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Access Type
WSU Access
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Mary Kay Pflum
Abstract
Post-translational modifications (PTMs) are important alterations made after translation that change protein interactions and functions in a cell. Phosphorylation is an important PTM that regulates cell biology by controlling the enzymatic function, localization, and molecular interactions of cellular proteins. Kinases catalyze the transfer of the γ-phosphoryl group from adenosine-5’-triphosphate (ATP) to a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of protein substrates (Figure 1).1, 2 Phosphorylation is a dynamic process and the phosphoryl can be cleaved by phosphatases, yielding the hydroxy residue. Together kinases and phosphatases regulate phosphorylation of their protein substrates. Kinase-mediated phosphorylation is highly regulated and crucial for proper cell function. Irregular kinase activity is linked to a variety of pathologies, including cancer and neurodegenerative diseases, making kinases a prominent drug target.3 Therefore, monitoring kinase mediated events in normal and diseased cellular conditions is essential to understand cell biology and identify potential drug targets. Due to the presence of more than 500 kinases and 20,000 phosphorylation sites, characterization of kinase-mediated signaling pathways remains a challenge. To remedy these challenges, The Pflum lab has developed γ-modified ATP analogs that can be used in kinase-catalyzed labeling to identify kinase-substrate pairs. K-CLIP is a method that utilizes γ-modified ATP analogs to identify upstream kinases of phosphoproteins of interest. K-CLIP was used in collaboration with two other labs to uncover unknown kinase biology. Next, K-CLIP was expanded with a new γ-modified ATP analog to identify the substrate scope of PKA. The new γ-modified ATP analogs allowed for a secondary enrichment step to separate upstream kinases and associated proteins from the kinase of interest and true substrates. Kinase-focused K-CLIP identified and validated SMC3 as a novel substrate of PKA. Finally, γ-modified ATP analogs were used in the K-BILDS and K-BIPS to identify substrate motifs of kinases and phosphatases respectively. Identifying substrate motifs in the context of full-length protein interactions will subsequently improve online databases that predict kinase-substrate pairs. In conclusion, γ-modified ATP analogs were developed and applied in methods to discover kinase-substrate pairs, which will expand the impact of kinase-mediated phosphorylation on cell biology.
Recommended Citation
Bremer, Hannah, "Development Of Chemical Tools To Identify Kinase-Substrate Pairs" (2024). Wayne State University Dissertations. 4049.
https://digitalcommons.wayne.edu/oa_dissertations/4049