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Vol. 20, Issue 13, 3077-3087, July 1, 2009

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

Ministry of Education Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

Submitted November 12, 2008; Revised March 9, 2009; Accepted May 4, 2009
Monitoring Editor: Thomas D. Fox

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.

Address correspondence to: Da Xing (xingda{at}scnu.edu.cn).

Abbreviations used: Bax, Bcl-2-associated X protein; BH, Bcl-2 homology domain; CCK-8, Cell Counting Kit-8; CFP, cyan fluorescent protein; DsRed, red fluorescent protein; GFP, green fluorescent protein; MOMP, mitochondrial outer membrane permeabilization; PUMA, p53 up-regulated modulator of apoptosis.