Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver

CDK4 Phosphorylates AMPKα2 to Inhibit Its Activity and Repress Fatty Acid Oxidation

The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4(-/-) mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK

Mechanistic insights into AMP-activated protein kinase-dependent gene expression

AMP-activated protein kinase (AMPK) is a fundamental enzyme that controls energy homeostasis, through orchestrating the cellular response to a reduction in energy availability. Under conditions of cellular energy stress AMPK senses the decrease in ATP levels and responds by activating catabolic pathways, which will generate ATP, and switching off ATP-consuming ones, in order to restore the energy balance. AMPK regulates several signaling cascades linked to metabolism, overall favoring cellular consumption of glucose and lipid, allowing the cell to adapt to sustained energetic challenges through modulation of gene transcription. The aim of this thesis is to investigate the role that AMPK plays in the adaptive reprogramming of metabolism through transcriptional control. To identify genes and pathways regulated in an AMPK-dependent mechanism, we performed a whole-genome transcriptome profiling using microarray technology and compared the effects of two small molecule AMPK activators acting via distinct mechanisms, namely 991, which binds at the allosteric drug and metabolite site, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). The impact on gene expression of 991 and AICAR was investigated using two cellular and genetic models, mouse embryonic fibroblasts (MEFs) and mouse primary hepatocytes, either wild-type or AMPK-deficient. Statistical analysis of differential gene expression, followed by pathway analysis, revealed compound- and model-specific gene expression signatures. Notably we found that in contrast to AICAR, 991 affected gene expression almost exclusively in an AMPK-dependent manner. Interestingly, we identified that 991 modulated genes involved in the metabolic and lysosomal pathways, and that a number of these genes are under the control of the sterol regulatory element-binding protein (SREBP) and transcription factor EB (TFEB). We identified the tumor suppressor folliculin (Flcn) and its binding partners, folliculin interacting protein (Fnip), as novel transcriptional targets of AMPK. We confirmed the upregulated expression of Flcn in response to pharmacological activation of AMPK in MEFs and primary hepatocytes. Furthermore, by taking advantage of a novel zebrafish whole-body knockout model of AMPK, we confirmed that physiological activation of AMPK partly mediates the increase in expression of Flcn and Fnip. We further identified TFEB as a mediator of the AMPK transcriptional response, accounting for the increase in Flcn expression through modulating its promoter activity. Moreover, we revealed the existence of a novel mechanism by which AMPK regulates TFEB through promoting dephosphorylation and nuclear translocation, independently of mammalian target of rapamycin complex 1 (mTORC1). Taken together, we identified several new AMPK-dependent/-regulated genes and pathways that are differentially modulated in a cell type- and compound-specific manner. Most importantly, we discovered a novel and conserved AMPK-TFEB-FLCN axis in cellular and in vivo models. This work contributes to advance our understanding of AMPK-mediated regulation of transcriptional programs, nevertheless future studies will be required to elucidate the physiological relevance of the AMPK-TFEB-FLCN cascade

Identification of novel PCTAIRE-1/CDK16 substrates using a chemical genetic screen

PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK) 16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been identified in human patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only a few cellular targets/substrates have been proposed with no clear functional significance. We and others recently showed that cyclin Y binds and activates PCTAIRE-1 via phosphorylation and 14-3-3 binding. In order to understand the physiological role that PCTAIRE-1 plays in brain, we have performed a chemical genetic screen in vitro using an engineered PCTAIRE-1/cyclin Y complex and mouse brain extracts. Our screen has identified potential PCTAIRE-1 substrates (AP2-Associated Kinase 1 (AAK1), dynamin 1, and synaptojanin 1) in brain that have been shown to regulate crucial steps of receptor endocytosis, and are involved in control of neuronal synaptic transmission. Furthermore, mass spectrometry and protein sequence analyses have identified potential PCTAIRE-1 regulated phosphorylation sites on AAK1 and we validated their PCTAIRE-1 dependence in a cellular study and/or brain tissue lysates. Our results shed light onto the missing link between PCTAIRE-1 regulation and proposed physiological functions, and provide a basis upon which to further study PCTAIRE-1 function in vivo and its potential role in neuronal/brain disorders

AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR

International audienceAMPK is a central regulator of energy homeostasis. AMPK not only elicits acute metabolic responses but also promotes metabolic reprogramming and adaptations in the long-term through regulation of specific transcription factors and coactivators. We performed a whole-genome transcriptome profiling in wild-type (WT) and AMPK-deficient mouse embryonic fibroblasts (MEFs) and primary hepatocytes that had been treated with 2 distinct classes of small-molecule AMPK activators. We identified unique compound-dependent gene expression signatures and several AMPK-regulated genes, including folliculin (Flcn), which encodes the tumor suppressor FLCN. Bioinformatics analysis highlighted the lysosomal pathway and the associated transcription factor EB (TFEB) as a key transcriptional mediator responsible for AMPK responses. AMPK-induced Flcn expression was abolished in MEFs lacking TFEB and transcription factor E3, 2 transcription factors with partially redundant function; additionally, the promoter activity of Flcn was profoundly reduced when its putative TFEB-binding site was mutated. The AMPK-TFEB-FLCN axis is conserved across species; swimming exercise in WT zebrafish induced Flcn expression in muscle, which was significantly reduced in AMPK-deficient zebrafish. Mechanistically, we have found that AMPK promotes dephosphorylation and nuclear localization of TFEB independently of mammalian target of rapamycin activity. Collectively, we identified the novel AMPK-TFEB-FLCN axis, which may function as a key cascade for cellular and metabolic adaptations.-Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., Lefebvre, G., Raymond, F., Parisi, A., Civiletto, G., Gut, P., Descombes, P., Sakamoto, K. AMPK promotes induction of the tumor suppressor FLCN through activation of TFEB independently of mTOR

Benzimidazole derivative small-molecule 991 enhances AMPK activity and glucose uptake induced by AICAR or contraction in skeletal muscle

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic alpha-subunit (alpha 1 and alpha 2) and regulatory beta (beta 1 and beta 2)- and gamma (gamma 1, gamma 2, gamma 3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the gamma 3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPK gamma 3 as well as ubiquitously expressed gamma 1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 mu M) stimulated a modest or negligible activity of both gamma 1- and gamma 3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPK gamma 1/gamma 3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle

AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis.

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1 MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1 MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1 phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1 MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate