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 to bind phorbol esters, is required during NF-xi B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by xi PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es), with xi PKC. The following evidence for interaction with the 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 molecular weight able to regulate I kappa B-alpha function. Activation of the I kappa B kinase was dependent on PKC enzymatic activity and ATP, suggesting that xi PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase. Importantly, xi PKC immunoprecipitates from TNF-alpha-stimulated NM-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings. We also show here that xi PKC associates with and activates MKK-MAPK in vitro, suggesting 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 is responsible for the putative I kappa B phosphorylating activity. These data provide a decisive step towards understanding the functions of xi PKC.