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Regulation of Snf1 Kinase

2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês

10.1074/jbc.m104418200

1083-351X

Rhonda R. McCartney, Martin C. Schmidt,

Plant nutrient uptake and metabolism

The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation. Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit. A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210. Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose. A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event. Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210. Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1. Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells. Finally, cells lacking the γ subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently. Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the γ subunit of the kinase itself. The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation. Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit. A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210. Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose. A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event. Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210. Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1. Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells. Finally, cells lacking the γ subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently. Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the γ subunit of the kinase itself. AMP-activated protein kinase hemagglutinin The Snf1 and AMP-activated protein kinases (AMPK)1 define a highly conserved family of serine-threonine protein kinases found in fungi, plants, Drosophila, Caenorhabditis elegans, and mammals. Members of the Snf1-AMPK family are central components of signal transduction pathways that are activated in response to nutrient stress (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1281) Google Scholar). The Snf1-AMPK enzymes are heterotrimers with a single catalytic α subunit and two regulatory subunits denoted β and γ (2Davies S.P. Hawley S.A. Woods A. Carling D. Haystead T.A. Hardie D.G. Eur. J. Biochem. 1994; 223: 351-357Crossref PubMed Scopus (131) Google Scholar, 3Schmidt M.C. McCartney R.R. EMBO J. 2000; 19: 4936-4943Crossref PubMed Google Scholar, 4Stapleton D. Gao G. Michell B.J. Widmer J. Mitchelhill K. Teh T. House C.M. Witters L.A. Kemp B.E. J. Biol. Chem. 1994; 269: 29343-29346Abstract Full Text PDF PubMed Google Scholar). In the yeast Saccharomyces cerevisiae, the Snf1 kinase plays a critical role when glucose becomes limiting. The net effect of Snf1 kinase activation is to down-regulate certain enzymes to conserve ATP energy and to relieve glucose repression of gene expression. Downstream targets of Snf1 include metabolic enzymes such as acetyl-CoA carboxylase (5Mitchelhill K.I. Stapleton D. Gao G. House C. Michell B. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1994; 269: 2361-2364Abstract Full Text PDF PubMed Google Scholar, 6Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19515Abstract Full Text PDF PubMed Google Scholar) and transcription factors such as Mig1 and Sip4 (3Schmidt M.C. McCartney R.R. EMBO J. 2000; 19: 4936-4943Crossref PubMed Google Scholar, 7Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (262) Google Scholar, 8Vincent O. Carlson M. EMBO J. 1998; 17: 7002-7008Crossref PubMed Scopus (106) Google Scholar). While there has been tremendous progress made in our understanding of glucose sensing and signal transduction (9Johnston M. Trends Genet. 1999; 15: 29-33Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), it is still unclear how glucose limitation is sensed and the information transduced to Snf1. Intracellular concentration of AMP, by analogy with the mammalian enzyme, is a good candidate for the signaling molecule, yet AMP has no effect on Snf1 kinase activity in vitro (6Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19515Abstract Full Text PDF PubMed Google Scholar,10Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). While AMP might not activate Snf1 directly, it is still possible that AMP might activate the Snf1 pathway by acting upstream of Snf1. To understand the regulation of Snf1 kinase, it is necessary to determine the number and identity of the regulators acting upstream of Snf1. A common regulatory switch