Mutations of tumor necrosis factor α-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability

Abstract

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.

Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL's in vitro transforming activity

“…We have recently shown that mutations at Ser460 or Ser471, which enhance REL's transforming ability, also affect MnSOD expression in opposite ways in A293 cells versus chicken spleen cells (Starczynowski et al, 2005). As discussed therein, the differences in MnSOD expression in transient transfection experiments in A293 cells versus in stably transformed chicken spleen cells may reflect the kinetics of REL-induced gene expression.…”

“…As a control, we also measured MnSOD protein levels in cells expressing REL-S533,536A, a mutant that behaves similarly to wild-type REL in transformation and transactivation assays (Martin et al, 2001;Starczynowski et al, 2005). The level of MnSOD protein in A293 cells overexpressing REL-S525P was approximately 4.5-fold higher than in cells transfected with the vector control, whereas the levels of MnSOD protein in cells overexpressing wild-type REL or REL-S533,536A were approximately 7.5-fold higher than in control cells ( Figure 5a).…”

“…Mutation of Ser525 to proline impairs TNFa-induced signaling to REL Ser525 has not been previously identified as a TNFaresponsive Ser residue in REL (Martin and Fresno, 2000;Martin et al, 2001;Starczynowski et al, 2005). To determine whether TNFa can enhance transactivation by REL-S525P, we performed GAL4-REL reporter gene assays in A293 cells in the presence and absence of TNFa.…”

“…Deletion of either C-terminal transactivation subdomain enhances the in vitro transforming activity of REL in chicken spleen cells, but such deletions reduce the overall transactivation ability of REL (Starczynowski et al, 2003). Mutation of certain tumor necrosis factor (TNF)a-responsive Ser residues in the transactivation domain also enhances REL's in vitro transforming ability and alters its ability to transactivate certain kB site-containing promoters (Starczynowski et al, 2005).…”

Mutations in the NF-κB signaling pathway: implications for human disease

“…Interestingly, in one of the lymphoma patients the S525P mutation is a germline mutation, suggesting that this alteration could be a predisposing mutation for lymphoma. It would not be surprising if other human B-cell lymphomas, with or without REL gene amplification, have activating mutations in REL, especially given that a variety of point mutations and deletions within the REL transactivation domain can enhance the in vitro transforming activity of REL (Starczynowski et al, 2003(Starczynowski et al, , 2005.…”