Transactivation by c-Rel (nuclear factor kappaB) was dependent on phosphorylation 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 activity; and 2) the 422-540 region, which responds to external stimuli as tumor necrosis factor (TNF) alpha or phorbol myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be required for TNFalpha-induced activation, whereas Ser between 492 and 519 were required for phorbol myristate acetate plus ionomycin activation. Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) zeta were identified as downstream signaling molecules of TNFalpha-activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKCzeta activation and dominant negative PKCzeta inhibited PI3K-mediated activation of c-Rel transactivating activity, indicating a cross-talk between both enzymes. We have identified the critical role of different Ser for PKCzeta- and PI3K-mediated responses. Interestingly, those c-Rel mutants not only did not respond to TNFalpha but also acted as dominant negative forms of nuclear factor kappaB activation.