Inhibition of p53 transcriptional activity by Bcl-2 requires its membrane-anchoring domain

We show here that the anti-apoptosis protein Bcl-2 potently inhibits p53-de pendent transcriptional activation of various p53-responsive promoters in r eporter gene co-transfection assays in human embryonic kidney 295 and MCF7 cells, without affecting nuclear accumulation of p53 protein. In contrast, Bcl-2(Delta transmembrane (TM)), which lacks a hydrophobic membrane-anchori ng domain, had no effect on p53 activity. Similarly, in MCF7 cells stably e xpressing either Bcl-2 or Bcl-2(Delta TM), nuclear levels of p53 protein we re up-regulated upon treatment with the DNA-damaging agents doxorubicin and UV radiation, whereas p53 responsive promoter activity and expression of p 21(CIP1/WAF1) were strongly reduced in MCF7-Bcl-2 cells but not in MCF7-Bcl -2(Delta TM) or control MCF7 cells. The issue of membrane anchoring was fur ther explored by testing the effects of Bcl-2 chimeric proteins that contai ned heterologous transmembrane domains from the mitochondrial protein ActA or the endoplasmic reticulum protein cytochrome b5, Both Bcl-2(ActA) and Bc l-2(Cytob5) suppressed p53-mediated transactivation of reporter gene plasmi ds with efficiencies comparable to wild-type Bcl-2. These results suggest t hat (a) Bcl-2 not only suppresses p53-mediated apoptosis but also interfere s with the transcriptional activation of p53 target genes at least in some cell lines, and (b) membrane anchoring is required for this function of Bcl -2. We speculate that membrane-anchored Bcl-2 may sequester an unknown fact or necessary for p53 transcriptional activity.