observed in protein kinases is the phosphorylation of one or more residues in the activation loop of the catalytic subunit. In the unphosphorylated state, the activation loop can block access of substrates to the active site (11Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). Phosphorylation of residues in the activation loop causes a large outward rotation of the activation loop, thus making the active site accessible to substrate and aligning active site residues for catalysis (12Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar, 13Sicheri F. Kuriyan J. Curr. Opin. Struct. Biol. 1997; 7: 777-785Crossref PubMed Scopus (332) Google Scholar). In many instances, the phosphorylation of one kinase is catalyzed by a distinct upstream kinase, thereby creating kinase cascades. The mitogen-activated protein kinases are held in a physical complex with their upstream activating kinases, thereby routing molecular signals through insulated cascades (14Elion E.A. Science. 1998; 281: 1625-1626Crossref PubMed Scopus (121) Google Scholar). Several members of the AGC group of protein kinases (protein kinase family including protein kinase A, protein kinase G, and protein kinase C), a group named after the protein kinases PKA, PKG, and PKC (15Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2296) Google Scholar), are known to be phosphorylated by the phosphotidylinositol-dependent protein kinase PDK-1 (16Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: R93-R96Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 17Peterson R.T. Schreiber S.L. Curr. Biol. 1999; 9: R521-R524Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). An alternative mechanism for the modification of activation loop residues is autophosphorylation, either intermolecular or intramolecular. While PDK-1 phosphorylates the activation loop of other kinases, the phosphorylation of its own activation loop is most likely due to autophosphorylation, since it is observed with recombinant protein in the absence of all other mammalian kinases (18Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (292) Google Scholar). Many of the Src family tyrosine kinases are activated by intermolecular autophosphorylation of residues in the activation loop (19Cooper J.A. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (137) Google Scholar, 20Sato K. Aoto M. Mori K. Akasofu S. Tokmakov A.A. Sahara S. Fukami Y. J. Biol. Chem. 1996; 271: 13250-13257Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In yet another variation on this theme, proteins can bind to the activation loop to exert control over kinase activity. In the case of TAK1, a mitogen-activated protein kinase kinase kinase (MAP3K), intramolecular autophosphorylation of the activation loop is stimulated by the binding of the TAB1 protein (21Sakurai H. Miyoshi H. Mizukami J. Sugita T. FEBS Lett. 2000; 474: 141-145Crossref PubMed Scopus (142) Google Scholar). In the case of the JAK family of kinases, binding of the activation loop by the JAB protein inhibits kinase activity (22Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar). Thus, many different mechanisms of regulating protein kinase activity are mediated through events at the activation loop. The activity of the Snf1 protein kinase, a member of the calcium/calmodulin-dependent protein kinase family of serine/threonine protein kinases (15Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2296) Google Scholar), is also regulated by phosphorylation (10Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). The conserved threonine residue in the activation loop of Snf1 kinase is at position 210. The first suggestion that this residue was important for Snf1 regulation came from the finding that a threonine to alanine mutation at position 210 inactivated the Snf1 kinase (23Estruch F. Treitel M.A. Yang X. Carlson M. Genetics. 1992; 132: 639-650Crossref PubMed Google Scholar). This study documented that threonine 210 was essential for Snf1 activity but did not address whether or not threonine 210 was modified. A subsequent study by Hardie and colleagues (10Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) showed that Snf1 kinase activity measured in vitro was greatly reduced by phosphatase treatment. This study indicated that the phosphorylation of Snf1 was essential for catalytic activity but did not identify the site(s) of modification. However, these studies and the numerous parallels with other protein kinases strongly suggested that the Snf1 kinase is activated by phosphorylation on threonine 210 and possibly other sites as well. The down-regulation of Snf1 kinase by dephosphorylation is almost certainly catalyzed by the PP1 phosphatase Glc7 in complex with the regulatory subunit Reg1 (24Ludin K. Jiang R. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6245-6250Crossref PubMed Scopus (159) Google Scholar, 25Tu J. Carlson M. EMBO J. 1995; 14: 5939-5946Crossref PubMed Scopus (195) Google Scholar). Studies of the mammalian orthologue of Snf1, AMPK, have shown that its regulation is mediated by multiple phosphorylation events, the most important one being at the conserved threonine residue in the activation loop (26Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar). Biochemical studies of rat liver extracts have identified an activity that is responsible for the phosphorylation of AMPK (27Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (1015) Google Scholar). The activating enzyme, called AMP-activated protein kinase kinase (AMPK kinase), is likely to be distinct from AMPK based on differences in chromatographic properties, response to allosteric regulation by AMP, and inactivation by phosphatase treatment. However, AMPK and AMPK kinase are very similar in size, and until the AMPK kinase is characterized at the molecular level, one cannot rule out the possibility that AMPK kinase is a modified form of AMPK itself. The focus of this study is on the Snf1 signaling pathway in yeast. While genetic and biochemical evidence suggest that the Snf1 kinase is regulated by phosphorylation on threonine 210, genetic screens for Snf mutants have failed to detect any kinases that could act upstream of Snf1 kinase. One possible explanation for the failure to identify the Snf1-activating kinase genetically is that it may not exist. The Snf1 protein could be activated by an autophosphorylation mechanism. For instance, the Snf1 reactivating factor identified by Wilson et al. (10Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) could represent a protein that binds Snf1 and stimulates an autophosphorylation event similar to the effect of TAB1 protein on TAK1 kinase (21Sakurai H. Miyoshi H. Mizukami J. Sugita T. FEBS Lett. 2000; 474: 141-145Crossref PubMed Scopus (142) Google Scholar). An alternative explanation for the failure to identify a Snf1-activating kinase genetically is that more than one kinase may be capable of phosphorylating Snf1. Thus, in genetic screens for loss of function alleles, redundant Snf1-activating kinases would complement each other and never be detected. To further define the Snf1 signaling pathway, we have developed an assay to measure the phosphorylation of Snf1 kinase on threonine 210. The combination of genetics with an immunological assay for threonine 210 phosphorylation has allowed us to unambiguously determine that the Snf1 kinase is regulated by an upstream kinase. Saccharomyces cerevisiae strains used in this study are described in Table I. Except where indicated, growth of yeast utilized standard medium at 30 °C (28Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990: 177-186Google Scholar). Glucose and raffinose were present at 2% (g/100 ml) except for derepressing medium, which contained 0.05% glucose (g/100 ml). Antimycin A was included at 1 μg/ml in all raffinose media. Transformation of yeast strains utilized the lithium acetate procedure (29Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1712) Google Scholar). Repressed cultures were grown in 2% glucose and harvested in mid-log phase at an A600 of 0.4–0.8. Derepressed cultures were prepared by resuspending repressed cells in medium containing 0.05% glucose and continuing growth for an additional 2 h.Table IS. cerevisiae strainsStrainGenotypeSource or referenceMSY182MATaura3–52 leu2Δ1 trp1Δ63 his3Δ200Ganster et al.(36Ganster R.W. McCartney R.R. Schmidt M.C. Genetics. 