Novel PKC signaling in Alzheimer’s Disease

Abstract

Protein kinase C (PKC) isozymes are abundantly expressed in the brain, where the enhanced activity of conventional isozymes is associated with neurodegenerative diseases such as Alzheimer’s Disease (AD) and spinocerebellar ataxia. Whether novel PKC isozymes contribute to neurodegeneration remains to be established. Here we identify a rare variant in the novel isozyme, PKCη, present exclusively in affected siblings in families with AD. This mutation, K65R, is present on the C2 domain, a module which participates in maintaining autoinhibition of the enzyme but is located on a solvent‐exposed face that is not predicted to be involved in interdomain interactions. Unsurprisingly, live‐cell imaging experiments using a genetically encoded biosensor CKAR (C kinase activity reporter), revealed that the K65R mutant, overexpressed in COS7 cells, had the same basal activity as wild‐type (WT) PKCη, consistent with intact autoinhibition. However, PKCη K65R displayed a 20% increase in agonist‐stimulated activity compared with WT. This increase did not result from improper folding or maturation of the protein: western blot analysis revealed that both the overexpressed WT and PKCη K65R proteins were properly processed by priming phosphorylations at the activation loop (Thr513) and the two C‐terminal sites (Thr656 and Ser675). In contrast to the enhanced activity observed in cells, in vitro kinase assays of PKCη WT or K65R (using protein purified from insect cells) revealed that the mutation did not alter the kinetics of activation by phosphatidylserine or diacylglycerol, nor did it alter the Km for phosphorylation of peptide substrate. Furthermore, studies in HEK‐293T comparing the overexpressed PKCη WT to K65R revealed that the mutation did not alter protein stability. Specifically, cycloheximide treatment resulted in loss of PKCη with a similar half‐life of approximately 40 hours for both. To ensure overexpression did not saturate degradation machinery, we examined the turnover of endogenous PKCη in the microglial cell line BV‐2. We chose this line because analysis of the brain by cell type‐specific enhancer‐promotor interactome map revealed that PRKCH is expressed predominantly in microglia. Endogenous PKCη was readily detectable in this cell line and displayed the same turnover half time as that of overexpressed protein. In summary, our data suggest that K65R AD‐associated variant of PKCη confers higher kinase activity in a cellular environment without affecting the intrinsic activity of the pure protein or cellular stability of the enzyme, suggesting that post‐translational modifications or altered protein:protein interactions may be responsible for the enhanced cellular activity. Taken together with reports that PKCη RNA expression is increased in microglia from AD brain, our identification of an AD‐associated variant in PKCη with enhanced activity poises this novel PKC isozyme in a prime spot to regulate microglial‐mediated inflammation, a key contributor to the progression of AD.