Doctor of Philosophy
Dr. David W. Litchfield
Protein kinase CK2 is a constitutively active, acidophilic protein serine/threonine kinase with an extensive array of protein substrates that have been implicated in the regulation of many cellular events including those that maintain the delicate balance between proliferation and survival. Although numerous substrates of CK2 have been identified, bioinformatic analysis predicts that 20% of the phosphoproteome can be attributed to CK2, suggesting that our understanding of CK2 is greatly underappreciated. Not surprisingly, CK2 levels are elevated in multiple cancers, and therapeutics targeting CK2 are in development. In order to realize the full potential of these therapies, it is essential to have robust methods to monitor CK2 activity as well as interrogate the specificity of CK2 to further understand the cellular role of CK2.
In this thesis, we developed three complementary chemical biology strategies to investigate the activity and substrate specificity of CK2. First, we generated a novel anti-Fc antibody to perform a detailed biochemical analysis of the ability of CK2 to utilize 5’-γ-ferrocenyl-ATP (FcATP), an electroactive ATP analog. CK2 effectively uses FcATP compared to ATP and our biochemical assays recapitulate electrochemical data, providing a strong foundation upon which to develop a multiplexed kinosensor to stratify patients. Second, to study the substrate specificity of CK2 we genetically engineered an analog-sensitive CK2 to utilize N6-(2-phenylethyl)ATP, to specifically phosphorylate and label a direct CK2 substrate, EEF1D. Finally, we performed a systematic analysis of hierarchical CK2 phosphorylation, where priming phosphorylation is required for subsequent CK2 phosphorylation. Phosphoserine oriented peptide arrays were used to define optimal consensus sequences for hierarchical CK2 phosphorylation. Subsequent bioinformatic analysis revealed over 2000 candidates involved in diverse signaling pathways, and a fraction of these candidates were known to be phosphorylated in cell.
Collectively, our results will greatly aid in the continued development and clinical usage of targeted-CK2 inhibitors in personalized medicine. These strategies have provided insight into the complexity of the signaling pathways that CK2 intricately regulates and using these strategies will increase our knowledge of the cellular roles and decipher the kinase-substrate relationships of CK2 to identify biomarkers for CK2 activity.
Gabriel, Michelle A., "Chemical Biology Approaches to Investigate the Activity and Substrate Specificity of Protein Kinase CK2" (2015). Electronic Thesis and Dissertation Repository. 3299.
Available for download on Thursday, September 14, 2017