FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080647804> ?p ?o ?g. }

• W2080647804 endingPage "19529" @default.
• W2080647804 startingPage "19521" @default.
• W2080647804 abstract "The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The γ subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (γ) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the β subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 β subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1. The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The γ subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (γ) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the β subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 β subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1. The SNF1/AMP-activated protein kinase (AMPK) 3The abbreviations used are: AMPK, AMP-activated protein kinase; CBS, cystathionine β-synthase; GBD, glycogen-binding domain; SC, synthetic complete; Suc, sucrose; 2DG, 2-deoxyglucose. 3The abbreviations used are: AMPK, AMP-activated protein kinase; CBS, cystathionine β-synthase; GBD, glycogen-binding domain; SC, synthetic complete; Suc, sucrose; 2DG, 2-deoxyglucose. family plays a central role in responses to metabolic stress and regulation of energy homeostasis in eukaryotes (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1265) Google Scholar, 2Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2293) Google Scholar). In mammals, AMPK regulates many metabolic processes, notably glucose and lipid metabolism, and controls transcription and protein synthesis to maintain energy balance. AMPK is activated by hormones and by stresses that deplete cellular ATP, thereby elevating the AMP:ATP ratio, and in humans, it is a target of drugs used in treatment of type 2 diabetes. AMPK is a heterotrimer comprising the catalytic α subunit and two regulatory subunits, β and γ, which exist as different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3). The kinase is activated by phosphorylation on Thr-172 in the activation loop of the α subunit by upstream kinases, including LKB1 (3Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (469) Google Scholar, 4Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 5Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1315) Google Scholar, 6Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1408) Google Scholar), Ca2+/calmodulin-dependent protein kinase kinase (7Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 8Hong S.P. Momcilovic M. Carlson M. J. Biol. Chem. 2005; 280: 21804-21809Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 9Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar, 10Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar), and possibly TAK1 (11Momcilovic M. Hong S.P. Carlson M. J. Biol. Chem. 2006; 281: 25336-25343Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). AMP activates AMPK allosterically, inhibits dephosphorylation, and may enhance phosphorylation, whereas ATP is inhibitory (4Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 12Hawley S.A. Selbert M.A. Goldstein E.G. Edelman A.M. Carling D. Hardie D.G. J. Biol. Chem. 1995; 270: 27186-27191Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 13Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (492) Google Scholar, 14Hawley 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 (995) Google Scholar, 15Suter M. Riek U. Tuerk R. Schlattner U. Wallimann T. Neumann D. J. Biol. Chem. 2006; 281: 32207-32216Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 16Sanders M.J. Grondin P.O. Hegarty B.D. Snowden M.A. Carling D. Biochem. J. 2007; 403: 139-148Crossref PubMed Scopus (517) Google Scholar).The effects of nucleotides are mediated by the γ subunit, which comprises four cystathionine β-synthase (CBS) repeats (17Bateman A. Trends Biochem. Sci. 1997; 22: 12-13Abstract Full Text PDF PubMed Scopus (439) Google Scholar) (Fig. 1). CBS repeats occur in a variety of proteins as tandem pairs, termed Bateman domains (18Adams J. Chen Z.P. Van Denderen B.J. Morton C.J. Parker M.W. Witters L.A. Stapleton D. Kemp B.E. Protein Sci. 2004; 13: 155-165Crossref PubMed Scopus (129) Google Scholar), and bind adenosine derivatives (19Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar). Biochemical studies suggested that each γ subunit can bind two AMP or ATP molecules (19Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar). Crystallographic analysis of a partial heterotrimer of rat AMPK, comprising the C-terminal domains of α1 and β2 and the complete γ1 subunit, revealed three nucleotide-binding sites, one of which contained tightly bound, nonexchangeable AMP (20Xiao B. Heath R. Saiu P. Leiper F.C. Leone P. Jing C. Walker P.A. Haire L. Eccleston J.F. Davis C.T. Martin S.R. Carling D. Gamblin S.J. Nature. 2007; 449: 496-500Crossref PubMed Scopus (429) Google Scholar). The first identification of a γ subunit mutation was the substitution R200Q in γ3 of pig, which increases glycogen levels in skeletal muscle (21Milan D. Jeon J.-T. Looft C. Amarger V. Robic A. Thelander M. Rogel-Gaillard C. Paul S. Iannuccelli N. Rask L. Ronne H. Lundstrom K. Reinsch N. Gellin J. Kalm E. Le Roy P. Chardon P. Andersson L. Science. 2000; 288: 1248-1251Crossref PubMed Scopus (605) Google Scholar). In humans, mutations in the γ2 subunit cause cardiac hypertrophy, glycogen accumulation, and ventricular pre-excitation associated with Wolff-Parkinson-White syndrome (19Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar, 22Gollob M.H. Green M.S. Tang A.S. Gollob T. Karibe A. Ali Hassan A.S. Ahmad F. Lozado R. Shah G. Fananapazir L. Bachinski L.L. Roberts R. N. Engl. J. Med. 2001; 344: 1823-1831Crossref PubMed Scopus (532) Google Scholar, 23Gollob M.H. Seger J.J. Gollob T.N. Tapscott T. Gonzales O. Bachinski L. Roberts R. Circulation. 2001; 104: 3030-3033Crossref PubMed Scopus (229) Google Scholar, 24Blair E. Redwood C. Ashrafian H. Oliveira M. Broxholme J. Kerr B. Salmon A. Ostman-Smith I. Watkins H. Hum. Mol. Genet. 2001; 10: 1215-1220Crossref PubMed Google Scholar, 25Arad M. Benson D.W. Perez-Atayde A.R. McKenna W.J. Sparks E.A. Kanter R.J. McGarry K. Seidman J.G. Seidman C.E. J. Clin. Investig. 2002; 109: 357-362Crossref PubMed Scopus (454) Google Scholar, 26Burwinkel B. Scott J.W. Buhrer C. van Landeghem F.K. Cox G.F. Wilson C.J. Hardie D.G. Kilimann M.W. Am. J. Hum. Genet. 2005; 76: 1034-1049Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Such mutations decrease the affinity of the γ subunit for AMP and ATP, reduce allosteric AMP activation, and despite some controversy, appear to increase the basal phosphorylation and activity of AMPK in cells expressing LKB1 (18Adams J. Chen Z.P. Van Denderen B.J. Morton C.J. Parker M.W. Witters L.A. Stapleton D. Kemp B.E. Protein Sci. 2004; 13: 155-165Crossref PubMed Scopus (129) Google Scholar, 19Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar, 26Burwinkel B. Scott J.W. Buhrer C. van Landeghem F.K. Cox G.F. Wilson C.J. Hardie D.G. Kilimann M.W. Am. J. Hum. Genet. 2005; 76: 1034-1049Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 27Hamilton S.R. Stapleton D. O'Donnell Jr., J.B. Kung J.T. Dalal S.R. Kemp B.E. Witters L.A. FEBS Lett. 2001; 500: 163-168Crossref PubMed Scopus (94) Google Scholar, 28Daniel T. Carling D. J. Biol. Chem. 2002; 277: 51017-51024Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Many of the altered residues are located close to nucleotide-binding sites in the mammalian enzyme structure (20Xiao B. Heath R. Saiu P. Leiper F.C. Leone P. Jing C. Walker P.A. Haire L. Eccleston J.F. Davis C.T. Martin S.R. Carling D. Gamblin S.J. Nature. 2007; 449: 496-500Crossref PubMed Scopus (429) Google Scholar).Structural analysis of AMPK from the fission yeast Schizosaccharomyces pombe suggested that the ability of the γ subunit to bind nucleotide is broadly conserved (29Townley R. Shapiro L. Science. 2007; 315: 1726-1729Crossref PubMed Scopus (151) Google Scholar, 30Jin X. Townley R. Shapiro L. Structure. 2007; 15: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The crystal structure of a partial heterotrimeric AMPK, lacking the catalytic domain, defined a binding site for AMP and ATP in the C-terminal Bateman domain of the γ subunit (29Townley R. Shapiro L. Science. 