AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.

[1]  G. Goshima,et al.  14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes , 2017, The Journal of cell biology.

[2]  E. Han,et al.  Crystal structures reveal a new and novel FoxO1 binding site within the human glucose-6-phosphatase catalytic subunit 1 gene promoter. , 2017, Journal of structural biology.

[3]  Bo-zhi Cai,et al.  Metformin inhibits estrogen‐dependent endometrial cancer cell growth by activating the AMPK–FOXO1 signal pathway , 2016, Cancer science.

[4]  B. Warscheid,et al.  A systems study reveals concurrent activation of AMPK and mTOR by amino acids , 2016, Nature Communications.

[5]  Wolfgang Link,et al.  Long live FOXO: unraveling the role of FOXO proteins in aging and longevity , 2015, Aging cell.

[6]  K. Nakazato,et al.  Activation of AMP-activated protein kinase induce expression of FoxO1, FoxO3a, and myostatin after exercise-induced muscle damage. , 2015, Biochemical and biophysical research communications.

[7]  Yuzhen Ning,et al.  FOXO1 silence aggravates oxidative stress-promoted apoptosis in cardiomyocytes by reducing autophagy. , 2015, The Journal of toxicological sciences.

[8]  P. Urbánek,et al.  Redox regulation of FoxO transcription factors , 2015, Redox biology.

[9]  Shu Chien,et al.  Identification of AMP-activated protein kinase targets by a consensus sequence search of the proteome , 2015, BMC Systems Biology.

[10]  K. Herrup,et al.  The Roles of Cdk5-Mediated Subcellular Localization of FOXO1 in Neuronal Death , 2015, The Journal of Neuroscience.

[11]  L. Kay,et al.  Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch , 2014, Nature.

[12]  Jongki Hong,et al.  AMP‐activated protein kinase mediates the antioxidant effects of resveratrol through regulation of the transcription factor FoxO1 , 2014, The FEBS journal.

[13]  S. Dakshinamurti,et al.  AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle , 2014, Pediatric pulmonology.

[14]  M. Mayzel,et al.  Tyrosine Phosphorylation within the Intrinsically Disordered Cytosolic Domains of the B-Cell Receptor: An NMR-Based Structural Analysis , 2014, PloS one.

[15]  Diana M. Mitrea,et al.  Structural polymorphism in the N-terminal oligomerization domain of NPM1 , 2014, Proceedings of the National Academy of Sciences.

[16]  David Carling,et al.  Structural basis of AMPK regulation by small molecule activators , 2013, Nature Communications.

[17]  Steven J. M. Jones,et al.  Analysis of FOXO1 mutations in diffuse large B-cell lymphoma. , 2013, Blood.

[18]  G. Elia,et al.  Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets. , 2012, Blood.

[19]  Yachen Wang,et al.  Applications of post-translational modifications of FoxO family proteins in biological functions. , 2011, Journal of molecular cell biology.

[20]  D. Hardie.  AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. , 2011, Genes & development.

[21]  Hui Chen,et al.  Protein Kinase A-α Directly Phosphorylates FoxO1 in Vascular Endothelial Cells to Regulate Expression of Vascular Cellular Adhesion Molecule-1 mRNA* , 2010, The Journal of Biological Chemistry.

[22]  Ladislav Stacho,et al.  Prediction of 492 human protein kinase substrate specificities , 2010, Proteome Science.

[23]  G. Leclerc,et al.  AMPK-induced activation of Akt by AICAR is mediated by IGF-1R dependent and independent mechanisms in acute lymphoblastic leukemia , 2010, Journal of molecular signaling.

[24]  C. Ottmann,et al.  Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling , 2010, Molecular and Cellular Biology.

[25]  Yu-gang Dong,et al.  Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro , 2010, Acta Pharmacologica Sinica.

[26]  Roland L. Dunbrack,et al.  PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. , 2010, Biochimica et biophysica acta.

[27]  M. Webb,et al.  Fluorescence changes , nucleotide affinities , and binding dynamics of different tetramethylrhodamine-labeled ParMmutants , 2010 .

[28]  D. Campbell,et al.  Bioinformatic and experimental survey of 14-3-3-binding sites , 2010, The Biochemical journal.

[29]  E. Greer,et al.  AMP‐activated Protein Kinase and FoxO Transcription Factors in Dietary Restriction–induced Longevity , 2009, Annals of the New York Academy of Sciences.

[30]  Victoria L. Murray,et al.  Practical protocols for production of very high yields of recombinant proteins using Escherichia coli , 2009, Protein science : a publication of the Protein Society.

[31]  Maria K. Lehtinen,et al.  Regulation of Neuronal Cell Death by MST1-FOXO1 Signaling* , 2009, Journal of Biological Chemistry.

[32]  R. Marmorstein,et al.  Structural basis for DNA recognition by FoxO1 and its regulation by posttranslational modification. , 2008, Structure.

[33]  Esther B. E. Becker,et al.  Activation of FOXO1 by Cdk1 in Cycling Cells and Postmitotic Neurons , 2008, Science.

[34]  Steven P Gygi,et al.  In situ observation of protein phosphorylation by high-resolution NMR spectroscopy , 2008, Nature Structural &Molecular Biology.

[35]  S. Gygi,et al.  An AMPK-FOXO Pathway Mediates Longevity Induced by a Novel Method of Dietary Restriction in C. elegans , 2007, Current Biology.

[36]  A. Fukamizu,et al.  Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. , 2007, Cellular signalling.

[37]  J. Marsh,et al.  Sensitivity of secondary structure propensities to sequence differences between α‐ and γ‐synuclein: Implications for fibrillation , 2006 .

[38]  D. Tindall,et al.  CDK2-Dependent Phosphorylation of FOXO1 as an Apoptotic Response to DNA Damage , 2006, Science.

[39]  Wayne Boucher,et al.  The CCPN data model for NMR spectroscopy: Development of a software pipeline , 2005, Proteins.

[40]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.

[41]  W. Biggs,et al.  Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Hitomi Matsuzaki,et al.  Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Martin Würtele,et al.  Structural view of a fungal toxin acting on a 14‐3‐3 regulatory complex , 2003, The EMBO journal.

[44]  Brian A. Hemmings,et al.  Crystal structure of an activated Akt/Protein Kinase B ternary complex with GSK3-peptide and AMP-PNP , 2002, Nature Structural Biology.

[45]  Ulf Reimer,et al.  Local structural changes caused by peptidyl-prolyl cis/trans isomerization in the native state of proteins. , 2002, Biophysical chemistry.

[46]  P. Cohen,et al.  Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion , 2002, The EMBO journal.

[47]  P. Cohen,et al.  Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting. , 2001, The Biochemical journal.

[48]  P. Cohen,et al.  Phosphorylation of the Transcription Factor Forkhead Family Member FKHR by Protein Kinase B* , 1999, The Journal of Biological Chemistry.

[49]  K. Guan,et al.  Negative Regulation of the Forkhead Transcription Factor FKHR by Akt* , 1999, The Journal of Biological Chemistry.

[50]  M. Greenberg,et al.  Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor , 1999, Cell.

[51]  J. Marsh,et al.  Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. , 2006, Protein science : a publication of the Protein Society.

[52]  Zoran Obradovic,et al.  Predicting intrinsic disorder from amino acid sequence , 2003, Proteins.