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Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Litchfield, David W.

Abstract

Apoptosis is a tightly regulated cellular process essential for normal development and tissue homeostasis. Perturbations to apoptotic signaling underscores numerous pathogenic processes emphasizing the importance of apoptotic regulation. During apoptosis, caspases orchestrate cellular degradation through proteolytic cleavage of key structural and enzymatic proteins. In a different manner, protein kinases regulate apoptosis by catalyzing the post-translational phosphorylation of substrate proteins to facilitate either pro- or anti-apoptotic signal transduction pathways. Emerging paradigms have indicated that bidirectional crosstalk between protein kinases and caspases serves to globally fine-tune the equilibrium between signals directing cell survival and cell death. In this regard, identifying points of intersection between the apoptotic phosphoproteome and degradome is valuable considering both enzyme families are targeted for therapeutic intervention in a variety of pathologies.

In this thesis, we employed diverse strategies to study the amplitude of phosphorylation and proteolysis in apoptotic cells. First, using a bioinformatics approach, we revealed that a considerable proportion of the human kinome and phosphatome is degraded in response to various apoptotic stimuli across distinct cell types. As a logical extension, we used quantitative proteomics strategies to explore both the apoptotic kinome and phosphoproteome of HeLa cells treated with doxorubicin. Using these strategies, we observed distinct variations within the kinome and extensive hypo- and hyper-phosphorylation of numerous phosphosites. In parallel, we investigated the complementary apoptotic degradome by applying a quantitative N-terminomics approach where we identified a number of previously validated protease cleavage products and indexed numerous novel neo-N-termini. Finally, to expand the biochemical toolbox for defining the spatial and temporal evolution of kinase–caspase networks, we investigated the impact of serine and phosphoserine determinants within caspase cleavage motifs and developed a number of prospective FRET biosensors to monitor the relationship between protein kinase CK2 phosphorylation and caspase-mediated proteolysis.

Collectively, using complementary strategies, we investigated the functional relationship between post-translational phosphorylation and proteolysis and provided insight into bidirectional communication between protein kinases and proteases during apoptosis.

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