2007; 315: 1726-1729Crossref PubMed Scopus (151) Google Scholar). This site corresponds to the site in the mammalian enzyme that contains nonexchangeable AMP and is not involved in AMP/ATP sensing (20Xiao B. Heath R. Saiu P. Leiper F.C. Leone P. Jing C. Walker P.A. Haire L. Eccleston J.F. Davis C.T. Martin S.R. Carling D. Gamblin S.J. Nature. 2007; 449: 496-500Crossref PubMed Scopus (429) Google Scholar). Additional studies of the S. pombe partial heterotrimer revealed a binding site for ADP in the N-terminal Bateman domain (30Jin X. Townley R. Shapiro L. Structure. 2007; 15: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, S. pombe AMPK has not been well characterized genetically or biochemically, and it is not known whether it is regulated by nucleotides.In the budding yeast Saccharomyces cerevisiae, SNF1 protein kinase is the ortholog of AMPK (for review, see Ref. 31Hedbacker K. Carlson M. Front. Biosci. 2008; 13: 2408-2420Crossref PubMed Scopus (384) Google Scholar). SNF1 is required for the adaptation of cells to metabolic stress, notably carbon stress (32Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (508) Google Scholar), but also starvation for other nutrients (33Thompson-Jaeger S. Francois J. Gaughran J.P. Tatchell K. Genetics. 1991; 129: 697-706Crossref PubMed Google Scholar, 34Kuchin S. Vyas V.K. Carlson M. Mol. Cell. Biol. 2002; 22: 3994-4000Crossref PubMed Scopus (171) Google Scholar, 35Orlova M. Kanter E. Krakovich D. Kuchin S. Eukaryot. Cell. 2006; 5: 1831-1837Crossref PubMed Scopus (72) Google Scholar) and various environmental stresses (36Alepuz P.M. Cunningham K.W. Estruch F. Mol. Microbiol. 1997; 26: 91-98Crossref PubMed Scopus (93) Google Scholar, 37Portillo F. Mulet J.M. Serrano R. FEBS Lett. 2005; 579: 512-516Crossref PubMed Scopus (46) Google Scholar, 38Platara M. Ruiz A. Serrano R. Palomino A. Moreno F. Arino J. J. Biol. Chem. 2006; 281: 36632-36642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 39Dubacq C. Chevalier A. Mann C. Mol. Cell. Biol. 2004; 24: 2560-2572Crossref PubMed Scopus (40) Google Scholar, 40Hong S.P. Carlson M. J. Biol. Chem. 2007; 282: 16838-16845Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Carbon metabolism is particularly important for yeast, and SNF1 is required for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose; the initial snf1 mutation was named for the sucrose-nonfermenting phenotype (41Carlson M. Osmond B.C. Botstein D. Genetics. 1981; 98: 25-40Crossref PubMed Google Scholar). The kinase is required to activate transcription of genes involved in the metabolism of sucrose and other carbon sources (42Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 43Young E.T. Dombek K.M. Tachibana C. Ideker T. J. Biol. Chem. 2003; 278: 26146-26158Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar); it is not required for growth on glucose, which inhibits its activity. In addition to regulating transcription, SNF1 controls the activity of metabolic enzymes involved in fatty acid metabolism and carbohydrate storage (44Hardy T.A. Huang D. Roach P.J. J. Biol. Chem. 1994; 269: 27907-27913Abstract Full Text PDF PubMed Google Scholar, 45Mitchelhill 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, 46Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar).Like other members of the AMPK family, SNF1 protein kinase is heterotrimeric, comprising the Snf1 catalytic (α) subunit, one of three β subunit isoforms (Gal83, Sip1, or Sip2), and the Snf4 (γ) subunit. Snf1 is phosphorylated on Thr-210 in the activation loop by the upstream kinases Sak1, Tos3, and Elm1 (3Hong S.-P. Leiper F.C. Woods A. Carling D. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8839-8843Crossref PubMed Scopus (469) Google Scholar, 47Sutherland C.M. Hawley S.A. McCartney R.R. Leech A. Stark M.J. Schmidt M.C. Hardie D.G. Curr. Biol. 2003; 13: 1299-1305Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 48Nath N. McCartney R.R. Schmidt M.C. Mol. Cell. Biol. 2003; 23: 3909-3917Crossref PubMed Scopus (125) Google Scholar) and is dephosphorylated by Reg1-Glc7 protein phosphatase 1 (49Sanz P. Alms G.R. Haystead T.A.J. Carlson M. Mol. Cell. Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (178) Google Scholar, 50McCartney R.R. Schmidt M.C. J. Biol. Chem. 2001; 276: 36460-36466Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). SNF1 is activated by glucose limitation (46Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 50McCartney R.R. Schmidt M.C. J. Biol. Chem. 2001; 276: 36460-36466Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 51Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 52Jiang R. Carlson M. Genes Dev. 1996; 10: 3105-3115Crossref PubMed Scopus (240) Google Scholar), but the regulatory signal(s) is not known. Acute glucose limitation depletes ATP and elevates the AMP:ATP ratio (51Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), but AMP does not allosterically activate SNF1 in vitro (45Mitchelhill 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, 46Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 51Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The possibility remains that AMP or ATP controls SNF1 activity in vivo.The β subunits control subcellular localization of SNF1 (53Vincent O. Townley R. Kuchin S. Carlson M. Genes Dev. 2001; 15: 1104-1114Crossref PubMed Scopus (218) Google Scholar) and interactions with substrates (54Vincent O. Carlson M. EMBO J. 1999; 18: 6672-6681Crossref PubMed Scopus (76) Google Scholar). Gal83 is the major isoform during growth on glucose (53Vincent O. Townley R. Kuchin S. Carlson M. Genes Dev. 2001; 15: 1104-1114Crossref PubMed Scopus (218) Google Scholar) and contributes the most to SNF1 activity in response to acute glucose limitation (55Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (72) Google Scholar). Gal83 and Sip2 contain a conserved glycogen-binding domain (GBD) and bind glycogen in vitro (56Wiatrowski H.A. van Denderen B.J.W. Berkey C.D. Kemp B.E. Stapleton D. Carlson M. Mol. Cell. Biol. 2004; 24: 352-361Crossref PubMed Scopus (34) Google Scholar).The Snf4 subunit, like mammalian γ subunits, consists of four CBS repeats (Fig. 1). Snf4 interacts directly with Snf1 and the β subunits (52Jiang R. Carlson M. Genes Dev. 1996; 10: 3105-3115Crossref PubMed Scopus (240) Google Scholar, 57Jiang R. Carlson M. Mol. Cell. Biol. 1997; 17: 2099-2106Crossref PubMed Scopus (203) Google Scholar, 58Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (130) Google Scholar) and is important for SNF1 function in vivo and in vitro; very little catalytic activity is detected in snf4 mutant cells, which exhibit a phenotype similar to that of snf1 cells (46Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 59Neigeborn L. Carlson M. Genetics. 1984; 108: 845-858Crossref PubMed Google Scholar, 60Celenza J.L. Eng F.J. Carlson M. Mol. Cell. Biol. 1989; 9: 5045-5054Crossref PubMed Scopus (145) Google Scholar). In contrast, Snf4 is not required for function of the truncated Snf1 catalytic domain (residues 1–309) (61Celenza J.L. Carlson M. Mol. Cell. Biol. 1989; 9: 5034-5044Crossref PubMed Scopus (174) Google Scholar) or of Snf1 lacking residues 381–488 (62Leech A. Nath N. McCartney R.R. Schmidt M.C. Eukaryot. Cell. 2003; 2: 265-273Crossref PubMed Scopus (40) Google Scholar), and Snf4 interacts directly with the C terminus of Snf1 (52Jiang R. Carlson M. Genes Dev. 1996; 10: 3105-3115Crossref PubMed Scopus (240) Google Scholar, 58Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (130) Google Scholar), suggesting that Snf4 counteracts autoinhibition by a C-terminal autoinhibitory sequence. However, several lines of evidence indicated that Snf4 is dispensable for glucose regulation. Various Snf4-independent mutant Snf1 proteins conferred glucose-regulated gene expression in the absence of Snf4 function (52Jiang R. Carlson M. Genes Dev. 1996; 10: 3105-3115Crossref PubMed Scopus (240) Google Scholar, 61Celenza J.L. Carlson M. Mol. Cell. Biol. 1989; 9: 5034-5044Crossref PubMed Scopus (174) Google Scholar, 62Leech A. Nath N. McCartney R.R. Schmidt M.C. Eukaryot. Cell. 2003; 2: 265-273Crossref PubMed Scopus (40) Google Scholar), and the phosphorylation of Thr-210 on full-length Snf1 remained glucose-regulated in snf4Δ mutants (50McCartney R.R. Schmidt M.C. J. Biol. Chem. 2001; 276: 36460-36466Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 63Elbing K. Rubenstein