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Date of Award
Mary K. Pflum
Protein phosphorylation is catalyzed by kinases and is an essential post-translationalmodification that plays a critical role in cellular events, such as signal transduction. Deregulated kinase activity results in various diseases, including cancer. Therefore, a number of drugs that target kinases have been developed to treat diseases. Development of effective therapeutics requires a deeper understanding of kinases, their substrates, and the signaling pathways they control. Unfortunately, current methods to study kinases are limited due to the low stoichiometry of phosphorylation, low abundance of protein substrates, and challenges in detecting dynamic phosphorylation events. Advanced chemical biology tools are needed to explore kinase biology related to disease formation. To address the challenges in studying dynamic signaling, we used the ability of protein kinases to accept γ-phosphate modified ATP analogs, such as ATP-biotin, as cosubstrates to label substrates and explore kinase biology. Using ATP-biotin, K-BMAPS (Kinase-catalyzed Biotinylation to MAP Signaling) was developed to map the well-characterized EGFR signaling pathway in vitro and in live cells. Pathway maps were developed considering EGFR-dependent functional categories and previously known protein interactions for in vitro and in cell K-BMAPS, separately.
Interestingly, more than 90% of the identified hits were included in the pathway mapsfrom both in vitro and in cell K-BMAPS studies, suggesting that K-BMAPS enriched dynamic EGFR pathway proteins. While in vitro K-BMAPS identified late EGFR pathway events, in cell K-BMAPS identified early EGFR pathway events. In addition, HDAC2 enriched in K-BMAPS was discovered as a novel direct EGFR substrate. Altogether, KBMAPS is a powerful tool to study dynamic signaling events. K-BILDS (Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates) was used to determine candidate substrates of LKB1 and AMPK in two collaborative projects. K-BILDS with LKB1 in HeLa lysates identified candidate substrates of LKB1 with a high confidence. PGD among the hits was validated as a direct substrate of LKB1 using a secondary assay. K-BILDS was also performed with AMPK in cardiomyocytes and identified previously known substrates with a high confidence. Collectively, K-BMAPS and K-BILDS are powerful tools to map dynamically changing kinase-dependent cellular signaling pathways and to identify kinase substrates, respectively. The new tools described in this study will provide a novel approach to understand the role of kinases in biology and thereby, will shed a new light to understand signaling pathways that governs disease.
Ramanayake-Mudiyanselage, Vindya Harshani Rathnayake, "Kinase-Catalyzed Biotinylation To Map Signaling Pathways And To Identify Kinase Substrates" (2020). Wayne State University Dissertations. 2505.