Abstract
SummaryThe SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
Highlights
Glucose repression is the process whereby high levels of this sugar repress the expression of specific genes (Gancedo, 1992)
In the budding yeast Saccharomyces cerevisiae, glucose depletion leads to activation of the SNF1 complex, which plays an essential role in derepression of glucose-repressed genes (Celenza and Carlson, 1986)
ADP Protects against Dephosphorylation of Thr210 in SNF1 We reported recently that ADP protects AMPK from dephosphorylation (Xiao et al, 2011)
Summary
Glucose repression is the process whereby high levels of this sugar repress the expression of specific genes (Gancedo, 1992). The activities of the upstream kinases in the cascade remain unaffected by a fall in glucose levels (Rubenstein et al, 2008) These findings suggest that activation of SNF1 is controlled at the level of the dephosphorylation step. This is similar to a mechanism previously described for the regulation of mammalian AMPK, where AMP protects against dephosphorylation of Thr172 by protein phosphatases (Davies et al, 1995; Sanders et al, 2007; Suter et al, 2006). Previous studies have shown that SNF1 is not regulated by AMP, either via an allosteric effect (Mitchelhill et al, 1994; Wilson et al, 1996; Woods et al, 1994) or by protecting it from dephosphorylation (Sanders et al, 2007)
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