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Date of Award
Mary Kay Pflum
Kinases play important roles in human metabolism, and dysregulation of signaling pathways involving kinases can lead to diseases such as cancer. Kinases modify hydroxy groups on protein substrates by replacing the hydrogen with a phosphate group. In order to fully understand kinase signaling pathways, we must develop a method of discovering kinase substrates. It is understood that kinases “read” a sequence of amino acids known as a consensus site in order to determine their substrates. The Pflum lab has previously reported a method for identifying kinase substrates using kinase-catalyzed labeling, but only full-length proteins have been identified to date. Expanding kinase-catalyzed labeling to identifying phosphosite-containing peptides would allow kinase consensus site mapping. To expand kinase-catalyzed labeling to peptide-based consensus site mapping, various methods of synthesizing the pan-kinase inhibitor FSBA were tested. Additionally, two methods of enriching biotin-labeled peptides were investigated. The synthesis of FSBA requires further optimization, while a pre-enrichment digest of biotinylated proteins was determined to be the better method for purifying biotinylated peptides via streptavidin resin. We have established that K-BILDS can be expanded to a peptide-based method, which will allow us to adapt it for kinase consensus site mapping.
Geisler, Pietro, "Developing Kinase-Catalyzed Labeling For Kinase Consensus Site Mapping" (2019). Wayne State University Theses. 752.