Author ORCID Identifier

https://orcid.org/0000-0003-4174-3612

Date of Award

2023

Document Type

Thesis (Ph.D.)

Department or Program

Molecular and Systems Biology

First Advisor

Dr. Scott A. Gerber

Abstract

Reversible phosphorylation is one of the most important post translational modifications that has allowed us as a species to quickly adapt to changing molecular environments due to external stimulation. This process is only capable through the activity of kinases to carry out the targeting of specific substrates defined by their recognition motif allowing for selective phosphorylation and activation and inactivation of distinct pathways as well as other changes that permit cell survival. By being so important for the maintenance of the cells disruption often leads to worsening of the cells, leading to various diseases like cancer, immunological and neurodegenerative disorders. This is why the comprehension of kinases, and their substrates is so important for making progress in the medical care of the patients.

For this exact purpose we chose Polo like kinase 2 (Plk2) as the target of study. Even though a great deal of knowledge exists about it, like the fact that it plays roles during the cell cycle, cell differentiation, ontogenesis, stress response, tumorigenesis, neurodegenerative diseases, and inflammation. The mechanisms at play i.e the specific interactions that permits its function in all these systems is not entirely known. As such, we employed the use CRISPR/Cas9 as the method of genetic modification for the endogenous tagging of Plk2 with the auxin inducible degron (AID) for the selective degradation of our target. After having observed through LC/MS the limitations of being a low abundant protein, we combined proximity labeling through Turbo-ID and targeted degradation as a method of accomplishing this task. We were successful in the identification of novel substrates of Plk2 in the cell cycle, and capable of study the interactome of Plk2 in asynchronous and G1/S arrested cells. Providing a sundry of new insight into Plk2 biology and the diversification of its interactions.

AID technology in combination with proximity labeling provides the next steps in the ability of study for any type of protein. Capable of gathering information about the context of the protein in regard to its environment, the interactions, and what the absence of that protein would have for specific pathways, while minimizing off-target effects.

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