1998; 150: 31-42PubMed Google Scholar)FY1193MATα ura3–52 leu2Δ1 trp1Δ63 his3Δ200 snf1Δ10Fred WinstonMSY563MATα ura3–52 leu2Δ1 his3Δ200 lys2–128δ snf1Δ10 snf4Δ1This studyMSY568MATα ura3–52 leu2Δ1 snf4Δ1This studyPY102MATa ura3–52 leu2Δ1 trp1Δ63 his4–917δ lys2–173R2 reg1Δ∷URA3Karen Arndt Open table in a new tab Mig1 protein was modified to contain three copies of the HA epitope at its C terminus (3Schmidt M.C. McCartney R.R. EMBO J. 2000; 19: 4936-4943Crossref PubMed Google Scholar). The Snf1 protein was epitope-tagged by polymerase chain reaction-amplifying three copies of the HA epitope present in plasmid pMR2307 (30Tyers M. Tokiwa G. Nash R. Futcher B. EMBO J. 1992; 11: 1773-1784Crossref PubMed Scopus (339) Google Scholar) with primers containingEcoRI sites at the termini. The tag was then inserted into the naturally occurring MunI site, which partially overlaps the Snf1 stop codon. The resulting construct encodes full-length Snf1 protein with these additional amino acids added to the C terminus: SYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAA. Antibodies directed against phosphorylated threonine 210 were purchased from Research Genetics (Huntsville, AL). A 13-mer peptide (DGNFLK[T-PO4]SCGSPN) corresponding to amino acids 204–216 of Snf1 protein was synthesized such that the central threonine residue was phosphothreonine. The peptide was coupled to keyhole limpet hemacyanin and used to immunize two rabbits. Sera were pooled, and antibodies were purified by two sequential steps of affinity chromatography. First, antibodies that bound to a phosphopeptide column were purified. Next, antibodies that bound to a column containing the unphosphorylated peptide were removed. The collection of antibodies resulting from these affinity selections are referred to as the α-PT210 antibodies. Protein extracts were prepared using a glass bead lysis procedure (31Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar) in NHTG buffer (40 mmHEPES, pH 7.3, 350 mm NaCl, 0.1% Tween 20, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each of benzamidine, pepstatin A, leupeptin, and aprotinin). Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard. For analysis of Thr210 phosphorylation, protein extracts were prepared using a modification of the NaOH extraction procedure developed by Kushnirov (32Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (679) Google Scholar). Cell cultures (25 ml) were grown to anA600 of 0.5–0.8. NaOH was added directly to the culture medium to a final concentration of 0.1 m, and cells were incubated for 5 min at room temperature. Cells were collected by centrifugation and proteins extracted by boiling in SDS sample buffer (10 μl per OD of cells) for 5 min. Debris was removed by centrifugation, and the supernatant fraction was recovered and extensively dialyzed against an excess of RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 50 mm Tris-HCl, pH 8.0) supplemented with 50 mmsodium fluoride and 5 mm sodium pyrophosphate. Protein recovery following dialysis was typically 1.75 mg of protein from a 25-ml culture. Western blots were performed using the method described previously (31Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar). Monoclonal antibodies against the HA epitope were purchased from Santa Cruz Biotechnology and used at a dilution of 1:1000. Affinity-purified α-PT210 antibodies were used at a dilution of 1:1000. For immunoprecipitations, 300–500 μg of protein was incubated with 2 μl of anti-HA for 90 min at 4 °C with gentle agitation in RIPA buffer supplemented with protease and phosphatase inhibitors (50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml pepstatin, and 2 μg/ml chymostatin). Immune complexes were collected by low speed centrifugation following incubation with 20 μl of protein A beads (Sigma) that had been washed in RIPA buffer. Bound proteins were eluted by boiling the beads in 15 μl of SDS sample buffer. Eluted proteins were resolved on an SDS 10% polyacrylamide gel and subjected to Western blotting with either anti-HA or α-PT210 antibodies (31Schmidt M.C. McCartney R.R. Zhang X. Tillman T.S. Solimeo H. Wolfl S. Almonte C. Watkins S.C. Mol. Cell. Biol. 1999; 19: 4561-4571Crossref PubMed Scopus (129) Google Scholar). All Western blotting experiments have been repeated at least twice with extracts from independent transformants and have produced comparable results. A representative exposure of one blot is shown. The Snf1 kinase is required by yeast for the fermentation of sucrose and the related trisaccharide raffinose (33Carlson M. Curr. Opin. Genet. Dev. 1998; 8: 560-564Crossref PubMed Scopus (72) Google Scholar). Strains that carry the deletion allele snf1Δ10 manifest a pronounced growth defect on raffinose medium that can be complemented by a plasmid-encoded SNF1 gene that bears three copies of the HA epitope at the C terminus (Fig.1A). The point mutation K84R eliminates the catalytic activity of Snf1 kinase by disrupting the ATP binding domain (34Celenza J.L. Carlson M. Mol. Cell. Biol. 1989; 9: 5034-5044Crossref PubMed Scopus (176) Google Scholar). The K84R mutation was engineered in the epitope-tagged SNF1 gene and tested for the ability to complement the snf1Δ10 deletion for growth on raffinose. The K84R mutation completely blocks the ability of the plasmid-encoded gene to complement; however, the accumulation of the K84R-Snf1 protein, as judged by Western blot directed against the HA tag, is unaffected (Fig. 1B). In a similar manner, a mutation that converts the activation loop threonine to the nonphosphorylatable residue alanine was engineered and tested. The T210A allele of SNF1 was also completely unable to complement the snf1Δ10 deletion for the fermentation of raffinose, but this mutation had no effect on protein levels as judged by Western blot. The affects of these two point mutations on Snf1 kinase activity have been reported previously (23Estruch F. Treitel M.A. Yang X. Carlson M. Genetics. 1992; 132: 639-650Crossref PubMed Google Scholar, 34Celenza J.L. Carlson M. Mol. Cell. Biol. 1989; 9: 5034-5044Crossref PubMed Scopus (176) Google Scholar). We show here that these two mutations inactivate the Snf1 kinase without affecting protein levels. Thus, these alleles are suitable for further studies to probe the regulation of Snf1 activity. To study the activation of the Snf1 kinase, we sought a reagent that could specifically detect Snf1 protein that had been phosphorylated on threonine 210. A 13-residue synthetic peptide, corresponding to amino acids 204–216 of Snf1 and containing phosphothreonine at position 210, was synthesized and used to immunize rabbits. Antibodies that recognized the phosphorylated peptide were affinity-purified and used in Western blotting experiments. A functional HA-tagged Snf1 protein was collected by immunoprecipitation and resolved on an SDS gel. When anti-HA monoclonal was used to probe the blot, equivalent levels of Snf1 protein were detected in extracts expressing wild type Snf1 or the T210A mutant (Fig.2B, lanes 1 and2). However, when the α-PT210 antibodies were used, the wild type, but not the T210A mutant, was detected. When the extracts were pretreated with either calf intestine alkaline phosphatase or λ-phosphatase, the α-PT210 antibodies were no longer able to detect the Snf1 protein (Fig. 2, lanes 3–7). Pretreatment with phosphatase did not alter Snf1 reactivity with the HA monoclonal antibody. Therefore the α-PT210 antibodies detected Snf1 protein only when threonine 210 was present and only when the endogenous phosphorylations were left intact. When combined with the knowledge of the immunogen, the simplest conclusion is that the α-PT210 antibodies specifically recognize Snf1 protein that is phosphorylated on threonine 210. The phosphorylation state of threonine 210 was assessed in cells that were grown under conditions of excess glucose (6%) or glucose limitation (0.05%) for 1 h. Since the mere act of centrifugation has been shown to be sufficient to activate Snf1 kinase (10Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), a procedure was devised to block cellular metabolism prior to harvest. Using a modification of a simple protein extraction procedure (32Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (679) Google Scholar), cell metabolism was arrested first by the addition of sodium hydroxide to a concentration of 0.1 m for 5 min prior to harvesting cells by centrifugation. Protein extracts were then isolated from cultures expressing both wild type Snf1 protein from the chromosome and an epitope-tagged Snf1 protein from a centromere plasmid. When an extract from glucose-repressed cultures was probed directly by Western blot with the α-PT210 antibodies, very little reactivity was observed (Fig.3A, lane 1). In contrast, extracts prepared from glucose-limited cultures readily detected a doublet with the α-PT210 antibodies. The upper band represents the epitope-tagged protein (75.6 kDa), and thelower band represents the chromosomal untagged Snf1 (72.0 kDa). Therefore, the phosphorylation state of the Snf1 threonine 210 correlates with glucose limitation. As a control, the same experiment was repeated using the T210A allele of SNF1 as the plasmid-encoded