E.M. McCartney R.R. Schmidt M.C. J. Biol. Chem. 2006; 281: 26170-26180Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These data indicate that at least one glucose regulatory mechanism operates independent of Snf4. The question remains: does Snf4 have any direct role in mediating regulatory signals?Structural analysis has not yet resolved the issue of whether Snf4 binds nucleotide. The crystal structure of the C-terminal Bateman (Bateman2) domain revealed a dimer with a pocket at its center, and residues corresponding to those altered by disease-causing mutations were located near this pocket (64Rudolph M.J. Amodeo G.A. Iram S.H. Hong S.P. Pirino G. Carlson M. Tong L. Structure. 2007; 15: 65-74Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The structure of a partial heterotrimer of SNF1, containing C-terminal fragments of Snf1 and Sip2 and full-length Snf4, was reported (58Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (130) Google Scholar). This structure lacked bound nucleotide, although AMP was included in the crystallization solution, and comparison with the S. pombe structure revealed side chain differences in Snf4 that would interfere with AMP binding; however, another crystal appeared to have AMP bound to Snf4 at a similar position as in the S. pombe subunit (58Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (130) Google Scholar).In this study, we used genetic analysis to address the regulatory role of Snf4. We first introduced substitutions at sites that, in mammalian γ subunits, contribute to nucleotide binding, including alterations analogous to those causing human disease, and assayed the effects of each mutant Snf4 protein on regulation of Thr-210 phosphorylation and activity of SNF1 in yeast cells. Some of these mutations relieved glucose inhibition of phosphorylation and activity, providing evidence that Snf4 participates in a glucose regulatory mechanism, which may involve ligand binding. Additional substitutions that lack mammalian counterparts and alter residues that are distant from putative nucleotide-binding sites also relieved glucose inhibition of SNF1. Inspection of the crystal structure suggested that interactions of Snf4 with the GBD of the β subunit were perturbed. We further showed that substitutions in the GBD and deletion of the GBD similarly relieve glucose inhibition of SNF1. Finally, we showed that glucose inhibition of SNF1 is normal in cells lacking glycogen. These findings provide evidence for novel roles of Snf4 and the GBD in glucose regulation of SNF1.EXPERIMENTAL PROCEDURESExpression of Mutant Snf4 Proteins—Centromeric plasmid pOV75 expresses Snf4 from the native promoter; this plasmid contains a NotI site at the C terminus of the open reading frame and differs from pOV76 (53Vincent O. Townley R. Kuchin S. Carlson M. Genes Dev. 2001; 15: 1104-1114Crossref PubMed Scopus (218) Google Scholar) in lacking green fluorescent protein sequence. Mutations were introduced into pOV75 by using the QuikChange II XL site-directed mutagenesis kit (Stratagene) and confirmed by sequencing. Wild-type and mutant Snf4 proteins were expressed from pOV75 and its mutant derivatives in snf4Δ mutant cells of S. cerevisiae strain MCY2634 (MATa snf4Δ2 his3 leu2 ura3), which has the S288C genetic background. The elc1Δ::KanMX4 mutant strain was a derivative of BY4741 (MATa his3 leu2 met15 ura3; Research Genetics).Expression of Mutant Gal83 Proteins—Centromeric plasmid pRT12 expresses Gal83 fused to green fluorescent protein from the native promoter (53Vincent O. Townley R. Kuchin S. Carlson M. Genes Dev. 2001; 15: 1104-1114Crossref PubMed Scopus (218) Google Scholar). pOV81 and pHW39 (56Wiatrowski H.A. van Denderen B.J.W. Berkey C.D. Kemp B.E. Stapleton D. Carlson M. Mol. Cell. Biol. 2004; 24: 352-361Crossref PubMed Scopus (34) Google Scholar) are derivatives of pRT12 that express Gal83G235R and Gal83W184A,R214Q, respectively. The GBD (codons 153–243) was deleted from pRT12 by site-directed mutagenesis, yielding pMM66. Wild-type and mutant Gal83 proteins were expressed from these plasmids in S. cerevisiae strain MCY4099 (MATα gal83Δ::TRP1 sip1Δ::kanMX6 sip2Δ::kanMX4 ade2 can1 