Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor(s)

Dr. David W. Litchfield

Abstract

The balance between cell survival and death is a crucial process in human development and tissue homeostasis, but is also misregulated in disease. In large part, apoptosis is controlled by caspases, a hierarchical series of cysteine aspartic acid proteases that demolish the cell by cleaving key structural and enzymatic proteins, but emerging paradigms have highlighted the ability of kinases to regulate caspase activity. One way in which kinases can control the progression of apoptosis is through phosphorylation of caspase substrates, which acts to prevent caspase cleavage of that target.

In this thesis, we develop new strategies to study this regulatory mechanism, and focus particularly on protein kinase CK2; a kinase with enigmatic regulation, an obvious, but undefined role in anti-apoptotic signaling, and demonstrated value as a candidate for targeted therapy of cancer. First, we used predictive strategies for identifying CK2 substrates that, when phosphorylated, regulate proteolysis by caspases. Interestingly, we predicted and validated that phosphorylation of caspase-3 itself by CK2 prevents its cleavage and activation, representing a putative, novel mechanism by which CK2 might control apoptosis in disease. We then utilized cell models that over-expressed different forms of CK2, and found that caspase-3 phosphorylation was specific only for the CK2a¢ subunit in the absence of CK2b - a level of isozyme specificity not before seen for CK2. Interestingly, documented misregulation of this isoform in disease suggests that the phenotypes generated by varied CK2 levels in cancer may well depend on the form of CK2 present. We also establish the utility of proteomic applications for identifying novel caspase substrates whose cleavage is regulated by phosphorylation. Interestingly, we noted that phosphorylation can also promote caspase substrate cleavage, likely through a mechanism in which phosphorylation alters scissile bond accessibility.

Collectively, our work provides insight into the regulation of protein kinase CK2 and demonstrates that the specialized functions of different forms may define cellular phenotypes in disease. Furthermore, we identified a number of caspase substrates whose cleavage is regulated by phosphorylation, thereby validating our novel approaches and delineating putative apoptotic control points.


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