RESUMO
Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by hexokinases or glucokinases. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2, and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, yeast Hxk2 has been proposed to shuttle into the nucleus in glucose-replete conditions, where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve its role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 and requires an N-terminal nuclear localization sequence (NLS). We used high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. Countering previous yeast studies, we find that Hxk2 is largely excluded from the nucleus under glucose-replete conditions but is retained in the nucleus under glucose-limiting conditions. We find that the Hxk2 N-terminus does not contain an NLS but instead is necessary for nuclear exclusion and regulating multimerization. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Alanine substation at nearby lysine 13 affects dimerization and maintenance of nuclear exclusion in glucose-replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 regulates Hxk2 localization. RNAseq analyses of the yeast transcriptome dispels the idea that Hxk2 moonlights as a transcriptional regulator of glucose repression, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose-replete and limiting conditions. Our studies define a new model of cis- and trans-acting regulators of Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, which aligns well with the nuclear regulation of mammalian orthologs. Our results lay the foundation for future studies of Hxk2 nuclear activity.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/genética , Serina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.
Assuntos
Desoxiglucose , Hexoquinase , Desoxiglucose/metabolismo , Glucose/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Previous studies of adaptation to the glucose analog, 2-deoxyglucose, by Saccharomyces cerevisiae have utilized haploid cells. In this study, diploid cells were used in the hope of identifying the distinct genetic mechanisms used by diploid cells to acquire drug resistance. While haploid cells acquire resistance to 2-deoxyglucose primarily through recessive alleles in specific genes, diploid cells acquire resistance through dominant alleles, haploinsufficiency, gene duplication and aneuploidy. Dominant-acting, missense alleles in all three subunits of yeast AMP-activated protein kinase confer resistance to 2-deoxyglucose. Dominant-acting, nonsense alleles in the REG1 gene, which encodes a negative regulator of AMP-activated protein kinase, confer 2-deoxyglucose resistance through haploinsufficiency. Most of the resistant strains isolated in this study achieved resistance through aneuploidy. Cells with a monosomy of chromosome 4 are resistant to 2-deoxyglucose. While this genetic strategy comes with a severe fitness cost, it has the advantage of being readily reversible when 2-deoxyglucose selection is lifted. Increased expression of the two DOG phosphatase genes on chromosome 8 confers resistance and was achieved through trisomies and tetrasomies of that chromosome. Finally, resistance was also mediated by increased expression of hexose transporters, achieved by duplication of a 117 kb region of chromosome 4 that included the HXT3, HXT6 and HXT7 genes. The frequent use of aneuploidy as a genetic strategy for drug resistance in diploid yeast and human tumors may be in part due to its potential for reversibility when selection pressure shifts.
Assuntos
Alelos , Aneuploidia , Diploide , Farmacorresistência Fúngica/genética , Duplicação Gênica , Genes Dominantes , Haploinsuficiência , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Desoxiglucose/farmacologia , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Sequenciamento Completo do GenomaRESUMO
Yeast and cancer cells are metabolically similar as they use fermentation of glucose as a primary means of generating energy. Reliance on glucose fermentation makes both of these cell types highly sensitive to the toxic glucose analog, 2-deoxyglucose. Here we review the cellular and metabolic pathways that play a role in 2-deoxyglucose sensitivity and discuss how the modifications to these pathways result in acquisition of 2-deoxyglucose resistance. Insights gained from genetic and proteomic studies in yeast provide new ideas for the design of combinatorial therapies for cancer treatment.
Assuntos
Dano ao DNA/genética , Desoxiglucose/genética , Endocitose/genética , Proteômica , Glucose/genética , Glucose/metabolismo , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.
Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do GenomaRESUMO
Rapidly proliferating cells growing by glucose fermentation must first transport glucose into the cell. Both budding yeast and human tumor cells utilize members of a conserved family of glucose transporters. In this issue of The EMBO Journal, Stahl et al (2019) reveal that budding yeast cells confer a growth advantage to their daughters using a novel mechanism, the asymmetric distribution to the daughter cell of the mRNA for a specific glucose transporter.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose , Proteínas de Saccharomyces cerevisiae , Glucose , Humanos , RNA Mensageiro , Saccharomyces cerevisiae/genéticaRESUMO
The adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. Recent studies reveal a novel role for AMPK in the regulation of glucose and other carbohydrates flux by controlling the endocytosis of transporters. The first step in glucose metabolism is glucose uptake, a process mediated by members of the GLUT/SLC2A (glucose transporters) or HXT (hexose transporters) family of twelve-transmembrane domain glucose transporters in mammals and yeast, respectively. These proteins are conserved from yeast to humans, and multiple transporters-each with distinct kinetic properties-compete for plasma membrane occupancy in order to enhance or limit the rate of glucose uptake. During growth in the presence of alternative carbon sources, glucose transporters are removed and replaced with the appropriate transporter to help support growth in response to this environment. New insights into the regulated protein trafficking of these transporters reveal the requirement for specific α-arrestins, a little-studied class of protein trafficking adaptor. A defining feature of the α-arrestins is that each contains PY-motifs, which can bind to the ubiquitin ligases from the NEDD4/Rsp5 (Neural precursor cell Expressed, Developmentally Down-regulated 4 and Reverses Spt- Phenotype 5, respectively) family. Specific association of α-arrestins with glucose and carbohydrate transporters is thought to bring the ubiquitin ligase in close proximity to its membrane substrate, and thereby allows the membrane cargo to become ubiquitinated. This ubiquitination in turn serves as a mark to stimulate endocytosis. Recent results show that AMPK phosphorylation of the α-arrestins impacts their abundance and/or ability to stimulate carbohydrate transporter endocytosis. Indeed, AMPK or glucose limitation also controls α-arrestin gene expression, adding an additional layer of complexity to this regulation. Here, we review the recent studies that have expanded the role of AMPK in cellular metabolism to include regulation of α-arrestin-mediated trafficking of transporters and show that this mechanism of regulation is conserved over the ~150 million years of evolution that separate yeast from man.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Arrestina/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Animais , Arrestina/química , Sítios de Ligação , Endocitose , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Humanos , Transporte Proteico , UbiquitinaçãoRESUMO
The phosphorylation status of a protein is highly regulated and is determined by the opposing activities of protein kinases and protein phosphatases within the cell. While much is known about the protein kinases found in Saccharomyces cerevisiae, the protein phosphatases are much less characterized. Of the 127 protein kinases in yeast, over 90% are in the same evolutionary lineage. In contrast, protein phosphatases are fewer in number (only 43 have been identified in yeast) and comprise multiple, distinct evolutionary lineages. Here we review the protein phosphatase families of yeast with regard to structure, catalytic mechanism, regulation, and signal transduction participation.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosforilação , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de SequênciaRESUMO
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.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Álcalis/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sequência Conservada , Ativação Enzimática , Glucose/farmacologia , Histidina/metabolismo , Isoenzimas/metabolismo , Cinética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Subunidades Proteicas/química , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
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.
Assuntos
Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/química , Saccharomyces cerevisiae/enzimologia , Serina/química , Treonina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Domínios Proteicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Serina/metabolismo , Treonina/metabolismoRESUMO
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.
Assuntos
Arrestina/metabolismo , Desoxiglucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glucose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/fisiologia , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
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.
Assuntos
Desoxiglucose/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Farmacorresistência Fúngica , Epistasia Genética , Deleção de Genes , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
In yeast, the mating response pathway is activated when a peptide pheromone binds to a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor, which leads to the activation of a mitogen-activated protein kinase signaling cascade and the stimulation of mating behavior. However, when nutrients in the environment are limiting, stimulation of the mating response would be maladaptive. A study indicates that the signaling pathways that respond to nutrient availability dampen the mating response by directly phosphorylating Gpa1, the G protein α subunit that initiates the mating response pathway. Snf1, the yeast homolog of adenosine monophosphate-activated protein kinase, is a highly conserved kinase that maintains energy homeostasis in response to nutrient limitation. The study found that the upstream kinases and phosphatase that control the activity of Snf1 also act on Gpa1 and provide a direct means to coordinate cell behavior and integrate the mating response with nutrient sensing.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Fosforilação/fisiologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Regulação Enzimológica da Expressão Gênica , Schizosaccharomyces/metabolismo , Difosfato de Adenosina/química , Animais , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/química , Humanos , Cinética , Ligantes , Modelos Moleculares , Conformação Molecular , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Ratos , Schizosaccharomyces/química , Transdução de Sinais , Estaurosporina/farmacologia , Especificidade por SubstratoRESUMO
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.
Assuntos
Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Quinases Proteína-Quinases Ativadas por AMP , Difosfato de Adenosina/química , Difosfato de Adenosina/genética , Monofosfato de Adenosina/química , Monofosfato de Adenosina/genética , Domínio Catalítico , Humanos , Fosforilação/fisiologia , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Proteína Fosfatase 1/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/metabolismo , Sinais de Localização Nuclear , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Transativadores/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Difosfato de Adenosina/metabolismo , Ativação Enzimática/genética , Glucose/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Difosfato de Adenosina/química , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Sequência Conservada , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Treonina/metabolismoRESUMO
In Saccharomyces cerevisiae, Snf1 kinase, the ortholog of the mammalian AMP-activated protein kinase, is activated by an increase in the phosphorylation of the conserved threonine residue in its activation loop. The phosphorylation status of this key site is determined by changes in the rate of dephosphorylation catalyzed by the yeast PP1 phosphatase Glc7 in a complex with the Reg1 protein. Reg1 and many PP1 phosphatase regulatory subunits utilize some variation of the conserved RVxF motif for interaction with PP1. In the Snf1 pathway, the exact role of the Reg1 protein is uncertain since it binds to both the Glc7 phosphatase and to Snf1, the Glc7 substrate. In this study we sought to clarify the role of Reg1 by separating the Snf1- and Glc7-binding functions. We generated a series of Reg1 proteins, some with deletions of conserved domains and one with two amino acid changes in the RVxF motif. The ability of Reg1 to bind Snf1 and Glc7 required the same domains of Reg1. Further, the RVxF motif that is essential for Reg1 binding to Glc7 is also required for binding to Snf1. Our data suggest that the regulation of Snf1 dephosphorylation is imparted through a dynamic competition between the Glc7 phosphatase and the Snf1 kinase for binding to the PP1 regulatory subunit Reg1.
Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , Mutação , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , beta-Frutofuranosidase/metabolismoRESUMO
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.