AMPK Inhibits ULK1-Dependent Autophagosome Formation and Lysosomal Acidification via Distinct Mechanisms

ABSTRACT Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

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ATG9A shapes the forming autophagosome through Arfaptin 2 and phosphatidylinositol 4-kinase IIIβ

The Journal of Cell Biology ◽

10.1083/jcb.201901115 ◽

2019 ◽

Vol 218 (5) ◽

pp. 1634-1652 ◽

Cited By ~ 48

Author(s):

Delphine Judith ◽

Harold B.J. Jefferies ◽

Stefan Boeing ◽

David Frith ◽

Ambrosius P. Snijders ◽

...

Keyword(s):

Quantitative Analysis ◽

Amino Acid ◽

Membrane Protein ◽

Initiation Site ◽

Amino Acid Starvation ◽

Autophagosome Formation ◽

Metabolizing Enzymes ◽

Bar Domain ◽

Golgi Membranes ◽

Phosphatidylinositol 4

ATG9A is a multispanning membrane protein essential for autophagy. Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation. ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome. To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid–starved cells was performed. These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes. Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site. PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response. PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes.

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AMP-activated protein kinase pathway and bone metabolism

Journal of Endocrinology ◽

10.1530/joe-11-0306 ◽

2011 ◽

Vol 212 (3) ◽

pp. 277-290 ◽

Cited By ~ 58

Author(s):

J Jeyabalan ◽

M Shah ◽

B Viollet ◽

C Chenu

Keyword(s):

Protein Kinase ◽

Energy Homeostasis ◽

Bone Cells ◽

Peripheral Tissues ◽

Ampk Signaling ◽

Obesity And Diabetes ◽

Regulate Food Intake ◽

Ampk Activity ◽

Amp Activated Protein Kinase

There is increasing evidence that osteoporosis, similarly to obesity and diabetes, could be another disorder of energy metabolism. AMP-activated protein kinase (AMPK) has emerged over the last decade as a key sensing mechanism in the regulation of cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones on the metabolism of appetite, fat and glucose. Novel work demonstrates that the AMPK signaling pathway also plays a role in bone physiology. Activation of AMPK promotes bone formationin vitroand the deletion of α or β subunit of AMPK decreases bone mass in mice. Furthermore, AMPK activity in bone cells is regulated by the same hormones that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. AMPK is also activated by antidiabetic drugs such as metformin and thiazolidinediones (TZDs), which also impact on skeletal metabolism. Interestingly, TZDs have detrimental skeletal side effects, causing bone loss and increasing the risk of fractures, although the role of AMPK mediation is still unclear. These data are presented in this review that also discusses the potential roles of AMPK in bone as well as the possibility for AMPK to be a future therapeutic target for intervention in osteoporosis.

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Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000480184 ◽

2017 ◽

Vol 42 (6) ◽

pp. 2418-2429 ◽

Cited By ~ 6

Author(s):

Huiying Shi ◽

Xinyan Zhao ◽

Zhen Ding ◽

Chaoqun Han ◽

Ye Jiang ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Epithelial Cells ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Amino Acid Starvation ◽

Autophagic Flux ◽

Intestinal Epithelial ◽

Expression Levels ◽

Sequestosome 1

Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

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TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

The Journal of Cell Biology ◽

10.1083/jcb.201111079 ◽

2012 ◽

Vol 197 (5) ◽

pp. 659-675 ◽

Cited By ~ 232

Author(s):

Andrea Longatti ◽

Christopher A. Lamb ◽

Minoo Razi ◽

Shin-ichiro Yoshimura ◽

Francis A. Barr ◽

...

Keyword(s):

Amino Acid ◽

Golgi Complex ◽

Membrane Vesicles ◽

Degradation Process ◽

Amino Acid Starvation ◽

Negative Regulators ◽

Autophagosome Formation ◽

Double Membrane ◽

Recycling Endosomes ◽

Guanosine Triphosphatase

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain–containing Rab guanosine triphosphatase–activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)–positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.

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Contraction-mediated phosphorylation of AMPK is lower in skeletal muscle of adenylate kinase-deficient mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.00885.2005 ◽

2006 ◽

Vol 100 (2) ◽

pp. 406-413 ◽

Cited By ~ 32

Author(s):

Chad R. Hancock ◽

Edwin Janssen ◽

Ronald L. Terjung

Keyword(s):

Skeletal Muscle ◽

Adenylate Kinase ◽

Force Production ◽

Acetyl Coa Carboxylase ◽

Acetyl Coa ◽

Ampk Signaling ◽

Ampk Activity ◽

The Right ◽

Initial Force ◽

Amp Activated Protein Kinase

The activity of AMP-activated protein kinase (AMPK) increases during muscle contractions as a result of elevated AMP concentration. We tested whether activation of AMPK would be altered during contractions in adenylate kinase (AK) 1-deficient (AK1−/−) mice, because they have a reduced capacity to form AMP. The right gastrocnemius-soleus-plantaris muscle group was stimulated via the sciatic nerve at 2 Hz for 30 min in both wild-type (WT) and AK1−/− animals. Initial force production was not different between the two groups (129.2 ± 3.3 g vs. 140.9 ± 8.5 g for WT and AK1−/−, respectively); however, force production by AK1−/− mice was significantly greater over the 30-min stimulation period, and final tension was 85 ± 4.5% of initial in WT and 102 ± 3.2% of initial in AK1−/− mice. Western blot analysis showed that AMPK phosphorylation with contractions was clearly increased in WT muscles (4.0 ± 1.1 above resting values), but did not change noticeably with AK deficiency (1.6 ± 0.4 above WT resting values). However, increases in phosphorylation of acetyl CoA carboxylase were robust in both WT and AK1−/− muscles and not different between the two groups. These results suggest that reduced formation of AMP during contractions in skeletal muscle of AK1−/− mice results in reduced phosphorylation of AMPK. However, altered AMPK signaling was not apparent in the phosphorylation status of acetyl CoA carboxylase, a typical marker of AMPK activity.

