Author ORCID Identifier
Date of Award
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Biochemistry and Cell Biology
Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.
Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies on the expression of an AID fusion protein and the F-box protein Tir1. Addition of auxin drives binding of the AID-POI and Tir1, resulting in rapid ubiquitination and degradation. Recently, we demonstrated that this approach can be used to study kinase-substrate relationships in a manner analogous to small-molecule inhibition using the kinase Plk1 as a proof-of-concept. Based on the results of this study, we applied AID-Tir1 protein degradation to interrogate kinase-substrate relationships for the Polo-like kinase (Plk), p21-activated kinase (PAK), and Aurora kinase families. Additionally, we made significant improvements to the CRISPR-Cas9 workflow and improved efficiency of AID-Tir1 cell line generation for kinases of interest.
Targeted degradation of PAK1 revealed low PAK1 activity in HEK293 cells. Follow-up experiments showed that, while many phosphorylation sites are regulated by the group 1 PAKs, PAK1 does not regulate these pathways alone and likely has overlapping functions with the closely related kinase, PAK2. We applied AID-Tir1 to Aurora B and observed high correlation between Aurora B degradation and inhibition by the Aurora B inhibitor AZD1152, demonstrating that protein degradation is a selective approach to identify direct Aurora B substrates. We identified an uncharacterized truncated Aurora B isoform that is sufficient for Aurora B signaling in the absence of full-length Aurora B. Finally, we used fluorescent reporter proteins and Fluorescence Activated Cell Sorting (FACS) to greatly improve the efficiency of AID-Tir1 cell line generation for kinases of interest. These improvements make strides towards widespread implementation of targeted degradation as a tool to study kinase-substrate relationships.
Howarth, Charles Lincoln, "PROTEOMIC APPROACHES TO IDENTIFY UNIQUE AND SHARED SUBSTRATES AMONG KINASE FAMILY MEMBERS" (2023). Dartmouth College Ph.D Dissertations. 178.