Activation and inhibition of Snf1 kinase activity by phosphorylation within the activation loop.

The AMP-activated protein kinase is a metabolic regulator that transduces information about energy and nutrient availability. In yeast, the AMP-activated protein kinase, called Snf1, is activated when energy and nutrients are scarce. Earlier studies have demonstrated that activation of Snf1 requires the phosphorylation of the activation loop on threonine 210. Here we examined the regulation of Snf1 kinase activity in response to phosphorylation at other sites. Phosphoproteomic studies have identified numerous phosphorylation sites within the Snf1 kinase enzyme. We made amino acid substitutions in the Snf1 protein that were either non-phosphorylatable (serine to alanine) or phospho-mimetic (serine to glutamate) and examined the effects of these changes on Snf1 kinase function in vivo and on its catalytic activity in vitro. We found that changes to most of the phosphorylation sites had no effect on Snf1 kinase function. However, changes to serine 214, a site within the kinase activation loop, inhibited Snf1 kinase activity. Snf1-activating kinase 1 still phosphorylates Snf1-S214E on threonine 210 but the S214E enzyme is non-functional in vivo and catalytically inactive in vitro. We conclude that yeast have developed two distinct pathways for down-regulating Snf1 activity. The first is through direct dephosphorylation of the conserved activation loop threonine. The second is through phosphorylation of serine 214.

Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation.

The AMP-activated protein kinase (AMPK) is a conserved signaling molecule in a pathway that maintains adenosine triphosphate homeostasis. Recent studies have suggested that low energy adenylate ligands bound to one or more sites in the γ subunit of AMPK promote the formation of an active, phosphatase-resistant conformation. We propose an alternative model in which the kinase domain association with the heterotrimer core results in activation of the kinase catalytic activity, whereas low energy adenylate ligands bound in the kinase active site promote phosphatase resistance. Purified Snf1 α subunit with a conservative, single amino acid substitution in the kinase domain is protected from dephosphorylation by adenosine diphosphate in the complete absence of the ß and γ subunits. Staurosporine, a compound known to bind to the active site of many protein kinases, mediates strong protection from dephosphorylation to yeast and mammalian AMPK enzymes. The analog-sensitive Snf1-I132G protein but not wild type Snf1 exhibits protection from dephosphorylation when bound by the adenosine analog 2NM-PP1 in vitro and in vivo. These data demonstrate that ligand binding to the Snf1 active site can mediate phosphatase resistance. Finally, Snf1 kinase with an amino acid substitution at the interface of the kinase domain and the heterotrimer core exhibits normal regulation of phosphorylation in vivo but greatly reduced Snf1 kinase activity, supporting a model in which kinase domain association with the heterotrimer core is needed for kinase activation.

Subunit and domain requirements for adenylate-mediated protection of Snf1 kinase activation loop from dephosphorylation.

Members of the AMP-activated protein kinase (AMPK) family are activated by phosphorylation at a conserved threonine residue in the activation loop of the kinase domain. Mammalian AMPK adopts a phosphatase-resistant conformation that is stabilized by binding low energy adenylate molecules. Similarly, binding of ADP to the Snf1 complex, yeast AMPK, protects the kinase from dephosphorylation. Here, we determined the nucleotide specificity of the ligand-mediated protection from dephosphorylation and demonstrate the subunit and domain requirements for this reaction. Protection from dephosphorylation was highly specific for adenine nucleotides, with ADP being the most effective ligand for mediating protection. The full-length α subunit (Snf1) was not competent for ADP-mediated protection, confirming the requirement for the regulatory ß and γ subunits. However, Snf1 heterotrimeric complexes that lacked either the glycogen-binding domain of Gal83 or the linker region of the α subunit were competent for ADP-mediated protection. In contrast, adenylate-mediated protection of recombinant human AMPK was abolished when a portion of the linker region containing the α-hook domain was deleted. Therefore, the exact means by which the different adenylate nucleotides are distinguished by the Snf1 enzyme may differ compared with its mammalian ortholog.

Reg1 protein regulates phosphorylation of all three Snf1 isoforms but preferentially associates with the Gal83 isoform.

The phosphorylation status of the Snf1 activation loop threonine is determined by changes in the rate of its dephosphorylation, catalyzed by the yeast PP1 phosphatase Glc7 in complex with the Reg1 protein. Previous studies have shown that Reg1 can associate with both Snf1 and Glc7, suggesting substrate binding as a mechanism for Reg1-mediated targeting of Glc7. In this study, the association of Reg1 with the three Snf1 isoforms was measured by two-hybrid analysis and coimmunoprecipitation. We found that Reg1 association with Snf1 occurred almost exclusively with the Gal83 isoform of the Snf1 complex. Nonetheless, Reg1 plays an important role in determining the phosphorylation status of all three Snf1 isoforms. We found that the rate of dephosphorylation for isoforms of Snf1 did not correlate with the amount of associated Reg1 protein. Functional chimeric ß subunits containing residues from Gal83 and Sip2 were used to map the residues needed to promote Reg1 association with the N-terminal 150 residues of Gal83. The Gal83 isoform of Snf1 is the only isoform capable of nuclear localization. A Gal83-Sip2 chimera containing the first 150 residues of Gal83 was able to associate with the Reg1 protein but did not localize to the nucleus. Therefore, nuclear localization is not required for Reg1 association. Taken together, these data indicate that the ability of Reg1 to promote the dephosphorylation of Snf1 is not directly related to the strength of its association with the Snf1 complex.

Global analysis of protein phosphorylation in yeast.

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex.

BACKGROUND: The yeast SNF1 protein kinase and the mammalian AMP-activated protein kinase are highly conserved heterotrimeric complexes that are "metabolic master switches" involved in the switch from fermentative/anaerobic to oxidative metabolism. They are activated by cellular stresses that deplete cellular ATP, and SNF1 is essential in the response to glucose starvation. In both cases, activation requires phosphorylation at a conserved threonine residue within the activation loop of the kinase domain, but identifying the upstream kinase(s) responsible for this has been a challenging, unsolved problem. RESULTS: Using a library of strains that express 119 yeast protein kinases as GST fusions, we identified Elm1p as the sole kinase that could activate the kinase domain of AMP-activated protein kinase in vitro. Elm1p also activated the purified SNF1 complex, and this correlated with phosphorylation of Thr210 in the activation loop. Removal of the C-terminal domain increased the Elm1p kinase activity, indicating that it is auto-inhibitory. Expression of activated, truncated Elm1p from its own promoter gave a constitutive pseudohyphal growth phenotype that was rescued by deletion of SNF1, showing that Snf1p was acting downstream of Elm1p. Deletion of ELM1 does not give an snf- phenotype. However, Elm1p is closely related to Pak1p and Tos3p, and a pak1Delta tos3Delta elm1Delta triple mutant had an snf1- phenotype, i.e., it would not grow on raffinose and did not display hyperphosphorylation of the SNF1 target, Mig1p, in response to glucose starvation. CONCLUSIONS: Elm1p, Pak1p, and Tos3p are upstream kinases for the SNF1 complex that have partially redundant functions.

Yeast Pak1 kinase associates with and activates Snf1.

Members of the Snf1/AMP-activated protein kinase family are activated under conditions of nutrient stress by a distinct upstream kinase. Here we present evidence that the yeast Pak1 kinase functions as a Snf1-activating kinase. Pak1 associates with the Snf1 kinase in vivo, and the association is greatly enhanced under glucose-limiting conditions when Snf1 is active. Snf1 kinase complexes isolated from pak1Delta mutant strains show reduced specific activity in vitro, and affinity-purified Pak1 kinase is able to activate the Snf1-dependent phosphorylation of Mig1 in vitro. Purified Pak1 kinase promotes the phosphorylation of the Snf1 polypeptide on threonine 210 within the activation loop in vitro, and an increased dosage of the PAK1 gene causes increased Snf1 threonine 210 phosphorylation in vivo. Deletion of the PAK1 gene does not produce a Snf phenotype, suggesting that one or more additional protein kinases is able to activate Snf1 in vivo. However, deletion of the PAK1 gene suppresses many of the phenotypes associated with the deletion of the REG1 gene, providing genetic evidence that Pak1 activates Snf1 in vivo. The closest mammalian homologue of yeast Pak1 kinase, calcium-calmodulin-dependent protein kinase kinase beta, may play a similar role in mammalian nutrient stress signaling.

Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae.

Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.

The ß subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress.

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which ß subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms.

2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and α-arrestin-mediated trafficking of hexose transporters 1 and 3.

The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.

Genetic analysis of resistance and sensitivity to 2-deoxyglucose in Saccharomyces cerevisiae.

Aerobic glycolysis is a metabolic pathway utilized by human cancer cells and also by yeast cells when they ferment glucose to ethanol. Both cancer cells and yeast cells are inhibited by the presence of low concentrations of 2-deoxyglucose (2DG). Genetic screens in yeast used resistance to 2-deoxyglucose to identify a small set of genes that function in regulating glucose metabolism. A recent high throughput screen for 2-deoxyglucose resistance identified a much larger set of seemingly unrelated genes. Here, we demonstrate that these newly identified genes do not in fact confer significant resistance to 2-deoxyglucose. Further, we show that the relative toxicity of 2-deoxyglucose is carbon source dependent, as is the resistance conferred by gene deletions. Snf1 kinase, the AMP-activated protein kinase of yeast, is required for 2-deoxyglucose resistance in cells growing on glucose. Mutations in the SNF1 gene that reduce kinase activity render cells hypersensitive to 2-deoxyglucose, while an activating mutation in SNF1 confers 2-deoxyglucose resistance. Snf1 kinase activated by 2-deoxyglucose does not phosphorylate the Mig1 protein, a known Snf1 substrate during glucose limitation. Thus, different stimuli elicit distinct responses from the Snf1 kinase.

