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.

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle

2001 ◽  
Vol 280 (5) ◽  
pp. E677-E684 ◽  
Author(s):  
Nicolas Musi ◽  
Tatsuya Hayashi ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
Lee A. Witters ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Uptake ◽  
Muscle Contractions ◽  
Treadmill Running ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-β-d-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPKα1 and AMPKα2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-β-d-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3- O-methyl-d-glucose (3-MG) uptake. There were dose-dependent increases in AMPKα2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPKα1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPKα2 activity and 3-MG uptake but had little effect on AMPKα1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPKα1 and -α2 activity and 3-MG uptake. Although the AMPKα1 and -α2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPKα2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.

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 Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

1998 ◽  
Vol 9 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Juleen R. Zierath ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Kinase C ◽  
Dose Dependent ◽  
Α Subunits ◽  
K Activity ◽  
Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

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 Skeletal muscle AMP-activated protein kinase γ1H151R overexpression enhances whole body energy homeostasis and insulin sensitivity

2015 ◽  
Vol 309 (7) ◽  
pp. E679-E690 ◽  
Author(s):  
Milena Schönke ◽  
Martin G. Myers ◽  
Juleen R. Zierath ◽  
Marie Björnholm
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Protein Kinase ◽  
Transgenic Mice ◽  
Energy Homeostasis ◽  
Whole Body ◽  
Α Subunit ◽  
Peripheral Tissues ◽  
Amp Activated Protein Kinase ◽  
Body Energy

AMP-activated protein kinase (AMPK) is a major sensor of energy homeostasis and stimulates ATP-generating processes such as lipid oxidation and glycolysis in peripheral tissues. The heterotrimeric enzyme consists of a catalytic α-subunit, a β-subunit that is important for enzyme activity, and a noncatalytic γ-subunit that binds AMP and activates the AMPK complex. We generated a skeletal muscle Cre-inducible transgenic mouse model expressing a mutant γ1-subunit (AMPKγ1H151R), resulting in chronic AMPK activation. The expression of the predominant AMPKγ3 isoform in skeletal muscle was reduced in extensor digitorum longus (EDL) muscle (81–83%) of AMPKγ1H151R transgenic mice, whereas the abundance and phosphorylation of the AMPK target acetyl-CoA carboxylase was increased in tibialis anterior muscle. Glycogen content was increased 10-fold in gastrocnemius muscle. Whole body carbohydrate oxidation was increased by 11%, and whereas glucose tolerance was unaffected, insulin sensitivity was increased in AMPKγ1H151R transgenic mice. Furthermore, perigonadal white adipose tissue mass and serum leptin were reduced in female AMPKγ1H151R transgenic mice by 38 and 51% respectively. Conversely, in male AMPKγ1H151R transgenic mice, food intake was increased (14%), but body weight and body composition were unaltered, presumably because of increased energy expenditure. In conclusion, transgenic activation of skeletal muscle AMPKγ1 in this model plays an important sex-specific role in skeletal muscle metabolism and whole body energy homeostasis.

Metformin Selectively Suppressed the Proliferation of AML Stem Cells through Increased Phosphorylation of AMP-Activated Protein Kinase

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4771-4771
Author(s):  
Myung-Geun Shin ◽  
Ha-Young Eom ◽  
Hye-Ran Kim ◽  
Jong-Hee Shin ◽  
Soon-Pal Suh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Protein Kinase ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Dependent Manner ◽  
Cell Sorter ◽  
The Mean ◽  
Amp Activated Protein Kinase ◽  
Dose Dependent

Abstract Abstract 4771 Background: Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). AML stem cell (ASC) is known as CD34+CD38– leukemic cell population. Yet, despite their critical importance, much remains to be learned about selectively targeting ASC. Recently metformin selectively targets breast cancer stem cells, and acts together with chemotherapy to block tumor growth. Thus, the current study investigated whether metformin selectively inhibits AML cells and AML stem cells directly or indirectly. Materials and Methods: ASC showing CD34+CD38– phenotype were selectively sorted from primary bulk AML cells and KG-1 AML cell lines using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). ASC from primary AML samples and cell lines were cultured in serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF. Metformin (1,1-dimethylbiguanide hydrochloride) and idarubicin (IDA) were dissolved and diluted with high purity distilled water in various concentrations (0.1 mmol/L to 30 mmol/L). Morphological change, cell count, viability and apoptosis using annexin V flowcytometry were checked after treatment of metformin and IDA. Phosphorylation of AMPK was assayed by western blotting and mitochondrial membrane potential was measured using MitoTracker green dye. Results: The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of ASC from KG-1 cell lines was 78.6±6.2%. Total cell count of ASC was gradually decreased after treatment of metformin in dose-time dependent manner. Overall viability of AML cell lines and ASC disclosed significant decrease after treatment of metformin. The proportion of apoptosis was increased in time and dose dependent manner after 24-hr treatment of metformin. Mitochondrial membrane potentials were increased after metformin exposure at dose dependent manner. Moreover, when combined with lower concentration of IDA, more profound selective killing effect on AML cell lines and ASC was observed. Western blot analysis showed that the treatment of AML cell lines and ASC with 30 mmol/L of metformin resulting in the increased phosphorylation of AMP-activated protein kinase (AMPK) at approximately 1.5-fold over total AMPK. Conclusion: Metformin selectively kills AML cells as well as AML stem cells, and acts together with chemotherapeutic drugs to eradicate leukemic cells and their stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An inhibited conformation for the protein kinase domain of theSaccharomyces cerevisiaeAMPK homolog Snf1

