Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3.
The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappa B family. v-Rel causes rapidly fatal lymphomas in young chickens, and transforms and immortalizes chicken lymphoid cells in vitro. Several mutations that have enhanced the oncogenicity of v-Rel have been selected during in vitro and in vivo passage of v-Rel-containing retroviruses. In this report, we show that the C-terminal deletion and two point mutations (Asp-->Gly at residue 91 and Asp-->Asn at residue 437) in v-Rel make it resistant to cleavage by the cell-death protease caspase-3. In contrast, c-Rel, which has Asp residues at these sites, can be cleaved by caspase-3 in vitro as well as in vivo in cells induced to undergo apoptosis. We have characterized activities of v-Rel mutants with recreated single caspase-3 cleavage sites, two cleavage sites, or an introduced artificial cleavage site. All of these mutant v-Rel proteins are sensitive to caspase-3 cleavage in vitro, and show wild-type activity in terms of nuclear localization in chicken fibroblasts and DNA binding in vitro. Moreover, all caspase-3-sensitive v-Rel mutants transform chicken spleen cells in vitro and induce fatal lymphoid tumors in vivo to approximately the same extent as wild-type v-Rel. As with v-Rel mutants, caspase-3-resistant c-Rel mutants behave similarly to caspase-3-sensitive wild-type c-Rel in terms of DNA binding, transcriptional activation, in vitro transformation, and tumorigenicity. Mammalian c-Rel proteins can also be cleaved by caspase-3 in vitro, and a c-Rel mutant from a human pre-T lymphoma cell line is less sensitive than wild-type human c-Rel to cleavage by caspase-3. Taken together, these results demonstrate that specific mutations render oncogenic forms of Rel proteins resistant to cleavage by a cell-death caspase; however, the biological relevance of this resistance remains unclear. Nevertheless, to our knowledge, this is the first demonstration of mutations in caspase-3 recognition sites occurring during the evolution of an oncogenic protein.
Barkett
M
M
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Dooher
J E
JE
Lemonnier
L
L
Simmons
L
L
Scarpati
J N
JN
Wang
Y
Y
Gilmore
T D
TD
D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002645:Chickens; D019143:Evolution, Molecular; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020572:Oncogene Proteins v-rel / Q000235:genetics*; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D020572:Oncogene Proteins v-rel; D020569:Proto-Oncogene Proteins c-rel; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases