Deletion of either C-terminal transactivation subdomain enhances the in vitro transforming activity of human transcription factor REL in chicken spleen cells

Abstract

The REL gene is amplified in many human B-cell lymphomas and we have previously shown that expression of REL from a retroviral vector can malignantly transform chicken spleen cells in vitro. To identify REL protein functions necessary for malignant transformation, we have performed deletion analysis on REL sequences encoding residues of two C-terminal subdomains that are involved in transcriptional activation. We find that deletion of both Cterminal transactivation subdomains abolishes the ability of REL to transform chicken spleen cells in vitro. In contrast, deletion of either transactivation subdomain alone, which reduces the transactivation ability of REL, enhances the transforming activity of REL. Transforming REL mutants missing C-terminal sequences can also be selected at a low frequency in vitro. The REL transactivation domain can be functionally replaced in transformation assays by a portion of the VP16 transactivation domain that activates at a level similar to RELtransforming mutants. We also find that deletion of 29 C-terminal amino acids causes the subcellular localization of REL to change from cytoplasmic to nuclear in chicken embryo fibroblasts. In contrast, wild-type REL and all transforming REL mutants are located primarily in the cytoplasm of transformed spleen cells. Nevertheless, treatment of transformed spleen cells with leptomycin B causes wild-type REL and two REL mutants to relocalize to the nucleus, and nuclear extracts from these transformed cells contain REL DNA-binding activity. Taken together, these results suggest the following: (1) that REL must activate transcription to transform cells in vitro; (2) that a reduced level of transactivation enhances the oncogenicity of REL; (3) that REL shuttles from the cytoplasm to the nucleus in transformed chicken spleen cells; and (4) that mutations in REL, in addition to amplifications, could activate its oncogenicity in human lymphomas.

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

“…Indeed, overexpression of human REL is sufficient to transform and immortalize primary chicken lymphoid cells in culture (Gilmore et al, 2001;Starczynowski et al, 2003;Fan et al, 2004). Conversely, the primary defect in c-rel knockout mice is one of B-cell survival and proliferation, in that c-Rel null B cells do not proliferate in response to many mitogens and show increased apoptosis (reviewed by Gerondakis et al, 2006, this issue).…”

“…However, it is important to note that in in vitro-transformed chicken spleen cells REL (and v-Rel) appear to be primarily cytoplasmic by immunofluorescence (Starczynowski et al, 2003), and yet the evidence is quite convincing that these oncogenic proteins are continually entering the nucleus to drive the gene transcription that causes transformation and immortalization of these cells (Gilmore, 1999;Gilmore et al, 2004). Thus, one cannot assume that cytoplasmic REL staining of lymphoma samples dictates the presence of only inactive REL complexes.…”

“…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.…”

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

“…REL contains an N-terminal Rel homology domain, which mediates DNA-binding, dimerization, nuclear localization and binding to its inhibitor, IkB. The C-terminal half of REL (aa 296-587) contains a transactivation domain, which is comprised of at least two subdomains: subdomain I (aa 422-497) and subdomain II (aa 518-587) (Martin et al, 2001;Starczynowski et al, 2003). The REL C-terminal transactivation domain can also be regulated by phosphorylation of specific serine (Ser) residues (Fognani et al, 2000;Martin and Fresno, 2000;Martin et al, 2001;Yu et al, 2004;Lawrence et al, 2005;Harris et al, 2006).…”

“…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).…”