gene. In this case, the wild type (lower band) Snf1 protein was phosphorylated normally in response to glucose limitation, but the T210A protein (upper band) shows no reactivity with the α-PT210 antibodies (Fig. 3A,lanes 3 and 4). Equivalent levels of the epitope-tagged Snf1 protein was detected in all the extracts (lower panel) confirming that the T210A protein was present, although not detected by the α-PT210 antibodies. In a similar experiment, extracts were prepared from cells expressing only the epitope-tagged Snf1 protein. The Snf1 protein was immunoprecipitated with anti-HA antibody prior to resolution by SDS gel and Western blot with both α-PT210 antibody and anti-HA antibody (Fig. 3B). The Snf1 protein is present in both extracts at comparable levels as judged by reactivity with the HA antibody. However, the reactivity with the α-PT210 antibodies is only found in derepressed cultures. Therefore, the phosphorylation of threonine 210 directly correlates with glucose stress and the activity of the Snf1 kinase. Several different mechanisms can be responsible for the phosphorylation of kinase activation loop residues. We sought to determine directly whether the Snf1 kinase is activated by an upstream kinase or by one of two possible modes of autophosphorylation (Fig.4A). If Snf1 were activated by a distinct upstream kinase, then a mutation in the Snf1 active site should not affect the phosphorylation of the Thr210 site. However, if the Thr210 phosphorylation were the result of an autophosphorylation event, then an active site mutation in Snf1 would eliminate the Thr210 phosphorylation. Cells that contained either wild type SNF1 or the snf1Δ10allele on the chromosome were transformed with HA-tagged Snf1 that was either catalytically active or inactivated by the K84R mutation in the ATP binding domain (34Celenza J.L. Carlson M. Mol. Cell. Biol. 1989; 9: 5034-5044Crossref PubMed Scopus (176) Google Scholar). Snf1 protein was harvested by immunoprecipitation with HA antibody and the phosphorylation state of threonine 210 assessed with the α-PT210 antibodies (Fig.4B). The SNF1 cells expressing the K84R kinase dead mutant are still able to phosphorylate Thr210, thereby eliminating intermolecular autophosphorylation as a mechanism. Thesnf1Δ10 cells transformed with a plasmid expressing the K84R kinase dead mutant have no Snf1 kinase activity, and yet they showed levels of phosphorylated threonine 210 equivalent to cells expressing active Snf1 kinase (Fig. 4B, lanes 2and 4). This result eliminates intramolecular autophosphorylation as a mechanism. Cells lacking Snf1 kinase activity also exhibit regulated Thr210 phosphorylation in response to glucose limitation (Fig. 4C). These data demonstrate that the phosphorylation of Snf1 threonine 210 cannot be the result of autophosphorylation. The phosphorylation of Snf1 kinase on threonine 210 must be catalyzed by a distinct upstream kinase. Studies that examined the involvement of the Snf1 kinase in the response to sodium ion stress have arrived at different conclusions (35Alepuz P.M. Cunningham K.W. Estruch F. Mol. Microbiol. 1997; 26: 91-98Crossref PubMed Scopus (95) Google Scholar, 36Ganster R.W. McCartney R.R. Schmidt M.C. Genetics. 1998; 150: 31-42PubMed Google Scholar). We tested whether sodium ion stress resulted in the phosphorylation of threonine 210 by treating cells for 1 h with 0.8 m NaCl. Protein extracts were prepared, and the epitope-tagged Snf1 protein was collected by immunoprecipitation. The level of threonine 210 phosphorylation measured by Western blot with the α-PT210 antibodies is consistently higher in cells that have been subjected to sodium ion stress (Fig. 5A). This experiment has been repeated three times with independent transformants, and in all cases the phosphorylation of Thr210 was detected in sodium-stressed cells but was less prominent than in glucose-stressed cells. We also tested whether sodium ion stress resulted in the phosphorylation of Mig1, a known target of Snf1 kinase in the glucose derepression pathway (7Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (262) Google Scholar, 37Smith F.C. Davies S.P. Wilson W.A. Carling D. Hardie D.G. FEBS Lett. 1999; 453: 219-223Crossref PubMed Scopus (82) Google Scholar). Earlier studies have shown that the mobility shift of Mig1 protein is due to phosphorylation (7Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18