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Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET

Volume 11, Issue 19   October 1, 2012
Pages 3536 - 3542
http://dx.doi.org/10.4161/cc.21462
Keywords: ABT-263, ABT-737, BH3-only proteins, Bcl-2, Bcl-XL, Bim, live MCF-7 cells, mitochondria, protein-protein interactions
Authors: Qian Liu, Brian Leber and David W. Andrews

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Abstract:
Increased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initiation, progression and resistance to traditional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to chemotherapy. Recently, we applied fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pattern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the corresponding BimEL mutant showed no decrease in binding to these anti-apoptotic proteins. Therefore, in MCF-7 cells, Bim has unique binding properties compared with other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics.

Received: June 29, 2012; Accepted: July 11, 2012; Published Online: August 16, 2012

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