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.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/genetics , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Apoptosis , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , Humans , Interleukin-3/metabolism , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid , bcl-2-Associated X Protein , src Homology DomainsABSTRACT
DNA damage induces apoptosis through a signalling pathway that can be suppressed by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown. Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that RAD9, a human protein involved in the control of a cell-cycle checkpoint, interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x L, but not with the pro-apoptotic BAX and BAD. When overexpressed in mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x L. Conversely, antisense RAD9 RNA suppresses cell death induced by methyl methanesulphonate. These findings indicate that RAD9 may have a new role in regulating apoptosis after DNA damage, in addition to its previously described checkpoint-control and other radioresistance-promoting functions.