2010 ◽  
Vol 66 (9) ◽  
pp. 999-1002 ◽  
Author(s):  
Michael J. Rudolph ◽  
Gabriele A. Amodeo ◽  
Liang Tong
Keyword(s):  
Protein Kinase ◽  
Active Site ◽  
Energy Homeostasis ◽  
Kinase Domain ◽  
Α Subunit ◽  
Protein Kinase Domain ◽  
Cellular Energy ◽  
Glucose Depletion ◽  
Inhibitory Domain ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master metabolic regulator for controlling cellular energy homeostasis. Its homolog in yeast, SNF1, is activated in response to glucose depletion and other stresses. The catalytic (α) subunit of AMPK/SNF1 in yeast (Snf1) contains a protein Ser/Thr kinase domain (KD), an auto-inhibitory domain (AID) and a region that mediates interactions with the two regulatory (β and γ) subunits. Here, the crystal structure of residues 41–440 of Snf1, which include the KD and AID, is reported at 2.4 Å resolution. The AID is completely disordered in the crystal. A new inhibited conformation of the KD is observed in a DFG-out conformation and with the glycine-rich loop adopting a structure that blocks ATP binding to the active site.

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.

scholarly journals AMP-activated protein kinase – not just an energy sensor

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1724 ◽  
Author(s):  
David Grahame Hardie ◽  
Sheng-Cai Lin
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Energy Homeostasis ◽  
Kinase Inhibitors ◽  
Adenine Nucleotides ◽  
Mitochondrial Fission ◽  
Nucleotide Binding Sites ◽  
Energy Sensor ◽  
Cell Layers ◽  
Amp Activated Protein Kinase

Orthologues of AMP-activated protein kinase (AMPK) occur in essentially all eukaryotes as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. The canonical role of AMPK is as an energy sensor, monitoring levels of the nucleotides AMP, ADP, and ATP that bind competitively to the γ subunit. Once activated, AMPK acts to restore energy homeostasis by switching on alternate ATP-generating catabolic pathways while switching off ATP-consuming anabolic pathways. However, its ancestral role in unicellular eukaryotes may have been in sensing of glucose rather than energy. In this article, we discuss a few interesting recent developments in the AMPK field. Firstly, we review recent findings on the canonical pathway by which AMPK is regulated by adenine nucleotides. Secondly, AMPK is now known to be activated in mammalian cells by glucose starvation by a mechanism that occurs in the absence of changes in adenine nucleotides, involving the formation of complexes with Axin and LKB1 on the surface of the lysosome. Thirdly, in addition to containing the nucleotide-binding sites on the γ subunits, AMPK heterotrimers contain a site for binding of allosteric activators termed the allosteric drug and metabolite (ADaM) site. A large number of synthetic activators, some of which show promise as hypoglycaemic agents in pre-clinical studies, have now been shown to bind there. Fourthly, some kinase inhibitors paradoxically activate AMPK, including one (SU6656) that binds in the catalytic site. Finally, although downstream targets originally identified for AMPK were mainly concerned with metabolism, recently identified targets have roles in such diverse areas as mitochondrial fission, integrity of epithelial cell layers, and angiogenesis.

Polydatin Attenuates Carbachol-induced Contraction of Guinea Pig Gallbladder

2019 ◽  
Vol 18 (1) ◽  
pp. 34-38
Author(s):  
Chen Lei ◽  
Pan Xiang ◽  
Shen Yonggang ◽  
Song Kai ◽  
Zhong Xingguo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Smooth Muscles ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Dependent Manner ◽  
Underlying Mechanisms ◽  
Dose Dependent

The aim of this study was to determine whether polydatin, a glucoside of resveratrol isolated from the root of Polygonum cuspidatum, warranted development as a potential therapeutic for ameliorating the pain originating from gallbladder spasm disorders and the underlying mechanisms. Guinea pig gallbladder smooth muscles were treated with polydatin and specific inhibitors to explore the mechanisms underpinning polydatin-induced relaxation of carbachol-precontracted guinea pig gallbladder. Our results shown that polydatin relaxed carbachol-induced contraction in a dose-dependent manner through the nitric oxide/cyclic guanosine monophosphate/protein kinase G and the cyclic adenosine monophosphate/protein kinase A signaling pathways as well as the myosin light chain kinase and potassium channels. Our findings suggested that there was value in further exploring the potential therapeutic use of polydatin in gallbladder spasm disorders.

Sign in / Sign up

Export Citation Format

Share Document