UC San Diego

This thesis aims to elucidate the mechanisms governing protein kinase Cγ (PKCγ) autoinhibition and activity and how impairing these mechanisms in different ways leads to pathogenesis in the context of both neurodegeneration and cancer. The family of serine/threonine protein kinase C (PKC) isozymes transduce a multitude of signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional isozyme PKCγ is reversibly activated by Ca2+ and diacylglycerol, which allows the enzyme to adopt an open state in which downstream signaling can occur. Here, we show how impairing autoinhibition can result in either gain or loss of PKCγ activity. First, we use a variety of biochemical assays to show that PKCγ variants linked to the neurodegenerative disorder spinocerebellar ataxia type 14 (SCA14) enhance basal activity by impacting C1 domain autoinhibitory constraints, while evading quality control degradation mechanisms mediated by the phosphatase PHLPP. We also use a transgenic mutant mouse model of ataxia to establish that mice with enhanced PKCγ basal activity exhibit significant changes in their cerebellar phosphoproteome. Additionally, we show an inverse correlation between level of mutant biochemical defect and average age of symptom onset in patients, establishing that impaired PKCγ autoinhibition is a main driver of SCA14. Lastly, we use a variety of FRET-based approaches to examine a number of cancer-associated PKCγ mutants, all of which result in loss of PKC function by a variety of mechanisms. We also show that PKCγ expressed in cancer cells is not granted a stability advantage as it is with SCA14-associated mutants, and thus likely results in downregulation of PKC activity. Taken together, the work described herein serves to clarify the mechanisms by which PKCγ can become deregulated and provide insight into how to better target this unique enzyme in disease.