The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (ζPKC and λ/ιPKC) are involved in the control of nuclear factor κB (NF-κB) through IKKβ activation. Here we show that the previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKβ to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKβ. These observations define a novel pathway for the activation of NF-κB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant λ/ιPKC impairs RIP-stimulated NF-κB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-κB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-κB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-κB by the TNFα signaling pathway.

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