Date of Award

5-1-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Sergei Kuchin

Committee Members

Mark McBride, Daad Saffarini, Chuck Wimpee, Ching-Hong Yang

Abstract

Kinases of the AMP-activated protein kinase (AMPK) family are conserved in eukaryotes and play central roles in responses to reduced energy availability. AMPK, nicknamed the “fuel gauge” of the cell, monitors cellular energy status via the ratio of AMP to ATP nucleotides. AMPK restores energy homeostasis by reducing energy “spending” and increasing energy “income”. Correspondingly, defects in AMPK signaling have been implicated in diseases including type II diabetes, obesity, and cancer.

In yeast, the AMPK homolog is Snf1 protein kinase. Glucose is the preferred carbon/energy source of yeast, and thus limitation for glucose similarly activates Snf1. Snf1 activation requires phosphorylation of its T-loop threonine (Thr210) by upstream kinases. When glucose is abundant, Snf1 is inhibited by Thr210 dephosphorylation. The latter involves the function of type 1 protein phosphatase Glc7, which is targeted to Snf1 by a regulatory subunit, Reg1. The reg1 mutation causes increased Snf1 activity and mimics various aspects of glucose limitation, including slower growth. Reg2 is another Glc7 regulatory subunit encoded by a paralogous gene, REG2. The goal of our study was to determine if Reg2 has a role in Snf1 regulation. Indeed, we have found that Reg2 contributes to Snf1 Thr210 dephosphorylation. Consistent with this role, Reg2 interacts with wild-type Snf1 but not with non-phosphorylatable Snf1-T210A. Additionally, the ability of Reg2 to regulate Snf1 depends on the Reg2-Glc7 interaction. Reg2 accumulation increases in a Snf1-dependent manner during prolonged glucose deprivation, and glucose-starved cells lacking Reg2 exhibit delayed Snf1 Thr210 dephosphorylation and slower growth recovery upon glucose replenishment. Accordingly, cells lacking Reg2 are outcompeted by wild-type cells in the course of several glucose starvation/replenishment cycles. Collectively, our results support a model in which Reg2-Glc7 contributes to the negative control of Snf1 in response to glucose re-feeding after prolonged starvation. The competitive growth advantage provided by Reg2 underscores the evolutionary significance of this paralog for S. cerevisiae.

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