Mutations of tumor necrosis factor alpha-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability.
The human c-rel gene (REL), encoding an NF-kappaB transcription factor, is amplified or mutated in several human B-cell lymphomas and can transform chicken lymphoid cells in vitro. We have previously shown that certain deletions of C-terminal transactivation sequences enhance REL's transforming ability in chicken spleen cells. In this report, we have analysed the effect of single amino-acid changes at select serine residues in the C-terminal transactivation domain on REL's transforming ability. Mutation of either of two TNFalpha-inducible serine residues (Ser460 and Ser471) to nonphosphorylatable residues (alanine, asparagine, phenylalanine) made REL more efficient at transforming chicken spleen cells in vitro. In contrast, mutation of Ser471 to a phosphorylation mimetic aspartate residue impaired REL's transforming ability, even though it increased REL's inherent transactivation ability as a GAL4-fusion protein. Alanine mutations of several other serine residues within the transactivation domain did not substantially affect REL's transforming ability. Transactivation by GAL4-REL fusion proteins containing either transformation enhancing or nonenhancing mutations at serine residues was generally similar to wild-type GAL4-REL. However, more transforming mutants with mutations at either Ser460 or Ser471 differed from wild-type REL in their ability to transactivate certain kappaB-site reporter genes. In particular, the SOD2 promoter, encoding manganese superoxide dismutase, was activated less strongly by the more transforming REL mutant REL-S471N in transient assays, but REL-S471N-transformed chicken spleen cells had increased levels of MnSOD protein as compared to wild-type REL-transformed cells. Taken together, our results show that mutations of certain serine residues can enhance REL's transforming ability in vitro and suggest that these mutations increase REL-mediated transformation by altering REL's ability to modulate the expression of select target genes. Furthermore, phosphorylation of Ser471 may be involved in REL-mediated modulation of transformation-specific target gene expression. Lastly, these results suggest that similar mutations in the REL transactivation domain contribute to the development of certain human B-cell lymphomas.
Starczynowski
Daniel T
DT
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Reynolds
Joseph G
JG
Gilmore
Thomas D
TD
D000818:Animals; D015153:Blotting, Western; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002645:Chickens; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020569:Proto-Oncogene Proteins c-rel / Q000502:physiology*; D017384:Sequence Deletion; D012694:Serine / Q000737:chemistry / Q000235:genetics*; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D020569:Proto-Oncogene Proteins c-rel; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D008156:Luciferases; D013482:Superoxide Dismutase