Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 24, Pages E5497-E5505Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1805046115
Keywords
protein kinase C; PKC; Alzheimer's disease; signal transduction; enzyme mutation
Categories
Funding
- NIH [R35 GM122523, GM43154]
- Cure Alzheimer's Fund
- Cancer Research UK
- University of California, San Diego Graduate Training Program in Cellular and Molecular Pharmacology [T32 GM007752]
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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 ofmanymechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKC alpha 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 similar to 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 alpha-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 alpha-M489V mutation. Our results unveil how an AD-associated mutation in PKC alpha permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKC alpha.
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