Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Turk, Benjamin

Abstract

Kin1 and Kin2 (Kin1/2), the S. cerevisiae orthologs of the mammalian microtubule affinity-regulating kinases (MARKs), regulate a variety of important cellular functions, including exocytosis and the unfolded protein response (UPR). In this study, I examined the regulation of Kin1/2 kinase activity and localization, which are poorly understood. I determined the impact on Kin1/2 of interaction with Bud14, a regulatory subunit of the phosphatase Glc7, the budding yeast ortholog of PP1. Kin1/2 localization to sites of polarized growth was completely dependent on interaction with Bud14, although Kin1/2 kinase activity was not dependent on Bud14. I also examined the impact of the kinase-associated 1 (KA1) domain on both the kinase activity and localization of Kin1/2. Mutation of the KA1 domain had a partial effect on Kin1/2 localization. The KA1 domain also autoinhibits Kin1/2 catalytic activity, and mutation of the KA1 domain increases Kin1/2 activity nearly ten-fold. Kin1/2 kinase activity is also profoundly impacted by activation loop phosphorylation of a conserved threonine residue, which increases Kin1/2 kinase activity by 20-fold. I found that the protein kinases Elm1, Sak1 and Tos3, which activate related kinases, are responsible for Kin1/2 activation loop phosphorylation. The level of kinase activity, but not localization, is vital for Kin1/2 function in the unfolded protein response. Taken together, these results further elucidate the mechanisms controlling cellular Kin1/2 activity and localization.

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