A DOMINANT-NEGATIVE PROTEIN-KINASE-C ZETA-SUBSPECIES BLOCKS NF-KAPPA-B ACTIVATION

Nuclear factor kappaB (NF-kappaB) plays a critical role in the regulation of a number of genes. NF-kappaB is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein IkappaB. Following stimulation of cells, IkappaB dissociates from NF-kappaB, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappaB activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappaB. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zetaPKC, is a required step in the activation of NF-kappaB in response to ras p21. We demonstrate here that overexpression of zetaPKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappaB into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zetaPKC dramatically inhibits the kappaB-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zetaPKC plays a decisive role in NF-kappaB regulation in mammalian cells.