PUMA Promotes Bax Translocation by Both Directly Interacting with Bax and by Competitive Binding to Bcl-X_L during UV-induced Apoptosis

Abstract

Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. p53 up-regulated modulator of apoptosis (PUMA) has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which PUMA regulates apoptosis, especially how PUMA activates Bcl-2-associated X protein (Bax) in response to UV irradiation is still controversial. In this study, by using real-time single-cell analysis and fluorescence resonance energy transfer, we investigated the tripartite nexus among PUMA, Bax, and Bcl-X L in living human lung adenocarcinoma cells (ASTC-a-1) to illustrate how PUMA promotes Bax translocation to initiate apoptosis. Our results show that the interaction between PUMA and Bax increased gradually, with Bax translocating to mitochondria and colocalizing with PUMA after UV irradiation, indicating PUMA promotes Bax translocation directly. Simultaneously, the interaction increased markedly between PUMA and Bcl-X L and decreased significantly between Bcl-X L and Bax after UV treatment, suggesting PUMA competitively binds to Bcl-X L to activate Bax indirectly. The above-mentioned results were further confirmed by coimmunoprecipitation experiments. In addition, pifithrin-␣ (a p53 inhibitor) and cycloheximide (a protein synthesis inhibitor) could inhibit PUMA-mediated Bax translocation and cell apoptosis. Together, these studies create an important conclusion that PUMA promotes Bax translocation by both by directly interacting with Bax and by competitive binding to Bcl-X L in UV-induced apoptosis. INTRODUCTIONUV irradiation is a potent carcinogen that can impair cellular functions by directly damaging DNA to induce apoptosis. The cellular response to DNA damage is centered on p53, a transcription factor that exerts its tumor-suppressive function by inducing cell cycle arrest, cell senescence, or apoptosis (Vousden and Lu, 2002). p53 stimulates a wide network of signals to activate the caspases that mediate apoptosis (Strasser et al., 1995). It has been shown that the apoptosis induced by p53 is in large part due to its ability to transcriptionally activate target genes (Chao et al., 2000). Many genes have been identified that are implicated in p53-mediated apoptosis (Vousden and Lu, 2002), such as PUMA and p21. Among the candidate proapoptotic p53 target genes, those that encode mitochondrial proteins such as PUMA stand out because p53-initiated apoptosis seems to proceed through a mitochondrial pathway (Polyak et al., 1997;Li et al., 1999;Soengas et al., 1999;Schuler et al., 2000).p53 up-regulated modulator of apoptosis (PUMA), a member of the Bcl-2 homology (BH)3-only Bcl-2 family proteins, also known as Bcl-2 binding component 3, is a direct transcriptional target of the p53 (Han et al., 2001;Nakano and Vousden, 2001;Yu et al., 2001;Chipuk et al., 2005). PUMA is localized in the mitochondria and induces apoptosis by activating caspases through mitochondrial dysfunction (Nakano and Vousden, 2001;Yu et al., ...

Distinct target genes and effector processes appear to be critical for p53-activated responses to acute DNA damage versus p53-mediated tumour suppression

“…This is consistent with the finding that Puma can bind with high affinity and therefore neutralise all pro-survival Bcl-2 family members, whereas Noxa interacts only with Mcl-1 and A1 [88,101,102]. Another reason why Puma may be a more potent killer than Noxa is provided by several studies, which showed that only the former is able to bind to and directly activate Bax [86,87,103,104].…”

Regulation of memory B-cell survival by the BH3-only protein Puma

“…Puma induces apoptosis by binding and antagonizing all of the 5 pro-survival Bcl-2 family members or, possibly more controversially, by directly activating the proapoptotic multi-BH domain protein Bax. [37][38][39][40][41][42] The proapoptotic effect of Puma might therefore be counteracted by one or several antiapoptotic proteins of the Bcl-2 family. Mcl-1 is an outstanding candidate for this role because it has recently been shown to be critical for the formation and of the germinal center reaction and establishment of B-cell memory.…”

Nuclear Poly (A)-Binding Protein and Oculopharyngeal Muscular Dystrophy

“…Puma may also directly interact with Bax, promoting its mitochondrial translocation (Chipuk et. al., 2004;Zhang et. al., 2009).…”

“…The Puma/Bax dependent pathway is usually triggered by a variety of cellular stress such as heat shock and oxygen stress (Uo et. al., 2007;Zhang et. al., 2009).…”