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.
Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas 78363, USA.
Juan A
JA
Conde
Cheryl J
CJ
Claunch
Hannah E
HE
Romo
Ana
A
Benito-Martín
Rafael P
RP
Ballestero
Maribel
M
González-García
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D014162:Transfection
C502654:MRPL41 protein, human; D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012269:Ribosomal Proteins; D001224:Aspartic Acid