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Identification of a motif in BMRP required for interaction with Bcl‐2 by site‐directed mutagenesis studies

Bcl‐2 is an anti‐apoptotic protein that inhibits apoptosis elicited by multiple stimuli in a large variety of cell types. BMRP (also known as MRPL41) was identified as a Bcl‐2 binding protein and shown to promote apoptosis. Previous studies indicated that the amino‐terminal two‐thirds of BMRP contain the domain(s) required for its interaction with Bcl‐2, and that this region of the protein is responsible for the majority of the apoptosis‐inducing activity of BMRP. We have performed site‐directed mutagenesis analyses to further characterize the BMRP/Bcl‐2 interaction and the pro‐apoptotic activity of BMRP. The results obtained indicate that the 13–17 amino acid region of BMRP is necessary for its binding to Bcl‐2. Further mutagenesis of this motif shows that amino acid residue aspartic acid (D) 16 of BMRP is essential for the BMRP/Bcl‐2 interaction. Functional analyses conducted in mammalian cells with BMRP site‐directed mutants BMRP(13Ala17) and BMRP(D16A) indicate that these mutants induce apoptosis through a caspase‐mediated pathway, and that they kill cells slightly more potently than wild‐type BMRP. Bcl‐2 is still able to counteract BMRP(D16A)‐induced cell death significantly, but not as completely as when tested against wild‐type BMRP. These results suggest that the apoptosis‐inducing ability of wild‐type BMRP is blocked by Bcl‐2 through several mechanisms. J. Cell. Biochem. 113: 3498–3508, 2012. © 2012 Wiley Periodicals, Inc.