Activation of IκB kinase β by protein kinase C isoforms

The atypical protein kinase C (PKC) isotlvpes (lambda/tPKC and zeta PKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappa B by this cytokine through a mechanism that most probably involves the phosphorylation of I kappa B. The inability of these PKC isotypes to directly phosphorylate I kappa B led to the hypothesis that zeta PKC may use a putative I kappa B kinase to functionally inactivate I kappa B. Recently several groups have molecularly characterized and cloned two I kappa B kinases (IKK alpha and IKK beta) which phosphorylate the residues in the I kappa B molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappa B through the activation of the IKKs. We report here that alpha PKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zeta PKC positively modulates IKK beta activity but not that of IKK alpha, whereas the transfection of a zeta PKC dominant negative mutant severely impairs the activation of IKK beta but not IKK alpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKK beta, which is entirely dependent on the activity of alpha PKC but not that of the atypical isoforms. In contrast, the inhibition of alpha PKC does not affect the activation of IKK beta by TNF-alpha. Interestingly, recombinant active zeta PKC and alpha PKC are able to stimulate in vitro the activity of IKK beta but not that of IKK alpha. In addition, evidence is presented here that recombinant zeta PKC directly phosphorylates IKK beta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappa B pathway at the level of IKK beta activation and I kappa B degradation.