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![Allosteric Activation by AMP, and Inhibition at High AMP Concentrations, of Rat Liver AMPK and GST Fusions of the Isolated a 1 and a 2 Kinase Domains (A) Effect of increasing AMP concentrations on the activity of purified rat liver AMPK. Data points were generated at three different concentrations of ATP (circles, 0.2 mM; squares, 1 mM; triangles, 5 mM) and were fitted to the equation Y = basal + ([{activation 3 basal À basal} 3 X] / [EC 50 + X]) À](profile/David-Hardie-5/publication/257460650/figure/fig2/AS:297432891052039@1447924935800/Allosteric-Activation-by-AMP-and-Inhibition-at-High-AMP-Concentrations-of-Rat-Liver.png)
Allosteric Activation by AMP, and Inhibition at High AMP Concentrations, of Rat Liver AMPK and GST Fusions of the Isolated a 1 and a 2 Kinase Domains (A) Effect of increasing AMP concentrations on the activity of purified rat liver AMPK. Data points were generated at three different concentrations of ATP (circles, 0.2 mM; squares, 1 mM; triangles, 5 mM) and were fitted to the equation Y = basal + ([{activation 3 basal À basal} 3 X] / [EC 50 + X]) À
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While allosteric activation of AMPK is triggered only by AMP, binding of both ADP and AMP has been reported to promote phosphorylation and inhibit dephosphorylation at Thr172. Because cellular concentrations of ADP and ATP are higher than AMP, it has been proposed that ADP is the physiological signal that promotes phosphorylation and that allosteri...
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... address the question of whether AMP is capable of competing with ATP at the allosteric sites, we also conducted assays not only at 200 mM ATP as in the standard assay, but also at the more physiological ATP concen- trations of 1 and 5 mM. We obtained a family of bell-shaped curves in which AMP activated AMPK at low concentrations and then inhibited at higher concentrations ( Figure 3A). The inhibitory effects were due to competition of AMP with ATP at the catalytic site, because if we assayed bacterially expressed Figure 2. AMP, but Not ADP, Enhances Thr172 Phosphorylation and Activation of AMPK by LKB1, but Not CaMKKb (A) Effect of AMP and ADP on activation and Thr172 phosphorylation by LKB1 and CaMKKb. ...
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... S-transferase (GST) fusions of the isolated a1 or a2 kinase domains (after phosphorylation by LKB1), we no longer observed activation by AMP but still observed the inhibitory effects ( Figures 3B and 3C); these only occurred at AMP concen- trations above 1 mM, which are unlikely to be physiologically relevant. ...
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... bell-shaped curves in Figure 3A shifted to higher AMP concentrations as ATP increased, consistent with the idea that ATP competes with AMP both at the activating site(s) on the g subunit and at the catalytic site on the a subunit. When we fitted the data to a simple model (see Figure 3 legend) that assumed single activating and inhibitory sites for AMP, we obtained good fits (continuous curves in Figure 3A) with the following parameters (±SEM at 0.2, 1, and 5 mM ATP, respectively): basal activity = 372 ± 16, 266 ± 14, and 226 ± 23 mmol/min/mg; EC 50 for activation by AMP = 5.3 ± 0.4, 22 ± 1.1, and 137 ± 14 mM; IC 50 for inhibition by AMP = 1.9 ± 0.092, 7.1 ± 0.33, and 22 ± 3 mM; degree of activation by AMP = 5.4 ± 0.2, 10 ± 0.5, and 13 ± 1.3-fold. ...
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... bell-shaped curves in Figure 3A shifted to higher AMP concentrations as ATP increased, consistent with the idea that ATP competes with AMP both at the activating site(s) on the g subunit and at the catalytic site on the a subunit. When we fitted the data to a simple model (see Figure 3 legend) that assumed single activating and inhibitory sites for AMP, we obtained good fits (continuous curves in Figure 3A) with the following parameters (±SEM at 0.2, 1, and 5 mM ATP, respectively): basal activity = 372 ± 16, 266 ± 14, and 226 ± 23 mmol/min/mg; EC 50 for activation by AMP = 5.3 ± 0.4, 22 ± 1.1, and 137 ± 14 mM; IC 50 for inhibition by AMP = 1.9 ± 0.092, 7.1 ± 0.33, and 22 ± 3 mM; degree of activation by AMP = 5.4 ± 0.2, 10 ± 0.5, and 13 ± 1.3-fold. Thus, even when the ATP concentration was 5 mM (similar to the concentration estimated in unstressed cells; Imamura et al., 2009), AMP caused a large allosteric activation (13-fold) with a half-maximal effect at 140 mM. ...
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... bell-shaped curves in Figure 3A shifted to higher AMP concentrations as ATP increased, consistent with the idea that ATP competes with AMP both at the activating site(s) on the g subunit and at the catalytic site on the a subunit. When we fitted the data to a simple model (see Figure 3 legend) that assumed single activating and inhibitory sites for AMP, we obtained good fits (continuous curves in Figure 3A) with the following parameters (±SEM at 0.2, 1, and 5 mM ATP, respectively): basal activity = 372 ± 16, 266 ± 14, and 226 ± 23 mmol/min/mg; EC 50 for activation by AMP = 5.3 ± 0.4, 22 ± 1.1, and 137 ± 14 mM; IC 50 for inhibition by AMP = 1.9 ± 0.092, 7.1 ± 0.33, and 22 ± 3 mM; degree of activation by AMP = 5.4 ± 0.2, 10 ± 0.5, and 13 ± 1.3-fold. Thus, even when the ATP concentration was 5 mM (similar to the concentration estimated in unstressed cells; Imamura et al., 2009), AMP caused a large allosteric activation (13-fold) with a half-maximal effect at 140 mM. ...
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... AMP concentra- tions did not change in response to A23187 or A769662 but increased 6-to 7-fold in response to berberine (from 42 ± 3 to 270 ± 50 mM). This corresponds well with the range of concentra- tions over which we found that AMP causes inhibition of dephos- phorylation (see Figure 1A, where the estimated change in ADP is also shown) and allosteric activation (see Figure 3A) when these effects were measured in cell-free assays in the presence of 5 mM ATP. It also corresponds with the concentrations at which AMP promoted phosphorylation (see Figure 2B), although that was only measured at 200 mM ATP. ...
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... inspection of Figure 1A (where we have added dashed vertical lines to indicate the estimated changes in AMP and ADP concentrations induced by berberine in G361 cells), suggests that the changes in AMP would have a larger effect on Thr172 dephosphorylation than would the changes in ADP. AMP also increases in response to berberine in G361 cells over a range where large effects on phosphoryla- tion by LKB1 ( Figure 2B) and on allosteric activation ( Figure 3A) were observed in cell-free assays. ...
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... allosteric activation is usually reported to be quite modest (often <2-fold), yet stoichiometric phosphoryla- tion of Thr172 can produce >100-fold activation. However, we could demonstrate 13-fold allosteric activation by AMP (Fig- ure 3A), even using an ATP concentration (5 mM) within the phys- iological range for unstressed cells (Imamura et al., 2009). In addition, the results in Figures 7A-7C show that the 4-fold in- crease in response to A23187 in G361 cells represents a change from only z4% to z16% of maximal, stoichiometric phosphor- ylation, whereas in HEK293 cells, where the basal phosphoryla- tion was much higher (25%), the increases in response to A23187 and berberine were only 1.5-fold and 2-fold. ...
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... we observed 13-fold allosteric activation by AMP even when conducting assays at 5 mM ATP. Under these condi- tions, allosteric activation by AMP occurred from 50-500 mM, corresponding nicely with our estimated increase (40-270 mM, see Figure 3A) in AMP in G361 cells treated with berberine. Thus, AMP can compete with ATP at the allosteric binding site(s), even when its concentration is one to two orders of magnitude lower than that of ATP. ...
