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Updated: Aug 13, 2026
Bimolecular Fluorescence Complementation
Published on: April 15, 2011
Molecular cloning and characterization of Bif-1. A novel Src homology 3 domain-containing protein that associates
S M Cuddeback1, H Yamaguchi, K Komatsu
1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Pharmacology and Therapeutics, University of South Florida, Tampa, Florida 33612, USA.
Abstract:
Bax is a proapoptotic member of the Bcl-2 protein family that commits the cell to undergo programmed cell death in response to apoptotic stimuli. To gain further insights into Bax mechanisms, we have identified a novel Bax-binding protein, termed Bif-1, by using a yeast two-hybrid cloning technique. Bif-1 is an evolutionarily conserved cytoplasmic protein that contains a predicted Src homology 3 (SH3) domain located near its C terminus but shares no significant homology with members of the Bcl-2 family. A Northern blot analysis indicates that Bif-1 is expressed in most tissues with abundant expression in heart and skeletal muscle. Bif-1 is capable of interacting with Bax as demonstrated by yeast two-hybrid, coimmunoprecipitation, and immunofluorescence studies. Induction of apoptosis in murine pre-B hematopoietic cells FL5.12 by interleukin-3 withdrawal results in increased association of Bax with Bif-1, which is accompanied by a conformational change in the Bax protein. Overexpression of Bif-1 promotes Bax conformational change, caspase activation, and apoptotic cell death in FL5.12 cells following interleukin-3 deprivation. Bif-1 thus represents a new type of regulator of Bax-mediated signaling pathways for apoptosis.
Insights
Researchers discovered Bif-1, a novel protein that binds to Bax. Bif-1 regulates Bax-mediated apoptosis by inducing conformational changes, promoting cell death. This finding reveals a new pathway in programmed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Bax is a key proapoptotic protein in the Bcl-2 family, crucial for initiating programmed cell death.
- Understanding Bax's regulatory mechanisms is vital for insights into apoptosis.
- Novel protein interactions influencing Bax function remain an area of active research.
Purpose of the Study:
- To identify novel proteins that interact with Bax.
- To characterize the function of a newly identified Bax-binding protein, Bif-1.
- To elucidate Bif-1's role in Bax-mediated apoptosis.
Main Methods:
- Yeast two-hybrid cloning to identify Bax-interacting proteins.
- Coimmunoprecipitation and immunofluorescence assays to confirm protein interactions.
- Northern blot analysis to determine Bif-1 expression patterns.
- Apoptosis induction in FL5.12 cells to study Bif-1's functional effects.
Main Results:
- A novel Bax-binding protein, Bif-1, was identified.
- Bif-1 interacts with Bax and is expressed in various tissues, notably muscle.
- Interleukin-3 withdrawal induced apoptosis, leading to increased Bax-Bif-1 association and Bax conformational changes.
- Bif-1 overexpression promoted Bax conformational changes, caspase activation, and apoptosis.
Conclusions:
- Bif-1 is a novel, evolutionarily conserved regulator of Bax.
- Bif-1 influences Bax conformation and signaling pathways, promoting apoptosis.
- This discovery offers new perspectives on the regulation of programmed cell death.
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