Differences in κB DNA-binding properties of v-Rel and c-Rel are the result of oncogenic mutations in three distinct functional regions of the Rel protein

Abstract

The oncogene v-rel of Reticuloendotheliosis virus, strain T, is derived from an avian c-rel proto-oncogene. c-rel encodes a member of the Rel/NF-kB family of transcription factors. The highly oncogenic v-Rel di ers from c-Rel which has low transforming potential by the acquisition of numerous mutations. In this manuscript, we demonstrate that the oncogenic mutations in v-Rel directly alter the ability of this protein to bind to DNA. Electrophoretic mobility shift analysis with Rel proteins synthesized in vitro as well as isolated from nuclei of Rel expressing cells showed that three mutation clusters, present in the N-terminus, the center and the C-terminus of v-Rel, altered three di erent aspects of DNA binding. In contrast, the oncogenic C-terminal deletion of 118 amino acids present in v-Rel had almost no in¯uence on its DNA binding. The N-terminal mutation cluster altered the kB DNA-binding speci®city of the v-Rel oncoprotein relative to c-Rel. The mutation Met-20?Thr was found to be principally responsible for this alteration. The second mutation cluster was responsible for increased binding of v-Rel to all the kB sites examined presumably because it stabilized v-Rel homodimers. This alteration in DNA binding was mapped to the group of two mutations within the cluster. In contrast, the third mutation cluster in the C-terminus of v-Rel destabilized the binding of v-Rel to all of the kB sites examined. This is the ®rst indication that regions outside the Rel Homology Region can participate in the control of binding of the c-Rel protein to DNA. The three mutation clusters examined contributed to the tumorigenic potential of v-Rel with the relative strength decreasing with their position from the N-terminus to the C-terminus. These results suggest that the oncogenic mutations in v-Rel cooperate and enable v-Rel to form nuclear complexes with aberrant DNA-binding properties that may directly alter gene expression and DNA replication resulting in the transformation of the cell.

Aberrant Production of IL-12 by Macrophages from Several Autoimmune-Prone Mouse Strains Is Characterized by Intrinsic and Unique Patterns of NF-κB Expression and Binding to the IL-12 p40 Promoter

“…Taken together, these findings suggest that the precise composition of the heterodimer may be regulated by the stimulus encountered by the M . Additionally, it has been shown that 1) mutation of the 5Ј region of the B site leads to a shift in binding preference from p50/p65 to p50/c-Rel (25), and 2) mutated c-Rel proteins display unique affinities for different native B sites (26). Thus, subtle differences in the B site or in flanking sequences in different promoters may also contribute to the composition of the B/Rel heterodimer that binds this sequence.…”

Mutant envelope residues confer a transactivation function onto N-terminal sequences of the v-Rel oncoprotein

“…The Env mutations in v-Rel appear to contribute cooperatively to the transforming function of v-Rel, as no single aa change is su cient to endow a Rev-A Env Rel protein with a transforming ability equal to that of v-Rel; however, the two Phe residues at positions three and nine of v-Rel appear to be especially important for the It is clear that the N-terminal mutant Env aa are not absolutely essential for the transforming activity of mutant Rel proteins. That is, chicken c-Rel proteins with only a C-terminal deletion can transform chicken spleen cells fairly e ciently (HrdlicÏ kova et al, 1994;Kamens et al, 1990;Sachdev and Hannink, 1998) and removal of the Env aa from the N terminus of v-Rel lessens, but does not abolish, its transforming activity (Bhat and Temin, 1990;Garson et al, 1990;Nehyba et al, 1997). However, the original Rev-T isolate almost certainly did contain Rev-A Env aa at the N terminus of v-Rel, and thus, the Env mutations were likely selected based on their ability to enhance the oncogenicity of the original Env-Rel oncoprotein.…”

Regulation of DNA binding by Rel/NF-κB transcription factors: structural views

“…Previous experimental evidence has shown that Met20Thr is one of four residues which are responsible for di erent DNAbinding speci®cities between p50 and RelA (Coleman et al, 1993). Furthermore, Nehyba et al (1997) have also shown that Met20Thr in v-Rel alters DNA-binding speci®cities and have provided additional evidence that the Arg250Gly and Glu269Ala mutations in v-Rel cause promiscuous kB site binding. Regarding phosphorylation, it is unclear what e ect, if any, results from the mutation or loss of phosphorylatable residues in c-Rel, namely the change of a serine 285 in c-Rel to a proline in v-Rel, and the deletion of Ser-352 from cRel in v-Rel.…”