Date of Award
Master of Science
Sergei V. Kuchin
Charles F. Wimpee, Ching-Hong Yang
Glucose, PKA, Quiescence, Snf1/AMPK, Stress Response, Yeast
AMPK, the fuel gauge of the cell, and its upstream kinase, LKB1, have been implicated in cancer prevention and stress response associated with energy exhaustion. In the yeast Saccharomyces cerevisiae, Snf1 is the ortholog of mammalian AMPK. In S. cerevisiae, Snf1 is activated by phosphorylation of its T–loop at Thr210, primarily by its upstream kinase Sak1, in absence of the preferred carbon source, glucose, or during some other stress responses. Cyclic AMP–dependent protein kinase A, PKA, is involved in nutrient signaling largely antagonistically to Snf1. Using yeast strains of the Sigma 1278b genetic background, which have a high basal level of cAMP signaling, PKA was previously suggested to downregulate Snf1 by a mechanism that involves phosphorylation of Sak1 on two consensus PKA recognition sites in the non–dashcatalytic C-terminal domain. Sequence analysis suggests that Snf1 or its immediate regulators are also targets for negative regulation by PKA in other genetic lineages of S. cerevisiae and even in other yeast species. Here, we have investigated the possible existence of an antagonistic relationship between PKA and Snf1 in another S. cerevisiae strain lineage, W303, which has a relatively low basal level of cAMP signaling. In addition to short-term glucose limitation, we monitored Snf1 activation under conditions of long–term carbon stress that normally leads to exit from the mitotic cycle and entry into a quiescent state. We observed that W303 ira1 mutant cells with increased PKA signaling have significantly reduced levels of Snf1 activation after long-term carbon stress. The quiescence-associated trait of stress resistance, specifically heat-shock survival, was also evaluated. W303 lacking Snf1 or with significantly higher levels of PKA activity due to the ira1 mutation, did not acquire normal heat-shock resistance, suggesting failure to enter quiescence. This suggests that downregulation of Snf1 represents one mechanism by which PKA inhibits entry into quiescence. Since the ability to enter quiescence offers significant evolutionary advantages, similar relationships between PKA and Snf1 are likely to exist in various fungal species. Moreover, since PKA and Snf1–AMPK are conserved in eukaryotes from yeast to humans, the PKA–AMPK pathway could also regulate quiescence as it pertains to development and tumorigenesis.
Doughty, Leah Bernadette, "Prolonged Glucose Deprivation Sensitizes Snf1 to Negative Regulation By PKA to Delay Entry into Quiescence" (2013). Theses and Dissertations. 682.