Direct Activation of Bax by p53 Mediates Mitochondrial Membrane Permeabilization and Apoptosis

Abstract

The tumor suppressor p53 exerts its anti-neoplastic activity primarily through the induction of apoptosis. We found that cytosolic localization of endogenous wild-type or trans-activation-deficient p53 was necessary and sufficient for apoptosis. p53 directly activated the proapoptotic Bcl-2 protein Bax in the absence of other proteins to permeabilize mitochondria and engage the apoptotic program. p53 also released both proapoptotic multidomain proteins and BH3-only proteins [Proapoptotic Bcl-2 family proteins that share only the third Bcl-2 homology domain (BH3)] that were sequestered by Bcl-xL. The transcription-independent activation of Bax by p53 occurred with similar kinetics and concentrations to those produced by activated Bid. We propose that when p53 accumulates in the cytosol, it can function analogously to the BH3-only subset of proapoptotic Bcl-2 proteins to activate Bax and trigger apoptosis.

Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress

“…Importantly, however, expression of high amounts of p53 without additional DNA-damage did not induce apoptosis in NIH3T3 cells (Figure 4a, b), indicating that damage-induced activation of p53 is required for the pro-apoptotic effect. In accordance with our observation that higher p53 levels do not correlate with higher expression levels of p53 targets (see before), the higher p53 amounts in HA-p53 transfected and Cytosolic p53 accumulates in pre-apoptotic cells and correlates with Bax-activation Recent studies have linked non-transcriptional proapoptotic activities of p53 to a cytosolic or mitochondrial localization of the protein (Chipuk et al, 2003(Chipuk et al, , 2004Dumont et al, 2003;Mihara et al, 2003;Leu et al, 2004). To test whether such a non-transcriptional activity of p53 might account for 100 J/m 2 UV-induced apoptosis in NIH3T3 cells, we monitored differently irradiated NIH3T3 cells for the presence of cytosolic p53.…”

“…Further analysis assigned the pro-apoptotic activity of high amounts of activated p53 to cytosolic p53, which was able to trigger Bax-activation. Previous in vitro studies had already shown that cytosolic p53 can activate Bax in a transcription-independent manner analogous to that of BH3-only-proteins, and thereby lead to apoptosis (Chipuk et al, 2003(Chipuk et al, , 2004. Our data demonstrate for the first time the relevance of this mechanism in vivo.…”

“…However, cytosolic p53 accumulated to high amounts upon apoptosis-inducing 100 J/m 2 UV-irradiation (Figure 5b, right panel), probably reflecting the high amounts of total p53 in these cells. Cytosolic accumulation of p53, achieved by inhibiting nuclear import, was reported to directly activate Bax, thereby leading to induction of mitochondria-mediated apoptosis (Chipuk et al, 2004). We therefore investigated whether intrinsically generated high cytosolic p53 Dissection of p53 apoptotic activities D Speidel et al levels in 100 J/m 2 UV-irradiated NIH3T3 cells are coupled to activation of Bax.…”

“…Elegant in vitro studies demonstrated the ability of p53 to directly activate Bax in a transcription-independent manner (Chipuk et al, 2003(Chipuk et al, , 2004. To prove that p53 and not another factor is responsible for Bax-activation also in our in vivo system, we analysed isogenic mouse fibroblasts based on the p53-deficient cell line 10-1 (Harvey and Levine, 1991;Ziegler, 1998).…”

“…Possible mechanisms for non-transcriptional p53 apoptosis, however, were only recently reported. While some studies suggest that translocation of the p53 protein itself to or into the mitochondria is responsible for its pro-apoptotic effect (Marchenko et al, 2000;Dumont et al, 2003;Mihara et al, 2003;Leu et al, 2004), others report a transcription-independent, direct activation of the pro-apoptotic protein Bax by cytosolic p53 (Chipuk et al, 2003(Chipuk et al, , 2004. However, neither the physiological impact of transcription-independent mechanisms for the induction of apoptosis in vivo nor the coordination between transcriptional and non-transcriptional p53 activities has been sufficiently clarified.…”