Arnason, Terra G2017-06-232019-06-232017-102017-06-23October 20http://hdl.handle.net/10388/7924The heterotrimeric AMP-dependent protein kinase (AMPK) family is activated upon low energy states, conferring a switch towards energy-conserving metabolic pathways through immediate kinase actions on enzyme targets, and delayed alterations in gene expression through its nuclear re-localization. This family has known roles in stress resistance, lifespan, and nutrient adaptation, making it a relevant target for improving human health. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) AMPK, the SNF1 kinase, is readily activated by low glucose growth conditions making it a simple model to study the activation and activity of this enzyme class. These 3-subunit enzymes contain a highly conserved ubiquitin-associated (UBA) motif in most catalytic subunits whose function we have confirmed to be inhibitory. We demonstrated that UBA motif mutations significantly enhanced SNF1 kinase activation and biological activity including oxidative stress resistance and lifespan. Significantly, the enhanced UBA-dependent longevity and oxidative stress response is dependent on the Forkhead 1 (Fkh1) and Fkh2 stress response transcription factors, which in turn influence Snf1 catalytic subunit expression. The catalytic subunits are poly-ubiquitinated in yeast and humans with negative regulatory impacts on enzyme activity, yet the ubiquitin cascade enzymes involved are unknown. S. cerevisiae has 13 ubiquitin conjugating enzymes (E2) and a simplified repertoire of ubiquitin ligases (E3) that were screened for their impact on SNF1 kinase activity. We discovered that a discrete cluster of stress-responsive E2s (Ubc1, Ubc4, and Ubc5) and their associated E3 enzyme, Rsp5, affected discrete aspects of SNF1 kinase regulation, none through proteolytic degradation. The deletion of Ubc1 lowered catalytic subunit abundance, due to a synchronized decrease in Fkh1/2 proteins resulting in impaired SNF1 gene transcription. Furthermore, the Fkh1/2 cognate transcription factor, Hcm1, failed to enter the nucleus in the absence of Ubc1. This implies that Ubc1 acts indirectly through transcriptional effects to modulate SNF1 kinase activity. We also determined that yeast strains disrupted for Ubc4/5 and Rsp5 functions were markedly impaired for nuclear import of the SNF1 kinase despite its full phosphorylation and activity, all without effecting protein stability. Together, this study supports a positive role for ubiquitin enzyme cascade in SNF1 kinase activation at multiple steps and is not limited to proteolytic degradation.application/pdfAMPKSNF1 kinaseubiquitinyeastUBAallosteric regulationThesis2017-06-23