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... S-transferase (GST) fusions of the isolated a 1 or a 2 kinase domains (after phosphorylation by LKB1), we no longer observed activation by AMP but still observed the inhibitory effects (Figures 3B and 3C); these only occurred at AMP concentrations above 1 mM, which are unlikely to be physiologically relevant. The bell-shaped curves in Figure 3A shifted to higher AMP concentrations as ATP increased, consistent with the idea that ATP competes with AMP both at the activating site(s) on the g subunit and at the catalytic site on the a subunit. When we fitted the data to a simple model (see Figure 3 legend) that assumed single activating and inhibitory sites for AMP, we obtained good fits (continuous curves in Figure 3A) with the following parameters (±SEM at 0.2, 1, and 5 mM ATP, respectively): basal activity = 372 ± 16, 266 ± 14, and 226 ± 23 m mol/min/mg; EC 50 for activation by AMP = 5.3 ± 0.4, 22 ± 1.1, and 137 ± 14 m M; IC 50 for inhibition by AMP = 1.9 ± 0.092, 7.1 ± 0.33, and 22 ± 3 mM; degree of activation by AMP = 5.4 ± 0.2, 10 ± 0.5, and 13 ± 1.3-fold. Thus, even when the ATP concentration was 5 mM (similar to the concentration estimated in unstressed cells; Imamura et al., 2009), AMP caused a large allosteric activation (13-fold) with a half-maximal effect at 140 m M. To study the relative importance of allosteric activation versus Thr172 phosphorylation in intact cells, we initially utilized G361 cells, a human melanoma line that lacks LKB1. In LKB1 null cells, agents that increase cellular AMP do not increase Thr172 phosphorylation (Hawley et al., 2003), so they should work entirely via allosteric mechanisms, while agents that increase intracellular Ca 2+ and activate CaMKK b work entirely via increased Thr172 phosphorylation. As expected, in G361 cells the natural product berberine (an inhibitor of respiratory chain complex I; Hawley et al., 2010) did not cause increased phosphorylation of Thr172 and failed to increase AMPK activity measured in washed immunoprecipitates (in which any effects of allosteric activation in the intact cell would have been lost). By contrast, the Ca 2+ ionophore A23187 markedly increased Thr172 phosphorylation and kinase activity (Figure 4A). Despite this, berberine increased phosphorylation of the downstream target acetyl-CoA carboxylase (ACC) to a larger extent than A23187 did. As expected, the CaMKK inhibitor STO609 blocked the effect of A23187 on AMPK activation and Thr172 phosphorylation. It also reduced the low Thr172 phosphorylation and AMPK activities observed under basal conditions and after berberine and reduced the effect of 100 m M berberine on ACC phosphorylation, suggesting that the low, basal CaMKK b activity was sufficient to generate some Thr172 phosphorylation even without A23187 treatment. We carried out similar experiments using another AMPK activator, A769662, which does not increase cellular AMP or ADP but acts instead by direct binding to AMPK at site(s) distinct from those used by adenine nucleotides, causing both allosteric activation and inhibition of Thr172 dephosphorylation (Go ̈ ransson et al., 2007; Hawley et al., 2010; 2012). The effects of A23187 and two different concentrations of A769662 on ACC phosphorylation were very similar and were reduced by STO609 (Figure 4B). However, the results with A769662 bore similarities to those with berberine in that A769662 had no ...
Citations
... This activation prevents phosphatase from binding to α-172, inhibiting multiple pathways of dephosphorylation [46], and enhancing AMPK activity [47,48]. As a result, it reduces the inactivation of AMPK [49]. (Fig. 2: Structure, function, and main regulatory factors of AMPK). ...
Immunotherapy has now garnered significant attention as an essential component in cancer therapy during this new era. However, due to immune tolerance, immunosuppressive environment, tumor heterogeneity, immune escape, and other factors, the efficacy of tumor immunotherapy has been limited with its application to very small population size. Energy metabolism not only affects tumor progression but also plays a crucial role in immune escape. Tumor cells are more metabolically active and need more energy and nutrients to maintain their growth, which causes the surrounding immune cells to lack glucose, oxygen, and other nutrients, with the result of decreased immune cell activity and increased immunosuppressive cells. On the other hand, immune cells need to utilize multiple metabolic pathways, for instance, cellular respiration, and oxidative phosphorylation pathways to maintain their activity and normal function. Studies have shown that there is a significant difference in the energy expenditure of immune cells in the resting and activated states. Notably, competitive uptake of glucose is the main cause of impaired T cell function. Conversely, glutamine competition often affects the activation of most immune cells and the transformation of CD4 ⁺ T cells into inflammatory subtypes. Excessive metabolite lactate often impairs the function of NK cells. Furthermore, the metabolite PGE2 also often inhibits the immune response by inhibiting Th1 differentiation, B cell function, and T cell activation. Additionally, the transformation of tumor-suppressive M1 macrophages into cancer-promoting M2 macrophages is influenced by energy metabolism. Therefore, energy metabolism is a vital factor and component involved in the reconstruction of the tumor immune microenvironment. Noteworthy and vital is that not only does the metabolic program of tumor cells affect the antigen presentation and recognition of immune cells, but also the metabolic program of immune cells affects their own functions, ultimately leading to changes in tumor immune function. Metabolic intervention can not only improve the response of immune cells to tumors, but also increase the immunogenicity of tumors, thereby expanding the population who benefit from immunotherapy. Consequently, identifying metabolic crosstalk molecules that link tumor energy metabolism and immune microenvironment would be a promising anti-tumor immune strategy. AMPK (AMP-activated protein kinase) is a ubiquitous serine/threonine kinase in eukaryotes, serving as the central regulator of metabolic pathways. The sequential activation of AMPK and its associated signaling cascades profoundly impacts the dynamic alterations in tumor cell bioenergetics. By modulating energy metabolism and inflammatory responses, AMPK exerts significant influence on tumor cell development, while also playing a pivotal role in tumor immunotherapy by regulating immune cell activity and function. Furthermore, AMPK-mediated inflammatory response facilitates the recruitment of immune cells to the tumor microenvironment (TIME), thereby impeding tumorigenesis, progression, and metastasis. AMPK, as the link between cell energy homeostasis, tumor bioenergetics, and anti-tumor immunity, will have a significant impact on the treatment and management of oncology patients. That being summarized, the main objective of this review is to pinpoint the efficacy of tumor immunotherapy by regulating the energy metabolism of the tumor immune microenvironment and to provide guidance for the development of new immunotherapy strategies.
... It is a heterotrimeric complex that consists of a catalytic subunit α and two regulatory subunits β and γ. When cellular ATP is low, AMP (adenosine monophosphate) or ADP (adenosine diphosphate) can directly interact with the γ subunit, leading to conformational changes that promote the phosphorylation of the α subunit at the T172 residue (Gowans et al., 2013;Xiao et al., 2007). The regulatory function of AMPKβ requires its αγ-subunit-binding sequence (αγ-SBS) that mediates interactions with the α and γ subunits. ...
... AMPKβ contains sequences (α, γ-SBS) that bind to the α and γ subunits, thus acts as a scaffold of the complex. The γ subunit contains cystathionineβ-synthase domains (CBS) that can bind AMP or ADP nucleotides to sense the energy status of the cell, which subsequently alters the activity of the AMPK complex and the activation of AMPKα (Gowans et al., 2013;Ross et al., 2016). In a simple model, one would predict that disruption of any subunit of the complex would lead to similar cellular consequences. ...
