Protein kinase C? gain-of-function variant in Alzheimer's disease displays enhanced catalysis by a mechanism that evades down-regulation.

ABSTRACT: Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca2+ and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKC? in Alzheimer's disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ?30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca2+ or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKC?-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKC?-M489V mutation. Our results unveil how an AD-associated mutation in PKC? permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKC?.

SUBMITTER: Callender JA 

PROVIDER: S-EPMC6004447 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature