scholarly journals Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

2009 ◽  
Vol 9 (1) ◽  
pp. 173-183 ◽  
Author(s):  
Simmanjeet Mangat ◽  
Dakshayini Chandrashekarappa ◽  
Rhonda R. McCartney ◽  
Karin Elbing ◽  
Martin C. Schmidt
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Constitutive Activity ◽  
Glucose Limitation ◽  
Α Subunit ◽  
Snf1 Kinase ◽  
Binding Domains ◽  
Conserved Domain ◽  
Amp Activated Protein Kinase

ABSTRACT 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 α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β 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 β 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 β 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.

scholarly journals Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1

2011 ◽  
Vol 10 (3) ◽  
pp. 313-319 ◽  
Author(s):  
Yang Liu ◽  
Xinjing Xu ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Glucose Limitation ◽  
Content Type ◽  
Snf1 Kinase ◽  
Phosphorylation State ◽  
Glucose Depletion ◽  
C Terminus ◽  
Amp Activated Protein Kinase

ABSTRACT The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.

scholarly journals Snf1 Protein Kinase and the Repressors Nrg1 and Nrg2 Regulate FLO11, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation

2002 ◽  
Vol 22 (12) ◽  
pp. 3994-4000 ◽  
Author(s):  
Sergei Kuchin ◽  
Valmik K. Vyas ◽  
Marian Carlson
Keyword(s):  
Protein Kinase ◽  
Surface Glycoprotein ◽  
Invasive Growth ◽  
Glucose Limitation ◽  
Cell Surface Glycoprotein ◽  
Snf1 Kinase ◽  
Repressor Proteins ◽  
A Cell ◽  
Pseudohyphal Differentiation

ABSTRACT The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

scholarly journals AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform

1998 ◽  
Vol 334 (1) ◽  
pp. 177-187 ◽  
Author(s):  
Ian SALT ◽  
Jakub W. CELLER ◽  
Simon A. HAWLEY ◽  
Alan PRESCOTT ◽  
Angela WOODS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Significant Proportion ◽  
Atp Depletion ◽  
Detection Methods ◽  
Α Subunit ◽  
Snf1 Kinase ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric αβγ complexes, where the catalytic subunit has two isoforms (α1 and α2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the α2 rather than the α1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein–α-subunit fusions. By all three methods a significant proportion of α2, but not α1, is localized in the nucleus. Like SNF1, AMPK-α2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of α1 and α2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

Role of the yeast Snf1 protein kinase in invasive growth

2003 ◽  
Vol 31 (1) ◽  
pp. 175-177 ◽  
Author(s):  
S. Kuchin ◽  
V.K. Vyas ◽  
M. Carlson
Keyword(s):  
Protein Kinase ◽  
Nitrogen Limitation ◽  
Invasive Growth ◽  
Gene Encoding ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
A Cell ◽  
Developmental Responses ◽  
Pseudohyphal Differentiation

The sucrose non-fermenting 1 (Snf1) protein kinase of Saccharomyces cerevisiae is important for transcriptional, metabolic and developmental responses to glucose limitation. Here we discuss the role of the Snf1 kinase in regulating filamentous invasive growth. Haploid invasive growth occurs in response to glucose limitation and requires FLO11, a gene encoding a cell-surface adhesin. Snf1 regulates transcription of FLO11 by antagonizing the function of two repressors, Nrg1 and Nrg2. Snf1 and the Nrg repressors also affect diploid pseudohyphal differentiation, which is a response to nitrogen limitation, suggesting an unexpected signalling role for the Snf1 kinase.

scholarly journals The regulation of AMP-activated protein kinase by phosphorylation

2000 ◽  
Vol 345 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Silvie C. STEIN ◽  
Angela WOODS ◽  
Neil A. JONES ◽  
Matthew D. DAVISON ◽  
David CARLING
Keyword(s):  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Mutant Complex

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.

scholarly journals The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells

2014 ◽  
Vol 46 (5) ◽  
pp. 394-400 ◽  
Author(s):  
J. Xiao ◽  
G. Niu ◽  
S. Yin ◽  
S. Xie ◽  
Y. Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Induced Apoptosis ◽  
Amp Activated Protein Kinase

Role of the AMP-activated protein kinase in regulating fatty acid metabolism during exerciseThis paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference – Muscles as Molecular and Metabolic Machines, and has undergone the Journal’s usual peer review process.

2009 ◽  
Vol 34 (3) ◽  
pp. 315-322 ◽  
Author(s):  
Gregory R. Steinberg
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acids ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Protein Kinase ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Moderate Intensity ◽  
Amp Activated Protein Kinase

During moderate-intensity exercise, fatty acids are the predominant substrate for working skeletal muscle. The release of fatty acids from adipose tissue stores, combined with the ability of skeletal muscle to actively fine tune the gradient between fatty acid and carbohydrate metabolism, depending on substrate availability and energetic demands, requires a coordinated system of metabolic control. Over the past decade, since the discovery that AMP-activated protein kinase (AMPK) was increased in accordance with exercise intensity, there has been significant interest in the proposed role of this ancient stress-sensing kinase as a critical integrative switch controlling metabolic responses during exercise. In this review, studies examining the role of AMPK as a regulator of fatty acid metabolism in both adipose tissue and skeletal muscle during exercise will be discussed. Exercise induces activation of AMPK in adipocytes and regulates triglyceride hydrolysis and esterfication through phosphorylation of hormone sensitive lipase (HSL) and glycerol-3-phosphate acyl-transferase, respectively. In skeletal muscle, exercise-induced activation of AMPK is associated with increases in fatty acid uptake, phosphorylation of HSL, and increased fatty acid oxidation, which is thought to occur via the acetyl-CoA carboxylase-malony-CoA-CPT-1 signalling axis. Despite the importance of AMPK in regulating fatty acid metabolism under resting conditions, recent evidence from transgenic models of AMPK deficiency suggest that alternative signalling pathways may also be important for the control of fatty acid metabolism during exercise.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

2004 ◽  
Vol 287 (4) ◽  
pp. E739-E743 ◽  
Author(s):  
Burton F. Holmes ◽  
David B. Lang ◽  
Morris J. Birnbaum ◽  
James Mu ◽  
G. Lynis Dohm
Keyword(s):  
Skeletal Muscle ◽  
Dominant Negative ◽  
Treadmill Running ◽  
Wild Type ◽  
Α Subunit ◽  
Ampk Activity ◽  
Denervated Muscles ◽  
Amp Activated Protein Kinase ◽  
Response To Exercise

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK α-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

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