Analysis of the role of conserved cysteine residues in the bcl-2 oncoprotein.
The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.
Maser
R S
RS
Department of Medicine, University of Pittsburgh, University of Pittsburgh Cancer Institute, BST E1055, 211 Lothrop Street, Pittsburgh, Pennnsylvania, 15213-2582, USA.
Antoku
K
K
Scully
W J
WJ
Cho
R L
RL
Johnson
D E
DE
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017124:Conserved Sequence / Q000235:genetics*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D053938:DNA Fragmentation; D019281:Dimerization; D007377:Interleukin-3 / Q000494:pharmacology; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D007377:Interleukin-3; D007473:Ion Channels; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D012694:Serine; D011065:Poly(ADP-ribose) Polymerases; D003545:Cysteine