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The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation

Biochemical Journal ◽

10.1042/bj1380155 ◽

1974 ◽

Vol 138 (2) ◽

pp. 155-163 ◽

Cited By ~ 2

Author(s):

J. E. M. Midgley ◽

R. J. Smith

Keyword(s):

Amino Acid ◽

Ribonucleic Acid ◽

Normal Growth ◽

Acid Synthesis ◽

Amino Acid Starvation ◽

Starvation Period ◽

Bacterial Strains ◽

Cell Content ◽

Messenger Ribonucleic Acid ◽

Acid Deficiency

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNAfMet for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RCstr strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RCrel strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RCstr and RCrel cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.

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AKT Ser/Thr kinase increases V-ATPase–dependent lysosomal acidification in response to amino acid starvation in mammalian cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013223 ◽

2020 ◽

Vol 295 (28) ◽

pp. 9433-9444 ◽

Cited By ~ 1

Author(s):

Michael P. Collins ◽

Laura A. Stransky ◽

Michael Forgac

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Atpase Activity ◽

Mammalian Cells ◽

Amino Acid Starvation ◽

Akt Inhibitor ◽

Cellular Targets ◽

Amino Acid Availability ◽

Dependent Protein ◽

Amp Activated Protein Kinase

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by the regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.

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Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

10.1101/2021.10.11.463987 ◽

2021 ◽

Author(s):

Danielle L Schmitt ◽

Stephanie D Curtis ◽

Allen Leung ◽

Jin-fan Zhang ◽

Mingyuan Chen ◽

...

Keyword(s):

Dynamic Range ◽

High Sensitivity ◽

Dynamic Activity ◽

Ampk Signaling ◽

Upstream Kinase ◽

Cellular Energetics ◽

Liver Kinase B1 ◽

Ampk Activity ◽

Nuclear Shuttling ◽

Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

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Rapid and Reversible Nuclear Accumulation of Cytoplasmic tRNA in Response to Nutrient Availability

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0006 ◽

2007 ◽

Vol 18 (7) ◽

pp. 2678-2686 ◽

Cited By ~ 74

Author(s):

Michael L. Whitney ◽

Rebecca L. Hurto ◽

Hussam H. Shaheen ◽

Anita K. Hopper

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Amino Acid ◽

Nutrient Availability ◽

Amino Acid Starvation ◽

Nuclear Accumulation ◽

Signal Transduction Pathways ◽

Glucose Starvation ◽

Acid Versus ◽

Nucleocytoplasmic Distribution

Cytoplasmic tRNAs have recently been found to accumulate in the nucleus during amino acid starvation in yeast. The mechanism and regulation by which tRNAs return to the nucleus are unclear. Here, we show accumulation of cytoplasmic tRNA in the nucleus also occurs during glucose starvation. Nuclear accumulation of tRNA in response to acute glucose or amino acid starvation is rapid, reversible, requires no new transcription, and is independent of the aminoacylation status of tRNA. Gradual depletion of nutrients also results in the accrual of tRNA in the nucleus. Distinct signal transduction pathways seem to be involved in the accumulation of cytoplasmic tRNA in the nucleus in response to amino acid versus glucose starvation. These findings suggest tRNA nucleocytoplasmic distribution may play a role in gene expression in response to nutritional stress.

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Effects of the spoT and relA Mutation on the Synthesis and Accumulation of ppGpp and RNA during Glucose Starvation

Canadian Journal of Biochemistry ◽

10.1139/o78-041 ◽

1978 ◽

Vol 56 (4) ◽

pp. 264-272 ◽

Cited By ~ 13

Author(s):

Gillian Chaloner-Larsson ◽

Hiroshi Yamazaki

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Rna Synthesis ◽

Amino Acid Starvation ◽

Protein Accumulation ◽

Glucose Starvation ◽

Basal Rate ◽

Synthetic Rate ◽

K 12 ◽

Stimulation Of

The effects of glucose starvation on the accumulation and synthesis of guanosine 3′,5′-bis(diphosphate) (ppGpp) were compared in four Escherichia coli K-12 strains having four different combinations of the spoT and relA alleles: spoT+relA+, spoT relA+, spoT+relA, and spoT relA. Glucose starvation caused a rapid and complete inhibition of RNA and protein accumulation and severe inhibition of RNA synthesis in all four strains. However, ppGpp accumulated only gradually in the relaxed (relA) strains and rapidly in the stringent (relA+) strains. Thus, ppGpp accumulation is not obligatory to the inhibition of RNA synthesis and accumulation during glucose starvation. During growth, relA strains synthesized ppGpp at a rate comparable with that in their relA+ partners. Glucose starvation did not affect the basal rate of ppGpp synthesis in the relA strains, but caused a transient stimulation of ppGpp synthesis in the relA+ strains. This suggests that glucose starvation causes transient amino-acid starvation. Since ppGpp accumulated in the relA strain without a change in its synthetic rate, it is inferred that ppGpp degradation decreased during glucose starvation. During growth, the turnover of ppGpp was considerably slower in the spoT strains than in the spoT+ strains. This suggests that the slower degradation of ppGpp in the spoT strains is counterbalanced by the slower synthesis of ppGpp. This difference in the rate of ppGpp synthesis became apparent when the relA strains were starved for glucose: The spoT relA strain accumulated ppGpp more slowly than did the spoT+relA strain.

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