The Energy Sensor AMP-activated Protein Kinase Directly Regulates the Mammalian FOXO3 Transcription Factor

Abstract

The maintenance of homeostasis throughout an organism's life span requires constant adaptation to changes in energy levels. The AMP-activated protein kinase (AMPK) plays a critical role in the cellular responses to low energy levels by switching off energy-consuming pathways and switching on energy-producing pathways. However, the transcriptional mechanisms by which AMPK acts to adjust cellular energy levels are not entirely characterized. Here, we find that AMPK directly regulates mammalian FOXO3, a member of the FOXO family of Forkhead transcription factors known to promote resistance to oxidative stress, tumor suppression, and longevity. We show that AMPK phosphorylates human FOXO3 at six previously unidentified regulatory sites. Phosphorylation by AMPK leads to the activation of FOXO3 transcriptional activity without affecting FOXO3 subcellular localization. Using a genome-wide microarray analysis, we identify a set of target genes that are regulated by FOXO3 when phosphorylated at these six regulatory sites in mammalian cells. The regulation of FOXO3 by AMPK may play a crucial role in fine tuning gene expression programs that control energy balance and stress resistance in cells throughout life.The maintenance of cellular energy levels in response to changes in nutrient availability, exercise, or stress stimuli is pivotal for organismal homeostasis throughout life. Disruption of this balance is associated with a number of pathologies, including diabetes and cancer. The AMP-activated protein kinase (AMPK) 3 plays a crucial role in translating changes in energy levels into adaptive cellular responses (1, 2). AMPK is a heterotrimeric protein kinase composed of three subunits: a catalytic subunit (␣), a scaffolding subunit (␤), and an AMP-sensing subunit (␥). AMPK is activated by stimuli that increase the AMP/ ATP ratio in cells. Excess AMP activates AMPK by inhibiting the dephosphorylation of the ␣ catalytic subunit (3) and by inducing a change in conformation in the AMPK heterotrimeric complex that promotes the phosphorylation and activation of the ␣ catalytic subunit by the AMPK-activating protein kinases, LKB1 and calmodulin-dependent protein kinase kinase (4 -8).AMPK controls cell metabolism and growth in response to low energy levels by phosphorylating a variety of substrates in cells, including acetyl-CoA carboxylase (ACC), tuberous sclerosis complex 2, and p27 KIP1 (9 -11). AMPK also regulates gene expression by phosphorylating co-activators, such as transducer of regulated CREB, thyroid hormone receptor interactor 6, and the transcription factor p53 (12-16). The LKB1-AMPK pathway plays a pivotal role in tumor suppression (17, 18), in diabetes prevention (19), and in longevity (20). Thus, identifying novel AMPK substrates is important to understand how the LKB1-AMPK pathway mediates its effects in the organism.FOXO transcription factors are good candidates to be regulated by AMPK. The FOXO family of Forkhead transcription factors (FOXO1, FOXO3, FOXO4, and FOXO6 in mammals) plays an important role in the...

Long live FOXO: unraveling the role of FOXO proteins in aging and longevity

“…Conversely, in the absence of growth factor signaling or upon cellular stress, FOXOs translocate into the nucleus and activate FOXO‐dependent gene expression. A diverse set of posttranslational modifications in addition to phosphorylation, such as acetylation/deacetylation, methylation, or ubiquitination has been shown to promote changes of subcellular localization, protein levels, DNA binding, and transcriptional activity of FOXO factors (Calnan & Brunet, 2008; Webb & Brunet, 2014) The combinatorial result of FOXO posttranslational modifications has been proposed to lead to the recruitment of specific FOXO‐binding partners regulating different FOXO‐dependent gene expression programs (Greer et al ., 2007b; Calnan & Brunet, 2008; Hill et al ., 2014). Several mechanisms of how FOXO proteins promote longevity have been suggested.…”

Post-translational modifications of FOXO family proteins

“…In mammals, AMPK phosphorylates human FOXO3 at six regulatory sites (Table I) (55). The inactivation of p38α triggers nuclear translocation of FOXO3a in an AMPK-dependent manner, and leads to subsequent activation of FOXO3a targets genes, which induces autophagy, cell-cycle arrest and cell death (56).…”

AMP kinase–mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis

“…However, it should be noted that the regulation of bim expression is highly complex (Biswas et al, 2007), and other pathways independent of JNK signaling may well be required for Bim-dependent excitotoxic apoptosis. A recent study has shown that AMPK can phosphorylate and enhance the transcriptional activity of Foxo3a (Greer et al, 2007). In addition, given that AMPK can down-regulate mTOR (mammalian target of rapamycin) signaling, this may in turn result in decreased Akt phosphorylation, nuclear translocation of Foxo3a, and induction of bim.…”