Access Denied: Snf1 Activation Loop Phosphorylation Is Controlled by Availability of the Phosphorylated Threonine 210 to the PP1 Phosphatase

Abstract

Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

“…Although the possibility of regulation of the activating kinases is not excluded, there is no evidence to support this idea; these kinases had similar activity when prepared from cells grown on high or low glucose (9), and expression of AMPK-activating kinases in yeast sufficed for glucoseregulated activation of SNF1 (10).…”

“…Phosphorylation of the activation-loop Thr of the catalytic subunit activates SNF1/AMPK. For SNF1, the protein kinases Sak1, Tos3, and Elm1 phosphorylate Thr-210 on the Snf1/α subunit in response to glucose limitation and other stresses (5)(6)(7)(8), but there is no evidence that these Snf1-activating kinases are regulated (9,10). Less is understood about the protein phosphatases that deactivate SNF1.…”

“…Less is understood about the protein phosphatases that deactivate SNF1. Reg1-Glc7 protein phosphatase 1 (PP1) has been thought to be solely responsible for dephosphorylating Thr-210 (9)(10)(11)(12)(13)(14), and it has been proposed that access of Reg1-Glc7 to Thr-210 is restricted during growth on limiting glucose (9). However, recent evidence has also implicated the type 2A-like protein phosphatase Sit4 in Thr-210 dephosphorylation (15).…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…In support of such a crucial role for the phosphatase in the regulation of Snf1 activity, it was recently reported that the dephosphorylation rate of Snf1 is more subject to glucose regulation than the phosphorylation rate (Rubenstein et al 2008). However, it also appears that the catalytic activity of the Glc7-Reg1 phosphatase itself is not controlled by glucose, but rather Snf1 itself, in such a way that the ability of the Snf1 activation loop to act as a substrate for Glc7-Reg1 is adapted to glucose availability (Rubenstein et al 2008).…”

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

“…In yeast, the AS strategy has been useful in determining the function of many kinases including Cdc28 (10, 11), Ime2 (11), Cla4 (12), and Snf1 (13). Several discoveries using this approach have also been made in mammalian systems including a noncatalytic role for Zap70 in regulatory T-cells (14), and the dependence of transformed cells on aberrant oncogenic signaling by the EGFR kinase (15).…”