Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d

Joseph, Biny K.; Liu, Hsing-Yin; Francisco, Jamie; Pandya, Devanshi; Donigan, Melissa; Gallo-Ebert, Christina; Giordano, Caroline; Bata, A; Nickels, Joseph T.

doi:10.1074/jbc.m114.626259

Cited by 73 publications

(46 citation statements)

References 58 publications

(58 reference statements)

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“…CGs bind to the ␣1 subunit of the Na ϩ ,K ϩ /ATPase pump and trigger pump endocytosis (37). In contrast, AICAR activates the pump and localizes ␣1 to the plasma membrane (46). CG-mediated pump internalization leads to its lysosomal degradation and/or signaling induction (37).…”

Section: Discussionmentioning

confidence: 99%

Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Mukhopadhyay

Venkatadri

Katsnelson

et al. 2018

J Virol

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Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na,K/ATPase α1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the α1 subunit was required for autophagy induction, since in α1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (α1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition. Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the α1 subunit of the Na,K/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in α1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

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Section: Discussionmentioning

confidence: 99%

Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Mukhopadhyay

Venkatadri

Katsnelson

et al. 2018

J Virol

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“…and therefore PKLR levels [57], as reflected in our results described before. Other authors found that PPP2R1A amounts were increased by a diet high in saturated fat [58].…”

Section: Effect Of Fish Oils and Polyphenols On Protein And Amino Acimentioning

confidence: 90%

Changes in liver proteins of rats fed standard and high-fat and sucrose diets induced by fish omega-3 PUFAs and their combination with grape polyphenols according to quantitative proteomics

Méndez

Ciordia

Fernández

et al. 2017

The Journal of Nutritional Biochemistry

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This study considered the physiological modulation of liver proteins due to the supplementation with fish oils under two dietary backgrounds: standard or high in fat and sucrose (HFHS), and their combination with grape polyphenols. By using a quantitative proteomics approach, we showed that the capacity of the supplements for regulating proteins depended on the diet; namely, 10 different proteins changed into standard diets, while 45 changed into the HFHS diets and only scarcely proteins were found altered in common. However, in both contexts, fish oils were the main regulatory force, although the addition of polyphenols was able to modulate some fish oils' effects. Moreover, we demonstrated the ability of fish oils and their combination with grape polyphenols in improving biochemical parameters and reducing lipogenesis and glycolysis enzymes, enhancing fatty acid beta-oxidation and insulin signaling and ameliorating endoplasmic reticulum stress and protein oxidation when they are included in an unhealthy diet.

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“…Second, binding of AMP or ADP to the γ-subunit induces a conformational change that protects against Thr172 dephosphorylation by protein phosphatases (Gowans et al, 2013; Xiao et al, 2011). Notably, the phosphatases that normally dephosphorylate AMPK under physiological conditions remain largely unknown, though recent reports implicate roles for different phosphatases (Garcia-Haro et al, 2010; Joseph et al, 2015). Lastly, binding of AMP, but not ADP, results in up to 10 fold allosteric activation of AMPK (Gowans et al, 2013).…”

Section: Nucleotide-dependent and Nucleotide-independent Regulation Omentioning

confidence: 99%

AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

2017

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AMPK is a highly conserved master regulator of metabolism, which restores energy balance during metabolic stress both at the cellular and physiological levels. The identification of numerous AMPK targets has helped explain how AMPK restores energy homeostasis. Recent advancements, however, demonstrate that regulation of AMPK is also affected by novel contexts, such as subcellular localization and phosphorylation by non-canonical upstream kinases. Notably, the therapeutic potential of AMPK is widely recognized and heavily pursued for treatment of metabolic diseases such as diabetes, but also obesity, inflammation and cancer. Moreover, the recently solved crystal structure of AMPK has shed light both into how nucleotides activate AMPK but, importantly, also into the sites bound by small molecule activators, thus providing a path for improved drugs.

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Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d

Cited by 73 publications

References 58 publications

Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Changes in liver proteins of rats fed standard and high-fat and sucrose diets induced by fish omega-3 PUFAs and their combination with grape polyphenols according to quantitative proteomics

AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

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Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d

Cited by 73 publications

References 58 publications

Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Changes in liver proteins of rats fed standard and high-fat and sucrose diets induced by fish omega-3 PUFAs and their combination with grape polyphenols according to quantitative proteomics

AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance

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Product

Resources

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Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

Digitoxin Suppresses Human Cytomegalovirus Replication via Na ⁺ , K ⁺ /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation