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

Eric L. Greer, Philip R. Oskoui, Max R. Banko, Jay M. Maniar, Melanie P. Gygi, Steven P. Gygi, Anne Brunet
2007 Journal of Biological Chemistry  
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
more » ... O3, 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 hetero-trimeric 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 regulation of organismal glucose metabolism, tumor suppression, and longevity (21-26). In mammalian cells, FOXO family members up-regulate target genes involved in glucose metabolism, cell cycle arrest, stress resistance, and even cell death (27) (28) (29) (30) (31) (32) . FOXO transcription factors integrate cellular signals emanating from insulin, growth factors, cytokines, and oxidative stress via a combination of post-translational modifications, including phosphorylation, acetylation, and ubiquitination (30, 33-37). In response to insulin and growth factors, AKT phosphorylates FOXO proteins, thereby sequestering them in the cytoplasm and inhibiting their function (30, 34) . Stimuli that activate AMPK have been reported to inhibit FOXO1-dependent transcription (38). However, whether FOXO family members are direct substrates of AMPK and how they mediate the effects of AMPK on gene expression is not known. Here, we show that the energy-sensing AMPK directly phosphorylates human FOXO3 at six previously unidentified residues in vitro. At least three of these sites are phosphorylated in mammalian cells in response to stimuli that activate AMPK. AMPK is necessary and sufficient for the phosphorylation of FOXO3 at these sites in cells. We show that mutating the
doi:10.1074/jbc.m705325200 pmid:17711846 fatcat:gbb4vkgmejfhxbwj337rgpy6km