Regulation of Nuclear Factor k B Transactivation IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND PROTEIN KINASE C z IN c-Rel ACTIVATION BY TUMOR NECROSIS FACTOR a *

Transactivation by c-Rel (nuclear factor k B) was dependent on phosphorylation of several serines in the transactivation domain, indicating that it is a phospho-rylation-dependent Ser-rich domain. By Ser 3 Ala mu- tational 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) a or phorbol myristate acetate plus ionomycin. Ser from 454 to 473 were shown to be required for TNF a -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) z were identified as downstream signaling mol-ecules of TNF a -activation of c-Rel transactivating activity. Interestingly, dominant negative forms of PI3K inhibited PKC z activation and dominant negative PKC z 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 PKC z - and PI3K-mediated responses. Interest-ingly, those c-Rel mutants not only did not respond to TNF a but also acted as