... In a simple model, one would predict that disruption of any subunit of the complex would lead to similar cellular consequences. However, this is really not the case in most organisms studied so far (Gowans et al., 2013;Viollet et al., 2003). In T. gondii, depletion of TgAMPKα or TgAMPKγ was lethal. ...
Toxoplasma gondii is a zoonotic parasite infecting humans and nearly all warm‐blooded animals. Successful parasitism in diverse hosts at various developmental stages requires the parasites to fine tune their metabolism according to environmental cues and the parasite's needs. By manipulating the β and γ subunits, we have previously shown that AMP‐activated protein kinase (AMPK) has critical roles in regulating the metabolic and developmental programmes. However, the biological functions of the α catalytic subunit have not been established. T. gondii encodes a canonical AMPKα, as well as a KIN kinase whose kinase domain has high sequence similarities to those of classic AMPKα proteins. Here, we found that TgKIN is dispensable for tachyzoite growth, whereas TgAMPKα is essential. Depletion of TgAMPKα expression resulted in decreased ATP levels and reduced metabolic flux in glycolysis and the tricarboxylic acid cycle, confirming that TgAMPK is involved in metabolic regulation and energy homeostasis in the parasite. Sequential truncations at the C‐terminus found an α‐helix that is key for the function of TgAMPKα. The amino acid sequences of this α‐helix are not conserved among various AMPKα proteins, likely because it is involved in interactions with TgAMPKβ, which only have limited sequence similarities to AMPKβ in other eukaryotes. The essential role of the less conserved C‐terminus of TgAMPKα provides opportunities for parasite specific drug designs targeting TgAMPKα.
... It is also closely associated with the regulation of cell polarity and energy metabolism [8]. Previous studies have shown that it enables the activation of adenosine monophosphate-activated protein kinase (AMPK), which leads to the inhibition of the downstream mTOR signaling pathway [9]. In recent years, it has been found that STK11 has a high rate of mutation in NSCLC, occurring in approximately 15-35% of cases [10]. ...
Background
This study aimed to systematically analyze the effect of a serine/threonine kinase (STK11) mutation (STK11mut) on therapeutic efficacy and prognosis in patients with non-small cell lung cancer (NSCLC).
Methods
Candidate articles were identified through a search of relevant literature published on or before April 1, 2023, in PubMed, Embase, Cochrane Library, CNKI and Wanfang databases. The extracted and analyzed data included the hazard ratios (HRs) of PFS and OS, the objective response rate (ORR) of immune checkpoint inhibitors (ICIs), and the positive rates of PD-L1 expression. The HR of PFS and OS and the merged ratios were calculated using a meta-analysis. The correlation between STK11mut and clinical characteristics was further analyzed in NSCLC datasets from public databases.
Results
Fourteen retrospective studies including 4317 patients with NSCLC of whom 605 had STK11mut were included. The meta-analysis revealed that the ORR of ICIs in patients with STK11mut was 10.1% (95%CI 0.9–25.2), and the positive rate of PD-L1 expression was 41.1% (95%CI 25.3–57.0). STK11mut was associated with poor PFS (HR = 1.49, 95%CI 1.28–1.74) and poor OS (HR = 1.44, 95%CI 1.24–1.67). In the bioinformatics analysis, PFS and OS in patients with STK11 alterations were worse than those in patients without alterations (p < 0.001, p = 0.002). Nutlin-3a, 5-fluorouracil, and vinorelbine may have better sensitivity in patients with STK11mut than in those with STK11wt.
Conclusions
Patients with STK11-mutant NSCLC had low PD-L1 expression and ORR to ICIs, and their PFS and OS were worse than patients with STK11wt after comprehensive treatment. In the future, more reasonable systematic treatments should be explored for this subgroup of patients with STK11-mutant NSCLC.
