Doctor of Philosophy
Dr. David Litchfield
Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to interrogate CK2-dependent signaling events in cells. Despite its widespread applications, an understanding of the mechanism of action of CX-4945 on cells remains limited.
In this thesis, comparison of proteomic sample preparation strategies led to the development of a phosphoproteomic workflow that enabled the enrichment of phosphopeptides conforming to the CK2 consensus sequence. Using the optimized workflow, phosphoproteomic profiling was conducted in HeLa cells treated with CX-4945. Several phosphorylation sites conforming to the recognition motif for CK2 phosphorylation displayed significantly decreased phosphorylation in response to CX-4945. Kinase substrate enrichment analysis also revealed a broad impact of CX-4945 on several kinases other than CK2. Profiling of the kinome utilizing multiplexed inhibitor beads also revealed changes in the activity of other kinases including activation of the ERK MAPK pathway and inhibition of the PI3K/Akt/mTOR pathway. Studies with Inhibitor VIII, an unrelated CK2 inhibitor, also resulted in activation of the ERK MAPK pathway suggesting that CK2 has a role in regulating this pathway. By comparison, the PI3K/Akt/mTOR pathway was not affected by Inhibitor VIII indicating that the effects of CX-4945 on that pathway are independent of CK2.
Overall, this investigation provides valuable insight into the regulation of the phosphoproteome and the kinome in response to CX-4945 in HeLa cells. Dynamic markers of CK2 activity in cells were identified and putative CK2-independent effects of CX-4945 were revealed. Collectively, these studies illustrate the utility of global proteomic approaches to elucidate the cellular effects of clinical-stage kinase inhibitors.
Rabalski, Adam J., "Quantitative Proteomic Characterization of CX-4945, a Clinical Stage Inhibitor of Protein Kinase CK2" (2017). Electronic Thesis and Dissertation Repository. 4400.