Date of Award

May 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Madhusudan Dey

Committee Members

Charles Wimpee, Douglas Steeber, Sergei Kuchin, David Frick, Heather Owen

Keywords

ER stress, Kin1 Kin2, MARK, Pal2, Protein Homeostasis, Protein kinase

Abstract

Eukaryotic protein kinases catalyze the transfer of the -phosphate of an ATP to a serine/threonine/tyrosine residue present in a protein substrate. The phosphorylation of proteins has profound effects on their activity and protein-protein interactions, thus regulating a plethora of cellular processes, including cell growth, differentiation and protein homeostasis (or proteostasis). Our lab is the first to demonstrate that protein kinases Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae, orthologs of human microtubule affinity-regulating kinase (MARK), contribute to protein-folding homeostasis inside the endoplasmic reticulum (ER), in addition to their canonical roles in cellular exocytosis. The main aim of my studies is to fully understand the Kin kinase signaling pathway and how it contributes to the ER protein-folding homeostasis in the yeast Saccharomyces cerevisiae. Specifically, I study how Kin kinases are activated and what their upstream and downstream effectors are.

My studies have revealed that the N-terminal half of Kin1 or Kin2 protein containing the kinase domain (KD) with a short kinase extension region (KER) was sufficient to complement the function of full-length Kin1 or Kin2. I have also found that phosphorylation of a single residue in Kin1 (Thr-302) or Kin2 (Thr-281) was important for their kinase domain function. Furthermore, I have found that phosphorylation of Thr-302 or Thr-281 occurred in trans by an upstream kinase. These results are published in Molecular and Cellular Biology. Further studies are directed towards identifying the Thr-302 or Thr-281 upstream kinase.

One third of total cellular proteins fold and mature inside the E¬R. Due to abiotic or biotic stresses, unfolded proteins may accumulate inside the ER lumen, causing ER stress. During ER stress, a dual kinase RNase Ire1 is activated and it restores the ER protein-folding homeostasis in Saccharomyces cerevisiae as follows. The active Ire1 initiates a signaling pathway by removing an intervening sequence from the HAC1 mRNA by an unconventional splicing mechanism. Matured HAC1 mRNA then translates an active transcription factor Hac1, which enhances the expression of protein folding enzymes and chaperones that help mitigate ER stress. We and others have shown that HAC1 splicing requires co-localization of the HAC1 mRNA with the Ire1 protein, which is mediated by a bipartite element (BE) present in the 3’-UTR of the HAC1 mRNA. I have shown that the Kin kinases and a BE-RNA-protein complex (RNP) significantly contribute to HAC1 mRNA splicing. Here I have characterized and determined the role of a component of the proposed RNP, an uncharacterized protein Pal2.

Our collaborator Dr. Benjamin Turk at Yale University identified a list of putative substrates of Kin kinases, using a phospho-proteomics based approach1. We have shown that Kin2 specifically phosphorylates the Ser-222 residue of Pal2. Further, molecular genetic studies showed that the yeast strain lacking Pal2 and its paralog Pal1 was deficient in maintaining ER protein homeostasis, which could be restored by expressing a wild-type Pal2 protein, but not by its unphosphorylated form. These data suggest that both Kin kinases and its substrate Pal2 significantly contribute to ER protein homeostasis. Overall, my finding of Pal2 phosphorylation by Kin kinases provides a novel mechanistic insight into the physiological signaling pathways mediated by the Kin kinases.

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