Abstract
In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.
Highlights
In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest
Using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1)
We showed that p73 induces transcription-independent apoptosis via its TAD by a mitochondrial pathway and that this apoptotic activity is modulated by anti-apoptotic BclXL
Summary
In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). In response to apoptotic stimuli, p53 translocates to the mitochondria and activates the apoptotic effectors Bak or Bax, triggering the apoptotic signaling process via mitochondrial outer membrane permeabilization and cytochrome c release from the mitochondria This transcription-independent apoptosis is inhibited by the sequestration of cytoplasmic p53 by anti-apoptotic Bcl-2 family member Bcl-XL [15, 16]. Our results provide a molecular basis for the transcription-independent p73-mediated apoptosis through Bcl-XL
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