AMP-dependent protein kinase activators: not just for diabetes?
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ABSTRACT:
SUBMITTER: Facundo HT
PROVIDER: S-EPMC2713438 | biostudies-literature | 2009 Feb
REPOSITORIES: biostudies-literature
Publications
AMP-dependent protein kinase activators: not just for diabetes?
Facundo Heberty T F HT Jones Steven P SP
Circulation research 20090201 3
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Project description:The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.
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Project description:Protein kinase A (PKA), the main effector of cAMP in eukaryotes, is a paradigm for the mechanisms of ligand-dependent and allosteric regulation in signalling. Here we report the orthologous but cAMP-independent PKA of the protozoan Trypanosoma and identify 7-deaza-nucleosides as potent activators (EC50 ≥ 6.5 nM) and high affinity ligands (KD ≥ 8 nM). A co-crystal structure of trypanosome PKA with 7-cyano-7-deazainosine and molecular docking show how substitution of key amino acids in both CNB domains of the regulatory subunit and its unique C-terminal αD helix account for this ligand swap between trypanosome PKA and canonical cAMP-dependent PKAs. We propose nucleoside-related endogenous activators of Trypanosoma brucei PKA (TbPKA). The existence of eukaryotic CNB domains not associated with binding of cyclic nucleotides suggests that orphan CNB domains in other eukaryotes may bind undiscovered signalling molecules. Phosphoproteome analysis validates 7-cyano-7-deazainosine as powerful cell-permeable inducer to explore cAMP-independent PKA signalling in medically important neglected pathogens.
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Project description:Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism. Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.
| S-EPMC8386596 | biostudies-literature
Project description:Adenosine 5'-monophosphate-activated protein kinase (AMPK) is emerging as a promising drug target for its regulatory function in both glucose and lipid metabolism. Compound PT1 (5) was originally identified from high throughput screening as a small molecule activator of AMPK through the antagonization of the autoinhibition in α subunits. In order to enhance its potency at AMPK and bioavailability, structure-activity relationship studies have been performed and resulted in a novel series of AMPK activators based on an alkene oxindole scaffold. Following their evaluation in pharmacological AMPK activation assays, lead compound 24 was identified to possess improved potency as well as favorable pharmacokinetic profile. In the diet-induced obesity (DIO) mouse model, compound 24 was found to improve glucose tolerance and alleviate insulin resistance. The in vitro and in vivo data for these alkene oxindoles warrant further studies for their potential therapeutic medications in metabolic associated diseases.
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Project description:Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.
| S-EPMC4919462 | biostudies-literature
Project description:Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.
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Project description:PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.
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Project description:AimsPhenformin, resveratrol and AICAR stimulate the energy sensor 5'-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis.MethodsKidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting.ResultsAMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin.ConclusionsAMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.
| S-EPMC3907577 | biostudies-literature
Project description:The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.
| S-EPMC6313912 | biostudies-literature
Project description:AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the β-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including β2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1β2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1β1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.
| S-EPMC6341387 | biostudies-literature