UC San Diego

Atypical protein kinase C (aPKC) isozymes are key modulators of insulin signaling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isozymes and the functionally-related Akt family of kinases. Here we show that aPKC is constitutively phosphorylated and basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mTORC2 mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by PDK1. Live cell imaging reveals that global aPKC activity is constitutive and insulin-unresponsive. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation.

We additionally show that protein scaffolds not only localize, but also differentially control the catalytic activity of PKCζ, thus promoting activity towards localized substrates and restricting activity towards global substrates. Using cellular substrate readouts and scaffolded activity reporters in live cell imaging, we show that PKCζ has highly localized and differentially-controlled activity on the scaffolds p62 and Par6. Both scaffolds tether aPKC in an active conformation as assessed through pharmacological inhibition of basal activity, monitored using a genetically-encoded reporter for PKC activity. However, binding to Par6 is of higher affinity and more effective in locking PKCζ in an active conformation. FRET-based translocation assays reveal that insulin promotes the association of both p62 and aPKC with the insulin-regulated scaffold IRS-1. Using the aPKC substrate MARK2 as another readout for activity, we show that overexpression of IRS-1 reduces the phosphorylation of MARK2 and enhances its plasma membrane localization, indicating sequestration of aPKC by IRS-1 away from MARK2. These results are consistent with scaffolds serving as allosteric activators of aPKCs, tethering them in an active conformation near specific substrates. Thus, signaling of these intrinsically low activity kinases is kept at a minimum in the absence of scaffolding interactions, which position the enzymes for stoichiometric phosphorylation of substrates co-localized on the same protein scaffold.