... Genetic deletion of Lkb1 abrogates the activation of AMPK by agonists such as aminoimidazole-4-carboxamide ribonucleoside (AICAR), metformin or phenformin or in response to energy stress, revealing that LKB1 is responsible for the majority of AMPK activation 172 . Increases in AMP or ADP activate AMPK mainly by promoting the phosphorylation of LKB1 but also by inhibiting dephosphorylation by protein phosphatases or directly activating AMPK 173 . ...
Autophagy is an essential quality control mechanism for maintaining organellar functions in eukaryotic cells. Defective autophagy in pancreatic beta cells has been shown to be involved in the progression of diabetes through impaired insulin secretion under glucolipotoxic stress. The underlying mechanism reveals the pathologic role of the hyperactivation of mechanistic target of rapamycin (mTOR), which inhibits lysosomal biogenesis and autophagic processes. Moreover, accumulating evidence suggests that oxidative stress induces Ca ²⁺ depletion in the endoplasmic reticulum (ER) and cytosolic Ca ²⁺ overload, which may contribute to mTOR activation in perilysosomal microdomains, leading to autophagic defects and β-cell failure due to lipotoxicity. This review delineates the antagonistic regulation of autophagic flux by mTOR and AMP-dependent protein kinase (AMPK) at the lysosomal membrane, and both of these molecules could be activated by perilysosomal calcium signaling. However, aberrant and persistent Ca ²⁺ elevation upon lipotoxic stress increases mTOR activity and suppresses autophagy. Therefore, normalization of autophagy is an attractive therapeutic strategy for patients with β-cell failure and diabetes.
... These findings were also supported by no increase in intracellular free calcium. We further focused our study on the energy status of the cells, which can be directly sensed by PRKAA1 to induce autophagy 33,34 . We, therefore, checked the levels of two important energy metabolites that is adenosine monophosphate (AMP) and adenosine triphosphate (ATP). ...
... These changes are sensed by the competitive binding of AMP and ATP at CBS3, one of three adenine nucleotide-binding sites formed by the tandem CBS repeats on the AMPK-γ subunits [4,5]. The replacement of ATP by AMP at CBS3 triggers a major conformational change [6,7], causing the activation of AMPK through three complementary mechanisms: (i) the promotion of phosphorylation at Thr172 within the activation loop of the α subunit kinase domain by the upstream kinase, LKB1 [8,9], causing up to 100-fold activation [10]; (ii) inhibition of the dephosphorylation of Thr172 by protein phosphatases [8]; and (iii) the allosteric activation of AMPK complexes already phosphorylated on Thr172, causing further activation [8,11]. AMPK then phosphorylates downstream targets at serine or ity for AMPK than SBI-0206965, although it did potently inhibit all four isoforms of RSK (RSK1-4, also known as RPS6KA1-4, Figure S1A,C). ...
... These changes are sensed by the competitive binding of AMP and ATP at CBS3, one of three adenine nucleotide-binding sites formed by the tandem CBS repeats on the AMPK-γ subunits [4,5]. The replacement of ATP by AMP at CBS3 triggers a major conformational change [6,7], causing the activation of AMPK through three complementary mechanisms: (i) the promotion of phosphorylation at Thr172 within the activation loop of the α subunit kinase domain by the upstream kinase, LKB1 [8,9], causing up to 100-fold activation [10]; (ii) inhibition of the dephosphorylation of Thr172 by protein phosphatases [8]; and (iii) the allosteric activation of AMPK complexes already phosphorylated on Thr172, causing further activation [8,11]. AMPK then phosphorylates downstream targets at serine or ity for AMPK than SBI-0206965, although it did potently inhibit all four isoforms of RSK (RSK1-4, also known as RPS6KA1-4, Figure S1A,C). ...
