Abstract
Engagement of death receptors such as tumor necrosis factor-R1 and Fas brings about the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria to activate Bax/Bak, resulting in the release of cytochrome c. The mechanism underlying the activation, however, is not fully understood. Here, we have identified the anti-apoptotic Bcl-2 family member Mcl-1 as a potent tBid-binding partner. Site-directed mutagenesis reveals that the Bcl-2 homology (BH)3 domain of tBid is essential for binding to Mcl-1, whereas all three BH domains (BH1, BH2, and BH3) of Mcl-1 are required for interaction with tBid. In vitro studies using isolated mitochondria and recombinant proteins demonstrate that Mcl-1 strongly inhibits tBid-induced cytochrome c release. In addition to its ability to interact directly with Bax and Bak, tBid also binds Mcl-1 and displaces Bak from the Mcl-1-Bak complex. Importantly, overexpression of Mcl-1 confers resistance to the induction of apoptosis by both TRAIL and tumor necrosis factor-alpha in HeLa cells, whereas targeting Mcl-1 by RNA interference sensitizes HeLa cells to TRAIL-induced apoptosis. Therefore, our study demonstrates a novel regulation of tBid by Mcl-1 through protein-protein interaction in apoptotic signaling from death receptors to mitochondria.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis
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Apoptosis Regulatory Proteins / metabolism
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BH3 Interacting Domain Death Agonist Protein / genetics
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BH3 Interacting Domain Death Agonist Protein / metabolism*
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Cytochromes c / metabolism*
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HeLa Cells
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Humans
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Membrane Glycoproteins / metabolism
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Mice
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Mutagenesis, Site-Directed
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Myeloid Cell Leukemia Sequence 1 Protein
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism*
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Protein Binding
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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RNA Interference
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TNF-Related Apoptosis-Inducing Ligand
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Tumor Necrosis Factor-alpha / metabolism
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Two-Hybrid System Techniques
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bcl-2 Homologous Antagonist-Killer Protein / genetics
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bcl-2 Homologous Antagonist-Killer Protein / metabolism
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bcl-2-Associated X Protein / genetics
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bcl-2-Associated X Protein / metabolism
Substances
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Apoptosis Regulatory Proteins
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BH3 Interacting Domain Death Agonist Protein
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Mcl1 protein, mouse
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Membrane Glycoproteins
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Myeloid Cell Leukemia Sequence 1 Protein
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Neoplasm Proteins
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Proto-Oncogene Proteins c-bcl-2
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TNF-Related Apoptosis-Inducing Ligand
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TNFSF10 protein, human
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Tnfsf10 protein, mouse
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Tumor Necrosis Factor-alpha
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
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Cytochromes c