A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion

We have identified single genes encoding homologues of the α, β and γ subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative α, β or γ subunits ablated this activity, and abolished expression of the α subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the ‘activation loop’ of the α subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK α subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs.

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

Biochemical Journal ◽

10.1042/bj3340177 ◽

1998 ◽

Vol 334 (1) ◽

pp. 177-187 ◽

Cited By ~ 310

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.

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Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor α by AMP-activated protein kinase

Biochemical Journal ◽

10.1042/bj20040955 ◽

2004 ◽

Vol 384 (2) ◽

pp. 295-305 ◽

Cited By ~ 55

Author(s):

Myriam BRONNER ◽

Rachel HERTZ ◽

Jacob BAR-TANA

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Catalytic Domain ◽

Phosphorylation Site ◽

Atp Depletion ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Α Subunit ◽

Co Activation ◽

Amp Activated Protein Kinase

AMPK (AMP-activated protein kinase) responds to intracellular ATP depletion, while PPARα (peroxisome proliferator-activated receptor α) induces the expression of genes coding for enzymes and proteins involved in increasing cellular ATP yields. PPARα-mediated transcription is shown here to be co-activated by the α subunit of AMPK, as well as by kinase-deficient (Thr172Ala) and kinase-less (Asp157Ala, Asp139Ala) mutants of AMPKα. The Ser452Ala mutant of mPPARα mutated in its putative consensus AMPKα phosphorylation site is similarly co-activated by AMPKα. AMPKα or its kinase-less mutants bind to PPARα; binding is increased by MgATP, to a lesser extent by MgADP, but not at all by AMP or ZMP [AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) monophosphate]. ATP-activated binding of AMPKα to PPARα is mediated primarily by the C-terminal regulatory domain of AMPKα. PPARα co-activation by AMPKα may, however, require its secondary interaction with the N-terminal catalytic domain of AMPKα, independently of its kinase activity. While AMPK catalytic activity is activated by AICAR, PPARα co-activation and PPARα-controlled transcription are robustly inhibited by AICAR, with concomitant translocation of nuclear AMPKα or its kinase-less mutants to the cytosol. In conclusion, AMPKα, independently of its kinase activity, co-activates PPARα both in primary rat hepatocytes and in PPARα-transfected cells. The kinase and transcriptional co-activation modes of AMPKα are both regulated by the cellular ATP/AMP ratio. Co-activation of PPARα by AMPKα may transcriptionally complement AMPK in maintaining cellular ATP status.

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Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.6.4.1023-1031.1986 ◽

1986 ◽

Vol 6 (4) ◽

pp. 1023-1031

Author(s):

R Terracol ◽

N Prud'homme

Keyword(s):

Drosophila Melanogaster ◽

Gene Copy Number ◽

Rdna Locus ◽

Gene Copy ◽

Specific Gene ◽

Type I ◽

28S Rrna ◽

Wild Type ◽

Y Chromosomes ◽

Genes Encoding

In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus.

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Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

Journal of Molecular Endocrinology ◽

10.1530/jme-11-0165 ◽

2012 ◽

Vol 49 (1) ◽

pp. 47-55 ◽

Cited By ~ 5

Author(s):

H Tokuda ◽

K Kato ◽

H Natsume ◽

A Kondo ◽

G Kuroyanagi ◽

...

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Interleukin 6 ◽

P38 Map Kinase ◽

Thrombin Time ◽

Rt Pcr ◽

Α Subunit ◽

Compound C ◽

Kinase C ◽

Amp Activated Protein Kinase

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression ofIL6mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. TheIL6mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

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Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK)

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004101 ◽

2018 ◽

Vol 293 (44) ◽

pp. 16994-17007 ◽

Cited By ~ 3

Author(s):

Xin Gu ◽

Michael D. Bridges ◽

Yan Yan ◽

Parker W. de Waal ◽

X. Edward Zhou ◽

...

Keyword(s):

Protein Kinase ◽

Energy Homeostasis ◽

Metabolic Diseases ◽

Kinase Domain ◽

Carbohydrate Binding ◽

Conformational Heterogeneity ◽

Α Subunit ◽

Distance Distributions ◽

Double Electron Electron Resonance ◽

Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the β-subunit. Here, using double electron–electron resonance (DEER) spectroscopy, we demonstrate that the CBM–KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD–CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD–CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

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Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002579 ◽

2018 ◽

Vol 293 (44) ◽

pp. 17208-17217 ◽

Cited By ~ 77

Author(s):

Elizabeth C. Hinchy ◽

Anja V. Gruszczyk ◽

Robin Willows ◽

Naveenan Navaratnam ◽

Andrew R. Hall ◽

...

Keyword(s):

Protein Kinase ◽

Fluorescent Proteins ◽

Adenine Nucleotide ◽

Direct Action ◽

Cysteine Residues ◽

Ros Production ◽

Α Subunit ◽

Atp Production ◽

Ampk Activity ◽

Amp Activated Protein Kinase

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide–dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2. In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.

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Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00558.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. H2412-H2421 ◽

Cited By ~ 25

Author(s):

Markus Frederich ◽

Li Zhang ◽

James A. Balschi

Keyword(s):

Protein Kinase ◽

Metabolic Inhibitors ◽

Protein Kinase Activity ◽

Α Subunit ◽

Rat Hearts ◽

Upstream Kinase ◽

Ampk Activity ◽

Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

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

Eukaryotic Cell ◽

10.1128/ec.00267-09 ◽

2009 ◽

Vol 9 (1) ◽

pp. 173-183 ◽

Cited By ~ 20

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.

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