Thesis Format
Integrated Article
Degree
Doctor of Philosophy
Program
Biochemistry
Supervisor
Litchfield, David W.
Abstract
Catalyzed by protein kinases, the reversible phosphorylation of serine, threonine, or tyrosine residues is a critical eukaryotic post-translational modification for cellular homeostasis and is commonly perturbed in numerous human diseases. Protein kinase CK2 (CSNK2, formerly casein kinase II) is a pivotal regulatory player in numerous fundamental biological processes including cell proliferation, DNA damage response, protein translation, and mitosis, with aberrant activity implicated in cancers, neurologic diseases, and viral infections. CK2 has emerged as an attractive therapeutic target, which has led to the development of multiple inhibitors including the novel and selective compound SGC-CK2-1. Despite being considered “constitutively active”, CK2 raises a paradox regarding its participation in precisely-controlled biological processes without external modifications or interactions. The peptidyl-prolyl isomerase Pin1 has previously been shown to regulate the substrate specificity and subcellular localization of CK2 after it is phosphorylated at the onset of mitosis, but no additional studies have been performed to further characterize its role in regulating CK2-dependent phosphorylation.
In this thesis, quantitative SILAC-based tandem mass spectrometry was employed to identify proteomic and phosphoproteomic changes in response to treatment with SGC-CK2-1 or the novel Pin1 inhibitor Sulfopin. In asynchronous Sulfopin-treated U2-OS cells, significant phosphoproteomic perturbations consistent with Pin1 inhibition were identified. By comparing differentially-expressed phosphosites to an analogous evaluation where U2-OS cells were treated with SGC-CK2-1, we identified 18 unique CK2 substrates that may be regulated by Pin1. Next, we again performed proteomic and phosphoproteomic profiling of mitotic HeLa cells treated with SGC-CK2-1 or Sulfopin, and subsequent bioinformatic analyses including KSEA, sequence motif enrichment, and GO-BP were performed to increase our understanding of the roles of these proteins during cell division. Ultimately, 10 unique CK2 substrates were identified that may be regulated by Pin1 in mitotic cells, including 6 that are preferentially phosphorylated during mitosis.
The work presented in this thesis provides valuable insights into the temporal regulation of CK2 by Pin1, laying the foundation for additional investigations into how a constitutively-active kinase participates in fundamental biological processes.
Summary for Lay Audience
Proteins are tiny machines inside our bodies that have specific jobs that help keep it functioning properly. After proteins are created by our cells, a process occurs where they undergo reversible changes called phosphorylation, which are essential for helping proteins perform their jobs. One key player in this process is a protein called CK2, which is involved in important activities like helping cells grow and repairing damaged DNA. Malfunctioning CK2 is linked to various diseases including cancers and viral infections like SARS-CoV-2 (the virus responsible for COVID-19). CK2 has been identified as being always active, which raises a puzzling question about how it participates in precisely controlled cellular functions. Another protein called Pin1 has been known to influence CK2's behavior, but we still need more information about how these two proteins cooperate.
In this thesis, we used experimental drugs targeting CK2 and Pin1 and advanced techniques to study how the two proteins affect the phosphorylation process by blocking their activities in different types of cancer cells. We found some overlapping changes to the phosphorylation process, identifying 28 specific targets of CK2 that are controlled by Pin1. This study sheds light on how CK2 is regulated over time by Pin1, which may eventually contribute to the development of new therapies for diseases associated with CK2 dysregulation.
Recommended Citation
Roffey, Scott E., "Elucidating the temporal regulation of protein kinase CK2 by the peptidyl-prolyl isomerase Pin1" (2024). Electronic Thesis and Dissertation Repository. 9961.
https://ir.lib.uwo.ca/etd/9961
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