ZETA-PKC INDUCES PHOSPHORYLATION AND INACTIVATION OF I-KAPPA-B-ALPHA IN-VITRO

The xi isotype of protein kinase C (xi 3-PKC), a distinct PKC unable t o bind phorbol esters, is required during NF-xi B activation as well a s in mitogenic signalling in Xenopus oocytes and mammalian cells. To i nvestigate the mechanism(s) for control of cellular functions by xi PK C, this enzyme was expressed in Escherichia coli as a fusion protein w ith maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form co mplex(es), with xi PKC. The following evidence for interaction with th e NF-kappa B/I kappa B pathway was obtained. MBP-xi PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of -50 kDa mo lecular weight able to regulate I kappa B-alpha function. Activation o f the I kappa B kinase was dependent on PKC enzymatic activity and ATP , suggesting that xi PKC controls, directly or indirectly, the activit y of a functionally significant I kappa B kinase. Importantly, xi PKC immunoprecipitates from TNF-alpha-stimulated NM-3T3 fibroblasts displa yed a higher I kappa B phosphorylating activity than untreated control s, indicating the in vivo relevance of these findings. We also show he re that xi PKC associates with and activates MKK-MAPK in vitro, sugges ting that one of the mechanisms whereby overexpression of xi PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex. However, neither MKK nor MAPK i s responsible for the putative I kappa B phosphorylating activity. The se data provide a decisive step towards understanding the functions of xi PKC.