Tumor necrosis factor-alpha activation of NF-kappa B requires the phosphorylation of Ser-471 in the transactivation domain of c-Rel

Activation of the transcription factor NF-KB is controlled at two levels in resting T cells: an initial activation induced by the triggering of the Tc R.CD3 complex and a second phase controlled by paracrine- or autocrine-secr eted TNF alpha, The initial phase is regulated by p65 (ReLA), whereas the s econd one is mainly dependent on c-Rel, We describe here a mutant clone, D6 , derived from the parental T lymphoblastic line Jurkat that fails to activ ate NF-kappa B upon TNF alpha stimulation. This clone had no alteration in tumor necrosis factor-alpha (TNF alpha) signaling pathways nor in I kappa B alpha, -beta, or -is an element of expression and degradation. However, TN Fa induced an exacerbated apoptotic response in this clone compared with Ju rkat cells. This mutant clone showed a defect in the intermediate-late tran slocation of c-Rel to the nucleus promoted by TNF alpha stimulation, wherea s early translocation is not affected. Activation or translocation of p65-c ontaining complexes was not altered in this mutant clone. Sequencing of the c-Rel gene from this clone revealed a mutation of Ser-471 to Asn in the tr ansactivation domain. The mutant S471N transactivation domain fused to the Gal4 DNA binding domain could not be activated by TNF alpha, unlike the wil d type. Moreover, the overexpression of the mutant protein c-Rel S471N into Jurkat cells abolished TNF alpha-induced NF-kappa B activity, thus demonst rating that this mutation is responsible for the failure of TNF alpha stimu lation of NF-kappa B. Moreover, extracts from TNF alpha-stimulated Jurkat c ells phosphorylated in vitro recombinant wild type GST-c-Rel 464-481 but no t the GST-c-Rel mutant. Thus, TNFa-induced phosphorylation of Ser-471 seems to be absolutely necessary for TNF alpha activation of c-Rel.