... These changes are sensed by the competitive binding of AMP and ATP at CBS3, one of three adenine nucleotide-binding sites formed by the tandem CBS repeats on the AMPK-γ subunits [4,5]. The replacement of ATP by AMP at CBS3 triggers a major conformational change [6,7], causing the activation of AMPK through three complementary mechanisms: (i) the promotion of phosphorylation at Thr172 within the activation loop of the α subunit kinase domain by the upstream kinase, LKB1 [8,9], causing up to 100-fold activation [10]; (ii) inhibition of the dephosphorylation of Thr172 by protein phosphatases [8]; and (iii) the allosteric activation of AMPK complexes already phosphorylated on Thr172, causing further activation [8,11]. AMPK then phosphorylates downstream targets at serine or ity for AMPK than SBI-0206965, although it did potently inhibit all four isoforms of RSK (RSK1-4, also known as RPS6KA1-4, Figure S1A,C). ...
AMP-activated protein kinase (AMPK) is the central component of a signalling pathway that senses energy stress and triggers a metabolic switch away from anabolic processes and towards catabolic processes. There has been a prolonged focus in the pharmaceutical industry on the development of AMPK-activating drugs for the treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. However, recent findings suggest that AMPK inhibitors might be efficacious for treating certain cancers, especially lung adenocarcinomas, in which the PRKAA1 gene (encoding the α1 catalytic subunit isoform of AMPK) is often amplified. Here, we study two potent AMPK inhibitors, BAY-3827 and SBI-0206965. Despite not being closely related structurally, the treatment of cells with either drug unexpectedly caused increases in AMPK phosphorylation at the activating site, Thr172, even though the phosphorylation of several downstream targets in different subcellular compartments was completely inhibited. Surprisingly, the two inhibitors appear to promote Thr172 phosphorylation by different mechanisms: BAY-3827 primarily protects against Thr172 dephosphorylation, while SBI-0206965 also promotes phosphorylation by LKB1 at low concentrations, while increasing cellular AMP:ATP ratios at higher concentrations. Due to its greater potency and fewer off-target effects, BAY-3827 is now the inhibitor of choice for cell studies, although its low bioavailability may limit its use in vivo.
... Regulation of energy homeostasis is mediated principally through sensing of low cellular ATP levels through an evolutionarily conserved system mediated by AMP-activated protein kinase (AMPK) (20,21). AMPK can monitor energy availability and respond to changes in the ATP/ADP and ATP/AMP ratio through direct binding of adenine nucleotides (22,23) and activation of its kinase activity (23)(24)(25)(26). Once activated, AMPK reprograms cellular metabolism through regulation of the phosphoproteome toward decreased anabolism and increased catabolism (20,21). ...
... Regulation of energy homeostasis is mediated principally through sensing of low cellular ATP levels through an evolutionarily conserved system mediated by AMP-activated protein kinase (AMPK) (20,21). AMPK can monitor energy availability and respond to changes in the ATP/ADP and ATP/AMP ratio through direct binding of adenine nucleotides (22,23) and activation of its kinase activity (23)(24)(25)(26). Once activated, AMPK reprograms cellular metabolism through regulation of the phosphoproteome toward decreased anabolism and increased catabolism (20,21). ...
... In humans, multiple isoforms of each subunit are encoded by different genes: αsubunit (PRKAA1 and PRKAA2), βsubunit (PRKAB1 and PRKAB2), and γsubunit (PRKAG1, PRKAG2, and PRKAG3). The γsubunit binds AMP (and ADP to a lesser extent) to stimulate AMPK activity through a key phosphorylation on threonine 172 (T172) in the αsubunit that contains the kinase domain (22)(23)(24)(25)(26). The βsubunit allows AMPK to bind and respond to glycogen (33). ...
Diacylglycerol lipase-beta (DAGLβ) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLβ ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLβ in primary macrophages leads to LKB1–AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLβ blockade, thereby directly supporting DAGLβ–AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.
... Cellular ADP/ATP ratios were analyzed with ADP/ATP Ratio Assay Kit (Sigma). AMP/ATP ratios were determined by assuming AMP/ATP = Keq×(ADP/ATP) 2 , where Keq = 1.05, as previously described [25,27]. ...
