Identification of a tumor-associated mutant form of the NF-kappaB RelA gene with reduced DNA-binding and transactivating activities.
Alterations of NF-kappaB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for kappaB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-kappaB members, such as p50, cREL and Ikappa Balpha. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.
Trecca
D
D
Istituto di Scienze Mediche, Università di Milano, Ospedale Maggiore IRCCS, Milan, Italy.
Guerrini
L
L
Fracchiolla
N S
NS
Pomati
M
M
Baldini
L
L
Maiolo
A T
AT
Neri
A
A
D000818:Animals; D019556:COS Cells; D002471:Cell Transformation, Neoplastic; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D009101:Multiple Myeloma / Q000235:genetics*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D015534:Trans-Activators / Q000494:pharmacology*; D051996:Transcription Factor RelA
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D015534:Trans-Activators; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA