scholarly journals Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade

2007 ◽  
Vol 403 (1) ◽  
pp. 139-148 ◽  
Author(s):  
Matthew J. Sanders ◽  
Pascal O. Grondin ◽  
Bronwyn D. Hegarty ◽  
Michael A. Snowden ◽  
David Carling
Keyword(s):  
Protein Kinase ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Inhibitory Effect ◽  
Allosteric Activation ◽  
Α Subunit ◽  
Γ Subunit ◽  
New Findings ◽  
Amp Activated Protein Kinase ◽  
Dependent Pathway

AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr172 within the catalytic α subunit. Here we show that mutations in the regulatory γ subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr172 by PP (protein phosphatase)2Cα. Moreover, a mutation in the γ subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr172 phosphorylation by either LKB1 or CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase β) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Cα in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Cα, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr172 from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKKβ. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKKβ and an AMP-dependent pathway, mediated by LKB1.

Post-translational modifications of the β-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization

2001 ◽  
Vol 354 (2) ◽  
pp. 275-283 ◽  
Author(s):  
Scott M. WARDEN ◽  
Christine RICHARDSON ◽  
John O'DONNELL ◽  
David STAPLETON ◽  
Bruce E. KEMP ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Β Subunit ◽  
Α Subunit ◽  
Amp Activated Protein Kinase ◽  
The Impact

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic α subunit and regulatory β and γ subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the α subunit. The β subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the β-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the β subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24,25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the β subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.

scholarly journals Troglitazone and Δ2Troglitazone Enhance Adiponectin Expression in Monocytes/Macrophages through the AMP-Activated Protein Kinase Pathway

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Jaw-Shiun Tsai ◽  
Lee-Ming Chuang ◽  
Ching-Shih Chen ◽  
Chan-Jung Liang ◽  
Yuh-Lien Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mrna Expression ◽  
De Novo ◽  
Inhibitory Effect ◽  
Monocyte Adhesion ◽  
Compound C ◽  
Adiponectin Mrna ◽  
Mrna And Protein Expression ◽  
Amp Activated Protein Kinase ◽  
Dependent Pathway

Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for inflammation and cardiovascular disorders. Here, we tested the effect of troglitazone (TG) and its newly synthesized derivative, 5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl-methoxy)-benzylidene]-2,4-thiazolidinedione (Δ2troglitazone, (Δ2TG)), on the adiponectin expression in monocytes/macrophages and the relative mechanisms. The expression of adiponectin was located in macrophages of atherosclerotic lesions from patients and cholesterol-fed rabbits. TG and Δ2TG enhanced adiponectin mRNA and protein expression in THP-1 cells by quantitative real-time PCR, Western blot, and immunocytochemistry. TG induced adiponectin mRNA expression through a PPARγ-dependent pathway whereas Δ2TG enhanced adiponectin mRNA expression through a PPARγ-independent pathway in THP-1 cells. Both TG and Δ2TG enhanced adiponectin mRNA expression through AMP-activated protein kinase (AMPK) activation. TG and Δ2TG decreased the adhesion of THP-1 cells to TNF-α-treated HUVECs and the inhibitory effect was abolished by specific antiadiponectin antibodies. TG- and Δ2TG-induced suppression on monocyte adhesion were inhibited by a selective AMPK inhibitor compound C. Our data suggest that the inhibitory effect of TG and Δ2TG on monocyte adhesion might be at least in part throughde novoadiponectin expression and activation of an AMPK-dependent pathway, which might play an important role in anti-inflammation and antiatherosclerosis.

scholarly journals Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMP-activated Protein Kinase Signaling

2007 ◽  
Vol 18 (5) ◽  
pp. 1874-1886 ◽  
Author(s):  
Paul B. Bokko ◽  
Lisa Francione ◽  
Esther Bandala-Sanchez ◽  
Afsar U. Ahmed ◽  
Sarah J. Annesley ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Disease ◽  
Mammalian Cells ◽  
Energy Homeostasis ◽  
Mitochondrial Diseases ◽  
Dependent Manner ◽  
Α Subunit ◽  
Gene Dose ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK α subunit (AMPKαT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKα expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKα expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial “mass” and ATP levels were reduced by AMPKα antisense inhibition and increased by AMPKαT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKα antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.

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

2012 ◽  
Vol 49 (1) ◽  
pp. 47-55 ◽  
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.

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

2018 ◽  
Vol 293 (44) ◽  
pp. 16994-17007 ◽  
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.

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

2018 ◽  
Vol 293 (44) ◽  
pp. 17208-17217 ◽  
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.

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

2002 ◽  
Vol 367 (1) ◽  
pp. 179-186 ◽  
Author(s):  
David A. PAN ◽  
D. Grahame HARDIE
Keyword(s):  
Drosophila Melanogaster ◽  
Protein Kinase ◽  
Atp Depletion ◽  
Specific Gene ◽  
Specific Substrate ◽  
Intact Cells ◽  
Drosophila Cell ◽  
Α Subunit ◽  
Genes Encoding ◽  
Amp Activated Protein Kinase

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.

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

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
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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