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase.

The 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.

A chemical genomics study identifies Snf1 as a repressor of GCN4 translation.

The Saccharomyces cerevisiae Snf1 kinase plays a critical role in recalibrating cellular metabolism in response to glucose depletion. Hundreds of genes show changes in expression levels when the SNF1 gene is deleted. However, cells can adapt to the absence of a specific gene when grown in long term culture. Here we apply a chemical genetic method to rapidly and selectively inactivate a modified Snf1 kinase using a pyrazolopyrimidine inhibitor. By allowing cells to adjust to a change in carbon source prior to inhibition of the Snf1 kinase activity, we identified a set of genes whose expression increased when Snf1 was inhibited. Prominent in this set are genes that are activated by Gcn4, a transcriptional activator of amino acid biosynthetic genes. Deletion of Snf1 increased Gcn4 protein levels without affecting its mRNA levels. The increased Gcn4 protein levels required the Gcn2 kinase and Gcn20, regulators of GCN4 translation. These data indicate that Snf1 functions upstream of Gcn20 to regulate control of GCN4 translation in S. cerevisiae.

Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase.

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.

Regulatory domains of Snf1-activating kinases determine pathway specificity.

In Saccharomyces cerevisiae, the Snf1 kinase can be activated by any one of three upstream kinases, Sak1, Tos3, or Elm1. All three Snf1-activating kinases contain serine/threonine kinase domains near their N termini and large C-terminal domains with little sequence conservation and previously unknown function. Deletion of the C-terminal domains of Sak1 and Tos3 greatly reduces their ability to activate the Snf1 pathway. In contrast, deletion of the Elm1 C-terminal domain has no effect on Snf1 signaling but abrogates the ability of Elm1 to participate in the morphogenetic-checkpoint signaling pathway. Thus, the C-terminal domains of Sak1, Tos3, and Elm1 help to determine pathway specificity. Additional deletion mutants of the Sak1 kinase revealed that the N terminus of the protein is essential for Snf1 signaling. The deletion of 43 amino acids from within the N terminus of Sak1 (residues 87 to 129) completely blocks Snf1 signaling and activation loop phosphorylation in vivo. The Sak1 kinase domain (lacking both N-terminal and C-terminal domains) is catalytically active and specific in vitro but is unable to promote Snf1 signaling in vivo when expressed at normal levels. Our studies indicate that the kinase domains of the Snf1-activating kinases are not sufficient by themselves for their proper function and that the nonconserved N-terminal and C-terminal domains are critical for the biological activities of these kinases.

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

Snf1 kinase complexes with different beta subunits display stress-dependent preferences for the three Snf1-activating kinases.

Three upstream kinases, Pak1, Tos3 and Elm1, are able to activate the Snf1 kinase. Since the Snf1 kinase itself assembles into three complexes that differ in their beta subunit identity, the possibility exists that each upstream kinase might be dedicated to a single isoform of the Snf1 kinase. To test this dedicated activator hypothesis, we generated a series of yeast strains that lacked different combinations of upstream kinases and beta subunits. Cells expressing only one of the three upstream kinases exhibited distinct abilities to activate Snf1, depending on the beta subunit present in the Snf1 kinase complex and the stress imposed on the cells. Pak1 and Gal83 were the most promiscuous. Pak1 was able to activate all three isoforms of the Snf1 kinase under all stress conditions tested. The Gal83 isoform of Snf1 was able to be activated by any of the three upstream kinases under aerobic growth conditions but showed a preference for Pak1 during growth on raffinose. Our results indicate that the three Snf1-activating kinases are not dedicated to specific isoforms of the Snf1 kinase. Instead, the different isoforms of the Snf1 kinase display stress-dependent preferences for the Pak1, Tos3 and Elm1 kinases.

Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition.

The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein. The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active. Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels. Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells. Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates.

Isolation of mutations in the catalytic domain of the snf1 kinase that render its activity independent of the snf4 subunit.

Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined. Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain. In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4. Deletion of residues 381 to 488 from the Snf1 protein resulted in a kinase that was activated by glucose limitation even in the absence of the Snf4 protein. A larger deletion (amino acids 381 to 608) encompassing virtually the entire regulatory domain resulted in complete inactivation of the Snf1 kinase even in the presence of Snf4. A genetic screen for amino acid substitutions that conferred an Snf4-independent phenotype identified four point mutations in the Snf1 catalytic domain. One very conservative mutation, leucine 183 to isoleucine, conferred nearly wild-type levels of Snf1 kinase function in the absence of the Snf4 protein. Purified Snf1 kinase was inactive when isolated from snf4Delta cells, whereas the Snf1-L183I kinase exhibited significant activity in the absence of Snf4. Our data support the idea that Snf1 kinase activity is constrained in cis by an autoinhibitory domain and that the Snf4-mediated activation of Snf1 can be bypassed by subtle conformational changes in the catalytic domain of the Snf1 kinase.