UC San Diego

The focus of this thesis is to study the mechanisms that control the activity of Protein Kinase C α and how deregulation of these mechanisms leads to neurodegenerative pathology. Protein kinase C (PKC) is a family of enzymes whose members transduce a large variety of cellular signals instigated by the receptor-mediated hydrolysis of membrane phospholipids. 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 show that PKCα signaling contributes to the pathology of Alzheimer’s disease (AD), a neurodegenerative disease that remains incurable. We observe changes in electrophysiology in hippocampal brain slices to show that PKCα is necessary for Amyloid beta (Aβ)-mediated synaptic depression. We use FRET-based PKC activity reporters to show that PKCα is activated as a result of exposing primary astrocytes to Aβ and that AD-associated mutations in PKCα increase its signaling output in cells. We use a combination of biochemical, in silico, and in vivo approaches to conduct an in-depth characterization of one of these variants (PKCα-M489V), thus establishing the mechanism through which it confers enhanced PKCα activity without compromising stability. We also use live cell imaging assays to study in more detail the mechanism of PKCα signaling, showing that it is regulated by intramolecular interactions between its N-terminal and C-terminal domains. We investigate a previously unstudied tyrosine phosphorylation site in the C2 domain of PKCα and establish that phosphorylation at this site—as well as tyrosine phosphorylation in general—promotes PKCα activity. Taken together, the work presented within this thesis serves the ultimate goal of understanding the mechanisms that regulate PKCα signaling in order to effectively target PKCα in the treatment of neurodegenerative diseases.