While ectonucleotidase CD39 is a cancer therapeutic target in clinical trials, its direct effect on T-cell differentiation in human non-small-cell lung cancer (NSCLC) remains unclear. Herein, we demonstrate that human NSCLC cells, including tumor cell lines and primary tumor cells from clinical patients, efficiently drive the metabolic adaption of human CD4 ⁺ T cells, instructing differentiation of regulatory T cells while inhibiting effector T cells. Of importance, NSCLC-induced T-cell mal-differentiation primarily depends on cancer CD39, as this can be fundamentally blocked by genetic depletion of CD39 in NSCLC. Mechanistically, NSCLC cells package CD39 into their exosomes and transfer such CD39-containing exosomes into interacting T cells, resulting in ATP insufficiency and AMPK hyperactivation. Such CD39-dependent NSCLC-T cell interaction holds well in patients-derived primary tumor cells and patient-derived organoids (PDOs). Accordingly, genetic depletion of CD39 alone or in combination with the anti-PD-1 immunotherapy efficiently rescues effector T cell differentiation, instigates anti-tumor T cell immunity, and inhibits tumor growth of PDOs. Together, targeting cancer CD39 can correct the mal-differentiation of CD4 ⁺ T cells in human NSCLC, providing in-depth insight into therapeutic CD39 inhibitors.
... AMPK is activated by the binding of adenosine monophosphate (AMP) and adenosine diphosphate (ADP)indicators of metabolic stressto AMPK's regulatory γ-subunit where they promote phosphorylation and activation 59 . In rat skeletal muscle, 6-hours of fasting causes a rapid increase in the AMP:ATP ratio and a corresponding increase in p-AMPK 9 . ...
... During periods of metabolic stress (e.g., starvation, exercise, and ischemia), energy depletion leads to shifts in the ADP/ATP and AMP/ ATP ratios, which are detected by AMPK and activate the enzyme complex to bias the cell into catabolic processes for ATP generation. In energy-replete conditions (where ATP levels are high), AMPK is kept in its inactive form to stimulate anabolic events such as lipogenesis, gluconeogenesis, and protein synthesis (Gowans et al., 2013). ...
Multiple Sclerosis (MS) is a chronic disease characterized by immune-mediated destruction of myelinating oligodendroglia in the central nervous system. Loss of myelin leads to neurological dysfunction and, if myelin repair fails, neurodegeneration of the denuded axons. Virtually all treatments for MS act by suppressing immune function, but do not alter myelin repair outcomes or long-term disability. Excitingly, the diabetes drug metformin, a potent activator of the cellular “energy sensor” AMPK complex, has recently been reported to enhance recovery from demyelination. In aged mice, metformin can restore responsiveness of oligodendrocyte progenitor cells (OPCs) to pro-differentiation cues, enhancing their ability to differentiate and thus repair myelin. However, metformin’s influence on young oligodendroglia remains poorly understood. Here we investigated metformin’s effect on the temporal dynamics of differentiation and metabolism in young, healthy oligodendroglia and in oligodendroglia following myelin damage in young adult mice. Our findings reveal that metformin accelerates early stages of myelin repair following cuprizone-induced myelin damage. Metformin treatment of both isolated OPCs and oligodendrocytes altered cellular bioenergetics, but in distinct ways, suppressing oxidative phosphorylation and enhancing glycolysis in OPCs, but enhancing oxidative phosphorylation and glycolysis in both immature and mature oligodendrocytes. In addition, metformin accelerated the differentiation of OPCs to oligodendrocytes in an AMPK-dependent manner that was also dependent on metformin’s ability to modulate cell metabolism. In summary, metformin dramatically alters metabolism and accelerates oligodendroglial differentiation both in health and following myelin damage. This finding broadens our knowledge of metformin’s potential to promote myelin repair in MS and in other diseases with myelin loss or altered myelination dynamics.
