detected by immunocytochemistry and Western blot analysis. The suppressive effect of NPM on p53 mitochondrial localization is also observed in TPA-treated primary epithelial cells and in JB6 cells treated with doxorubicin. NPM enhances the expression of p53 target gene p21 and bax. However, the increase in Bax level in the absence of p53 in mitochondria did not lead to an increase in TPA-induced apoptosis, suggesting that the presence of p53 in mitochondria is important. Suppression of NPM by NPM small interfering RNA leads to an increase of p53 levels in mitochondria and apoptosis. Furthermore, suppression of NPM in tumor cells with a high constitutive level of NPM results in p53 translocation to mitochondria and enhances TPA-mediated apoptosis. The results demonstrate the effect of NPM on p53 localization in mitochondria and apoptosis. Together, the data indicate that the presence of p53 in mitochondria plays an important role in stress-induced apoptosis and suggest that NPM may protect cells from apoptosis by reducing the mitochondrial level of p53.

Highlights

The linkage between mitochondrial oxidative stress and p53-induced apoptosis has attracted considerable attention for the following reasons
Our study shows that bcl2 does not play a major role in NPMmediated protection from TPA2-induced apoptosis and that the antiapoptotic function of NPM is associated with its ability to suppress p53 localization to mitochondria
NPM Protects Cells from tetradecanoylphorbol 13-acetate (TPA)-induced Apoptosis—Because overexpression of NPM reduces the level of p53 in the mitochondria, we investigated the effect of NPM in TPA-induced apoptosis

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The mouse skin epithelial cell line (JB6 Pϩ, clone 41, promotable by TPA treatment) was originally provided by Dr Nancy H. The nuclear pellets were resuspended in buffer B containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 35% glycerol, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitor (pepstatin, aprotinin, and leupeptin) at a concentration of 1 ␮g/ml and incubated on ice for 20 min. After incubation with the antibody, 20 ␮l of protein A/G (Santa Cruz Biotechnology) was added to the reaction mixture of the antibody and nuclear extract and rotated for 2 h at 4 °C. After twice washing the cells with 1ϫ PBS (pH 7.4), TUNEL reaction mixture was added, and the slide was incubated for 60 min in the dark in a humidified atmosphere at 37 °C.

RESULTS

DISCUSSION