Regulation of nuclear factor kappa B transactivation - Implication of phosphatidylinositol 3-kinase and protein kinase C xi IN c-Rel activation by tumor necrosis factor alpha

Transactivation by c-Rel (nuclear factor kappaB) was dependent on phosphory lation of several serines in the transactivation domain, indicating that it is a phosphorylation-dependent Ser-rich domain. By Ser --> Ala mutational and deletion analysis, we have identified two regions in this domain: 1) a C-terminal region (amino acids 540-588), which is required for basal activi ty; and 2) the 422-540 region, which responds to external stimuli as tumor necrosis factor (TNF) alpha or phorbol myristate acetate plus ionomycin. Se r from 454 to 473 were shown to be required for TNF alpha -induced activati on, whereas Ser between 492 and 519 were required for phorbol myristate ace tate plus ionomycin activation. Phosphatidylinositol S-kinase (PI3K) and pr otein kinase C (PKC) zeta were identified as downstream signaling molecules of TNF alpha -activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKC zeta activation and dominant negative PKC zeta inhibited PI3K-mediated activation of c-Rel transactivat ing activity, indicating a cross-talk between both enzymes. We have identif ied the critical role of different Ser for PKC zeta- and PI3K-mediated resp onses. Interestingly, those c-Rel mutants not only did not respond to TNF a lpha but also acted as dominant negative forms of nuclear factor kappaB act ivation.