his3 leu2 trp1 ura3) (56Wiatrowski H.A. van Denderen B.J.W. Berkey C.D. Kemp B.E. Stapleton D. Carlson M. Mol. Cell. Biol. 2004; 24: 352-361Crossref PubMed Scopus (34) Google Scholar), which has the W303 genetic background.Assay of SNF1 Catalytic Activity by Phosphorylation of SAMS Peptide—Cultures were grown to exponential phase (A600 of 0.7–1.0) in selective synthetic complete (SC) medium containing 2% glucose. Cells (100 ml) were collected by rapid filtration and were either scraped from the filter and frozen in liquid nitrogen or resuspended in 0.05% glucose for 10 min, collected, and frozen. For some experiments, after incubation in 0.05% glucose, an aliquot of cells was incubated in 2% glucose for 10 min, collected, and frozen. Cell extracts were prepared from two to three independent cultures as described (55Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (72) Google Scholar). SNF1 was partially purified by chromatography on DEAE-Sepharose (Amersham Biosciences) and assayed for phosphorylation of a synthetic peptide (HMRSAMSGLHLVKRR, called the SAMS peptide) (65Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (371) Google Scholar) as described (46Woods A. Munday M.R. Scott J. Yang X. Carlson M. Carling D. J. Biol. Chem. 1994; 269: 19509-19516Abstract Full Text PDF PubMed Google Scholar, 55Hedbacker K. Hong S.P. Carlson M. Mol. Cell. Biol. 2004; 24: 8255-8263Crossref PubMed Scopus (72) Google Scholar), using different protein concentrations to confirm linearity. Protein concentrations were determined by Bio-Rad assay. Kinase activity is expressed as nanomoles of phosphate incorporated into the peptide/min/mg of protein (65Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (371) Google Scholar).Immunoblot Analysis—Proteins (2–4 μg) from assayed fractions were separated on 10% SDS-PAGE, except where noted otherwise, and analyzed by immunoblotting using anti-Thr(P)-172-AMPK (Cell Signaling Technologies), anti-polyhistidine (Sigma), anti-Snf1 (32Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (508) Google Scholar), and anti-Snf4 (66Estruch F. Treitel M.A. Yang X. Carlson M. Genetics. 1992; 132: 639-650Crossref PubMed Google Scholar) antibodies. Before the membrane was reprobed, it was incubated in 0.2 m glycine, pH 2, for 10 min. ECL Plus or ECL Advance (Amersham Biosciences) was used for visualization.Growth Assay for 2-Deoxyglucose Resistance—Fresh overnight cultures were grown in selective SC plus 2% glucose and diluted to A600 of 2 in selective SC. The cells were spotted with 2-fold dilutions on selective SC medium containing 2% sucrose, 2-deoxy-d-glucose (200 μg/ml), and antimycin A (1 μg/ml). The media contained the respiratory inhibitor antimycin A to increase the stringency of the assay. The control plates lacked 2-deoxyglucose. The plates were incubated at 30 °C and photographed.RESULTSStrategy to Assess Function of SNF1 Protein Kinase Containing a Mutant Snf4 Subunit—To determine whether alteration of Snf4 at sites that cause disease in humans would affect the regulation of SNF1, we introduced mutations into the SNF4 gene, under the control of its own promoter on a centromeric plasmid, and expressed the mutant proteins in snf4Δ yeast cells. The positions of the altered residues are shown in the Snf4 structure from the partial SNF1 heterotrimer, with AMP positioned as it is in the S. pombe γ subunit (58Amodeo G.A. Rudolph M.J. Tong L. Nature. 2007; 449: 492-495Crossref PubMed Scopus (130) Google Scholar) (Fig. 2). We used three assays to analyze the function of the mutant SNF1. First, we assessed the regulation of kinase activity in response to glucose availability. The cells were grown to mid-log phase in 2%" @default.
• W2080647804 created "2016-06-24" @default.
• W2080647804 creator A5023363049 @default.
• W2080647804 creator A5041341228 @default.
• W2080647804 creator A5079324651 @default.
• W2080647804 creator A5090963887 @default.
• W2080647804 date "2008-07-01" @default.
• W2080647804 modified "2023-09-30" @default.
• W2080647804 title "Roles of the Glycogen-binding Domain and Snf4 in Glucose Inhibition of SNF1 Protein Kinase" @default.
• W2080647804 cites W149589726 @default.
• W2080647804 cites W1535912426 @default.
• W2080647804 cites W1955808217 @default.
• W2080647804 cites W1958996383 @default.
• W2080647804 cites W1964327488 @default.
• W2080647804 cites W1966382091 @default.
• W2080647804 cites W1970205234 @default.
• W2080647804 cites W1972091198 @default.
• W2080647804 cites W1973816983 @default.
• W2080647804 cites W1974669825 @default.
• W2080647804 cites W1974870493 @default.
• W2080647804 cites W1977482668 @default.
• W2080647804 cites W1978729249 @default.
• W2080647804 cites W1978763812 @default.
• W2080647804 cites W1985978202 @default.
• W2080647804 cites W1987399378 @default.
• W2080647804 cites W1990278643 @default.
• W2080647804 cites W1994041994 @default.
• W2080647804 cites W2000233707 @default.
• W2080647804 cites W2005165257 @default.
• W2080647804 cites W2009826605 @default.
• W2080647804 cites W2013002765 @default.
• W2080647804 cites W2013947447 @default.
• W2080647804 cites W2021182257 @default.
• W2080647804 cites W2027815803 @default.
• W2080647804 cites W2033497590 @default.
• W2080647804 cites W2046304613 @default.
• W2080647804 cites W2047373064 @default.
• W2080647804 cites W2047879424 @default.
• W2080647804 cites W2051351724 @default.
• W2080647804 cites W2059825898 @default.
• W2080647804 cites W2067015511 @default.
• W2080647804 cites W2067346106 @default.
• W2080647804 cites W2067740358 @default.
• W2080647804 cites W2067979493 @default.
• W2080647804 cites W2068342714 @default.
• W2080647804 cites W2070380343 @default.
• W2080647804 cites W2072314593 @default.
• W2080647804 cites W2073125968 @default.
• W2080647804 cites W2073181999 @default.
• W2080647804 cites W2074258205 @default.
• W2080647804 cites W2074894911 @default.
• W2080647804 cites W2074932195 @default.
• W2080647804 cites W2091670176 @default.
• W2080647804 cites W2094825112 @default.
• W2080647804 cites W2095454212 @default.
• W2080647804 cites W2096258273 @default.
• W2080647804 cites W2097016419 @default.
• W2080647804 cites W2097572585 @default.
• W2080647804 cites W2098947405 @default.
• W2080647804 cites W2099788003 @default.
• W2080647804 cites W2102108703 @default.
• W2080647804 cites W2102891343 @default.
• W2080647804 cites W2104611938 @default.
• W2080647804 cites W2104865524 @default.
• W2080647804 cites W2105523638 @default.
• W2080647804 cites W2106338324 @default.
• W2080647804 cites W2107724201 @default.
• W2080647804 cites W2111921719 @default.
• W2080647804 cites W2115168068 @default.
• W2080647804 cites W2116719682 @default.
• W2080647804 cites W2117745265 @default.
• W2080647804 cites W2127421883 @default.
• W2080647804 cites W2128468829 @default.
• W2080647804 cites W2134314989 @default.
• W2080647804 cites W2139443530 @default.
• W2080647804 cites W2140586775 @default.
• W2080647804 cites W2143907477 @default.
• W2080647804 cites W2147594695 @default.
• W2080647804 cites W2153352656 @default.
• W2080647804 cites W2154836767 @default.
• W2080647804 cites W2158631571 @default.
• W2080647804 cites W2159620999 @default.
• W2080647804 cites W2159647919 @default.
• W2080647804 cites W2166714586 @default.
• W2080647804 cites W2170549904 @default.
• W2080647804 cites W2171885622 @default.
• W2080647804 cites W2324797060 @default.
• W2080647804 cites W4230496537 @default.
• W2080647804 doi "https://doi.org/10.1074/jbc.m803624200" @default.
• W2080647804 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2443652" @default.
• W2080647804 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18474591" @default.
• W2080647804 hasPublicationYear "2008" @default.
• W2080647804 type Work @default.
• W2080647804 sameAs 2080647804 @default.
• W2080647804 citedByCount "44" @default.
• W2080647804 countsByYear W20806478042012 @default.
• W2080647804 countsByYear W20806478042013 @default.
• W2080647804 countsByYear W20806478042014 @default.

• next