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, 19 (1), 307-16

Mapping of a Serine-Rich Domain Essential for the Transcriptional, Antiapoptotic, and Transforming Activities of the v-Rel Oncoprotein

Affiliations

Mapping of a Serine-Rich Domain Essential for the Transcriptional, Antiapoptotic, and Transforming Activities of the v-Rel Oncoprotein

C Chen et al. Mol Cell Biol.

Abstract

The v-Rel oncoprotein belongs to the Rel/NF-kappaB family of transcription factors and induces aggressive lymphomas in chickens and transgenic mice. Current models for cell transformation by v-Rel invoke the combined activation of gene expression and the dominant inhibition of transcription mediated by its cellular homologs. Here, we mapped a serine-rich transactivation domain in the C terminus of v-Rel that is necessary for its biological activity. Specific serine-to-alanine substitutions within this region impaired the transcriptional activity of v-Rel, whereas a double mutant abolished its function. In contrast, substitutions with phosphomimetic aspartate residues led to a complete recovery of the transcriptional potential. The transforming activity of v-Rel mutants correlated with their ability to inhibit programmed cell death. The transforming and antiapoptotic activities of v-Rel were abolished by defined Ser-to-Ala mutations and restored by most Ser-to-Asp substitutions. However, one Ser-to-Asp mutant showed wild-type transactivation ability but failed to block apoptosis and to transform cells. These results show that the transactivation function of v-Rel is necessary but not sufficient for cell transformation, adding an important dimension to the transformation model. It is possible that defined protein-protein interactions are also required to block apoptosis and transform cells. Since v-Rel is an acutely oncogenic member of the Rel/NF-kappaB family, our data raise the possibility that phosphorylation of its serine-rich transactivation domain may regulate its unique biological activity.

Figures

FIG. 1
FIG. 1
Effect of C-terminal deletions on the transcriptional and transforming activities of v-Rel. (A) Structures of v-Rel deletion mutants. RHR, Rel homology region; NLS, nuclear localization sequence; TA, transactivation domain. v-Rel sequences derived from the envelope gene of Rev-A are shown as hatched boxes. (B) Effect of deletion mutants on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected with 1.2 μg of CMV expression vectors for wild-type v-rel or mutant v-rel genes and 0.8 μg of reporter plasmid pIL6κBCAT. pCMV-b10 was used as a negative control. Assays were performed with 50 μg of protein for 2.5 h. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations.
FIG. 2
FIG. 2
Transcriptional activity of GAL4-Rel fusion proteins. (A) v-Rel subfragments fused to the yeast GAL4 DNA-binding domain. v-Rel sequences derived from the envelope gene of Rev-A are shown as hatched boxes. The positions of Ser-to-Ala mutations are indicated as black boxes (see Fig. 3A). TA, transactivation domain. (B) Transcriptional activity of GAL4-Rel fusion proteins. Undifferentiated Tera-2 cells were cotransfected with 1.2 μg of GAL4-Rel fusion genes and 0.8 μg of pG5BCAT. The pCG147 vector was used as a negative control. Assays were performed with 10 μg of protein for 2 h. Relative CAT activity from the average of three independent experiments is plotted. Error bars show standard deviations.
FIG. 3
FIG. 3
Transcriptional activity of serine mutants of v-Rel. (A) Serine mutations in the C-terminal half of v-Rel. “A” mutants contained substitutions of alanine for serine residues. “D” mutants contained substitutions of aspartate for serine residues. RHR, Rel homology region; TA, transactivation domain. (B) Effect of alanine substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected with 1.2 μg of CMV expression vectors for wild-type v-rel or mutant v-rel genes and 0.8 μg of pIL6κBCAT. pCMV-b10 was used as a negative control. Assays were performed with 50 μg of protein for 2.5 h. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations. (C) Effect of alanine and aspartate substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected and analyzed for CAT activity as described above. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations.
FIG. 3
FIG. 3
Transcriptional activity of serine mutants of v-Rel. (A) Serine mutations in the C-terminal half of v-Rel. “A” mutants contained substitutions of alanine for serine residues. “D” mutants contained substitutions of aspartate for serine residues. RHR, Rel homology region; TA, transactivation domain. (B) Effect of alanine substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected with 1.2 μg of CMV expression vectors for wild-type v-rel or mutant v-rel genes and 0.8 μg of pIL6κBCAT. pCMV-b10 was used as a negative control. Assays were performed with 50 μg of protein for 2.5 h. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations. (C) Effect of alanine and aspartate substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected and analyzed for CAT activity as described above. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations.
FIG. 3
FIG. 3
Transcriptional activity of serine mutants of v-Rel. (A) Serine mutations in the C-terminal half of v-Rel. “A” mutants contained substitutions of alanine for serine residues. “D” mutants contained substitutions of aspartate for serine residues. RHR, Rel homology region; TA, transactivation domain. (B) Effect of alanine substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected with 1.2 μg of CMV expression vectors for wild-type v-rel or mutant v-rel genes and 0.8 μg of pIL6κBCAT. pCMV-b10 was used as a negative control. Assays were performed with 50 μg of protein for 2.5 h. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations. (C) Effect of alanine and aspartate substitutions on the transcriptional activity of v-Rel. Undifferentiated Tera-2 cells were cotransfected and analyzed for CAT activity as described above. The average fold activation normalized to that for pCMV-v-rel from three independent experiments is plotted. Error bars show standard deviations.
FIG. 4
FIG. 4
Effect of C-terminal serine mutations on the DNA-binding activity of wild-type or mutant v-Rel. 35S-labeled v-Rel proteins were incubated with a 32P-labeled oligonucleotide containing an IL6-κB DNA-binding motif. DNA-protein complexes were resolved from the unbound probe on a 5% native polyacrylamide gel.
FIG. 5
FIG. 5
Competitive inhibition of c-Rel-mediated transcription by wild-type or mutant v-Rel proteins. COS-7 cells were cotransfected with 3 μg of pIL6κBCAT and 2 μg of CMV c-rel plasmid alone or together with 10 μg of CMV v-rel vectors. The total amount of transfected DNA (15 μg) was kept constant by the addition of pJDCMV19SV vector DNA. CAT assays were performed with 10 μg of total cellular protein for 1 h. Error bars show standard deviations.
FIG. 6
FIG. 6
Antiapoptotic activity of wild-type or mutant v-Rel proteins. (A) Immunoblot analysis of wild-type or mutant v-Rel protein expression in tetracycline-regulated HtTA-1-derived cell clones. Cells were maintained in the presence of tetracycline, and v-Rel expression was induced by the removal of the drug for 48 h. Extracts (20 μg) were resolved by SDS-PAGE and analyzed by enhanced chemiluminescence-immunoblotting with an antibody specific for the N terminus of v-Rel (1967). An antiactin antibody was used as a control. (B) Analysis of cell survival of TNF-α-induced apoptosis. The parental HtTA-1 cell clone (control) and HtTA-derived cells expressing wild-type or mutant v-Rel proteins were induced for protein production following the removal of tetracycline for 48 h. Cells were treated with CHX alone or together with TNF-α for 14 h. Cell survival was quantitated by crystal violet staining. Relative cell viability represents the ratio of the optical density of cells treated with TNF-α together with CHX to that of cells treated with CHX alone. The average viability observed in three independent experiments is plotted. Error bars show standard deviations.
FIG. 7
FIG. 7
Homology between the v-Rel transactivation domain and a portion of a serine-rich domain in human transcription factor Oct1/OTF1. Double lines indicate identical residues; single lines indicate conserved residues.

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