Functional dissection of the human Bcl2 protein: sequence requirements for inhibition of apoptosis.
Overexpression of the cytoplasmic oncoprotein Bcl2 blocks programmed cell death (apoptosis) in many cellular systems. To map the sequences in Bcl2 that are necessary for its activity, we created a library of deletion-scanning mutants of this 239-amino-acid protein and tested their abilities to block staurosporine-induced fibroblast apoptosis, using a novel transient-transfection assay. Phenotypes of informative mutants were then confirmed by assaying for inhibition of steroid-induced apoptosis in stably transfected T-lymphoid cells. In accordance with earlier results, we found that Bcl2 activity was only partially reduced after deletion of the hydrophobic tail that normally anchors it in cytoplasmic membranes. Essential sequences were found in the remainder of the protein and appeared to be organized in at least two discrete functional domains. The larger, more C-terminal region (within residues 90 to 203) encompassed, but extended beyond, two oligopeptide motifs called BH1 and BH2, which are known to mediate dimerization of Bcl2 and related proteins. The second, more N-terminal regions (within residues 6 to 31) was not required for protein dimerization in vivo, but its deletion imparted a dominant negative phenotype, yielding mutants that promoted rather than inhibited apoptotic death. Residues 30 to 91 were not absolutely required for function; by deleting most of this region along with the hydrophobic tail, we derived a 155-residue mini-Bcl2 that retains significant ability to inhibit apoptosis.
Hunter
J J
JJ
Department of Pathology, University of California, San Francisco 94143-0506, USA.
Bond
B L
BL
Parslow
T G
TG
D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D018014:Gene Transfer Techniques; D006801:Humans; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D017421:Sequence Analysis
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2