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

Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. 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 Bax in response to UV irradiation is still controversial. In this study, based on real-time single-cell analysis and fluorescence resonance energy transfer (FRET) technique, we investigated the tripartite nexus between PUMA, Bax and Bcl-XL in living human lung adenocarcinoma cells (ASTC-a-1) to illustrate how PUMA promotes Bax translocation to initiates apoptosis. Our results show that the interaction between PUMA and Bax increased gradually, with Bax translocating to mitochondria and co-localizing with PUMA after UV irradiation, indicating PUMA promotes Bax translocation directly. Simultaneously, the interaction increased markedly between PUMA and Bcl-XL while decreased significantly between Bcl-XL and Bax after UV treatment, suggesting PUMA competitive binds to Bcl-XL to activate Bax indirectly. The above results were further confirmed by the experiments of co-immunoprecipitation. In addition, Pifithrin-α (p53 inhibitor) and CHX (protein synthesis inhibitor) could inhibit PUMA mediated Bax translocation and cell apoptosis. Taken together, these studies create a concernful conclusion that PUMA promotes Bax translocation by both directly interacting with Bax and competitive binding to Bcl-XL in UV-induced apoptosis.