Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

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Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes

Biochemical Journal ◽

10.1042/bj20030502 ◽

2003 ◽

Vol 373 (2) ◽

pp. 505-513 ◽

Cited By ~ 8

Author(s):

Michael T. N. MØLLER ◽

Hamid R. SAMARI ◽

Monica FENGSRUD ◽

Per E. STRØMHAUG ◽

Anne C. ØSTVOLD ◽

...

Keyword(s):

Protein Kinase ◽

Okadaic Acid ◽

Rat Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Limited Region ◽

Flight Mass Spectrometry ◽

Methylation Capacity ◽

Dependent Protein ◽

Amp Activated Protein Kinase ◽

Isolated Rat

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

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Characterization of the Role of AMP-Activated Protein Kinase in the Regulation of Glucose-Activated Gene Expression Using Constitutively Active and Dominant Negative Forms of the Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6704-6711.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6704-6711 ◽

Cited By ~ 293

Author(s):

Angela Woods ◽

Dalila Azzout-Marniche ◽

Marc Foretz ◽

Silvie C. Stein ◽

Patricia Lemarchand ◽

...

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Fatty Acid Synthase ◽

Physiological Role ◽

Rat Hepatocytes ◽

Dominant Negative ◽

Ampk Activity ◽

Amp Activated Protein Kinase ◽

Constitutively Active

ABSTRACT In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (α1312) acts as a constitutively active kinase, while the other (α1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of α1312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of α1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

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

Biochemical Journal ◽

10.1042/bj3540275 ◽

2001 ◽

Vol 354 (2) ◽

pp. 275-283 ◽

Cited By ~ 103

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.

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AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00155.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. E710-E715 ◽

Cited By ~ 21

Author(s):

Denise Hurst ◽

Eric B. Taylor ◽

Troy D. Cline ◽

Lyle J. Greenwood ◽

Cori L. Compton ◽

...

Keyword(s):

Electrical Stimulation ◽

Protein Kinase ◽

Kinase Activity ◽

Protein A ◽

Covalent Modification ◽

Ampk Activity ◽

Gastrocnemius Muscles ◽

Amp Activated Protein Kinase ◽

Protein Kinase Kinase

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.

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Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity

Biochemical Journal ◽

10.1042/bj2840633 ◽

1992 ◽

Vol 284 (3) ◽

pp. 633-636 ◽

Cited By ~ 67

Author(s):

I Holen ◽

P B Gordon ◽

P O Seglen

Keyword(s):

Protein Kinase ◽

Okadaic Acid ◽

Kinase Inhibitors ◽

Specific Inhibitor ◽

Rat Hepatocytes ◽

Protein Kinase Inhibitors ◽

Disruptive Effect ◽

Isolated Rat Hepatocytes ◽

Calmodulin Antagonist ◽

General Protein

The protein phosphatase inhibitor okadaic acid suppressed autophagy completely in isolated rat hepatocytes, as measured by the sequestration of electroinjected [3H]raffinose into sedimentable autophagic vacuoles. Okadaic acid was effectively antagonized by the general protein kinase inhibitors K-252a and KT-5926, the calmodulin antagonist W-7, and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). These inhibitors also antagonized a cytoskeleton-disruptive effect of okadaic acid, manifested as the disintegration of cell corpses after breakage of the plasma membrane. CaMK-II, or a closely related enzyme, would thus seem to play a role in the control of autophagy as well as in the control of cytoskeletal organization.

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Glucagon stimulation of hepatic Na+-pump activity and α-subunit phosphorylation in rat hepatocytes

Biochemical Journal ◽

10.1042/bj3130983 ◽

1996 ◽

Vol 313 (3) ◽

pp. 983-989 ◽

Cited By ~ 6

Author(s):

Christopher J. LYNCH ◽

Kenneth M. McCALL ◽

Yuk-Chow NG ◽

Stacy A. HAZEN

Keyword(s):

Protein Kinase ◽

Rat Kidney ◽

Rat Hepatocytes ◽

Transport Activity ◽

Isolated Rat Hepatocytes ◽

Α Subunit ◽

Na Pump ◽

Pump Activity ◽

Stimulation Of

In this study the possible role of Na+ influx, arachidonate mediators and α-subunit phosphorylation in the stimulatory response of hepatic Na+/K+-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4(±1.3)×10-10 M. Analysis of the K+-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent K0.5. Unlike monensin, glucagon stimulation of Na+/K+-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K+-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome P-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb+ uptake, hepatocytes were metabolically radiolabelled with [32P]Pi. Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that co-migrates with Na+/K+-ATPase α-subunit immunoreactivity in two-dimensional gel electrophoresis. The α-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K+-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]Pi, the immunoprecipitated α-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197±21% (n = 6). Similar results were observed with a rabbit anti-peptide serum (‘anti-LEAVE’ serum) prepared against an amino acid sequence in the α-subunit. The EC50 for glucagon-stimulated phosphorylation of the α-subunit (1×10-10 M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K+-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na+-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the α-subunit.

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AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase

Biochemical Journal ◽

10.1042/bj20112026 ◽

2012 ◽

Vol 443 (1) ◽

pp. 193-203 ◽

Cited By ~ 61

Author(s):

Laurent Bultot ◽

Bruno Guigas ◽

Alexander Von Wilamowitz-Moellendorff ◽

Liliane Maisin ◽

Didier Vertommen ◽

...

Keyword(s):

Protein Kinase ◽

Glycogen Synthase ◽

Phosphorylation Site ◽

Rat Hepatocytes ◽

Liver Glycogen ◽

Enzyme Inactivation ◽

Dependent Manner ◽

Isolated Rat Hepatocytes ◽

Amp Activated Protein Kinase ◽

Specific Deletion

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.

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