PP2A Regulates the Pro-apoptotic Activity of FOXO1*

FOXO1, a member of the evolutionarily conserved forkhead family of transcription factors, regulates expression of a number of genes that play critical roles in cell cycle and apoptosis. A pivotal regulatory mechanism of FOXO is reversible phosphorylation, catalyzed by kinases and phosphatases. Phosphorylation of FOXO1 is associated with 14-3-3 binding and cytosolic localization, whereas dephosphorylated FOXO1 translocates to the nucleus and is transcriptionally active. Experiments were performed to identify the serine/threonine phosphatase that dephosphorylates FOXO1. PP2A inhibitors, okadaic acid and fostriecin, increased FOXO1 phosphorylation in vitro and in cells. Microcystin-agarose pull-downs suggested that a phosphatase binds to FOXO1, and PP2A catalytic subunit was identified in endogenous FOXO1 immunocomplexes, indicating that PP2A is a FOXO1 phosphatase. Purified PP2A interacted directly with FOXO1 and dephosphorylated FOXO1 in vitro. Silencing of PP2A protected FOXO1 from dephosphorylation and delayed FOXO1 nuclear translocation, confirming the physiologic role of PP2A in the regulation of FOXO1 function. Furthermore, inhibition of PP2A phosphatases rescued FOXO1-mediated cell death by regulating the level of the pro-apoptotic protein BIM. We conclude that PP2A is a physiologic phosphatase of FOXO1.

[1]  R. Honkanen,et al.  Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A , 1997, FEBS letters.

[2]  Jun Wu,et al.  B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A , 1997 .

[3]  S. Schreiber,et al.  Alpha 4 associates with protein phosphatases 2A, 4, and 6. , 1998, Biochemical and biophysical research communications.

[4]  E Ruoslahti,et al.  Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. , 1999, Biochemistry.

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

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

[7]  T. Nakazawa,et al.  Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. , 2000, The Biochemical journal.

[8]  R. Medema,et al.  AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1 , 2000, Nature.

[9]  J. Lammers,et al.  Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1 , 2000, Current Biology.

[10]  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.

[11]  Brian A. Hemmings,et al.  Protein Kinase SGK Mediates Survival Signals by Phosphorylating the Forkhead Transcription Factor FKHRL1 (FOXO3a) , 2001, Molecular and Cellular Biology.

[12]  Geert J. P. L. Kops,et al.  The Forkhead Transcription Factor FoxO Regulates Transcription of p27Kip1 and Bim in Response to IL-21 , 2002, The Journal of Immunology.

[13]  Rakesh Nagarajan,et al.  FOXO Proteins Regulate Tumor Necrosis Factor-related Apoptosis Inducing Ligand Expression , 2002, The Journal of Biological Chemistry.

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

[15]  J. Krieglstein,et al.  Serine/threonine protein phosphatases in apoptosis. , 2002, Current opinion in pharmacology.

[16]  D. Accili,et al.  Regulation of insulin action and pancreatic β-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1 , 2002, Nature Genetics.

[17]  B. Burgering,et al.  Cell cycle and death control: long live Forkheads. , 2002, Trends in biochemical sciences.

[18]  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.

[19]  Cindy L. Kanies,et al.  Protein Phosphatase 2A Dephosphorylation of Phosphoserine 112 Plays the Gatekeeper Role for BAD-Mediated Apoptosis , 2003, Molecular and Cellular Biology.

[20]  C. Thompson,et al.  Akt Activation Promotes Degradation of Tuberin and FOXO3a via the Proteasome* , 2003, The Journal of Biological Chemistry.

[21]  P. Coffer,et al.  Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. , 2001, Biochemical Society transactions.

[22]  P. Coffer,et al.  FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons , 2003, The Journal of cell biology.

[23]  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.

[24]  Michael Majeski,et al.  Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms. , 2004, The Biochemical journal.

[25]  Hao Jiang,et al.  Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Accili,et al.  FoxOs at the Crossroads of Cellular Metabolism, Differentiation, and Transformation , 2004, Cell.

[27]  Marten P Smidt,et al.  The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. , 2004, The Biochemical journal.

[28]  Laura A. Solt,et al.  The PP2A-Associated Protein α4 Is an Essential Inhibitor of Apoptosis , 2004, Science.

[29]  Hao Jiang,et al.  Triple Layer Control: Phosphorylation, Acetylation and Ubiquitination of FOXO Proteins , 2005, Cell cycle.

[30]  C. van Hoof,et al.  PP2A: the expected tumor suppressor. , 2005, Current opinion in genetics & development.

[31]  D. Virshup,et al.  Protein serine/threonine phosphatases: life, death, and sleeping. , 2005, Current opinion in cell biology.

[32]  S. de Vos,et al.  FOXO‐dependent expression of the proapoptotic protein Bim: pivotal role for apoptosis signaling in endothelial progenitor cells , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  E. Greer,et al.  FOXO transcription factors at the interface between longevity and tumor suppression , 2005, Oncogene.

[34]  K. Arden.  Multiple roles of FOXO transcription factors in mammalian cells point to multiple roles in cancer , 2006, Experimental Gerontology.

[35]  C. Hellberg,et al.  Protein-tyrosine phosphatases and cancer , 2006, Nature Reviews Cancer.

[36]  Jason A. Koutcher,et al.  Identification of a tumour suppressor network opposing nuclear Akt function , 2006, Nature.

[37]  Yigong Shi,et al.  Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins , 2006, Cell.

[38]  W. Hahn,et al.  Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. , 2006, Cell.

[39]  K. Heidenreich,et al.  FoxO1 Regulates Multiple Metabolic Pathways in the Liver , 2006, Journal of Biological Chemistry.

[40]  Yigong Shi,et al.  Structure of the Protein Phosphatase 2A Holoenzyme , 2006, Cell.

[41]  E. Lam,et al.  FOXO transcription factors: key regulators of cell fate. , 2006, Biochemical Society transactions.

[42]  S. Armstrong,et al.  FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress , 2007, Cell.

[43]  L. Trinkle-Mulcahy,et al.  Emerging roles of nuclear protein phosphatases , 2007, Nature Reviews Molecular Cell Biology.

[44]  Na Wang,et al.  FoxO transcription factors activate Akt and attenuate insulin signaling in heart by inhibiting protein phosphatases , 2007, Proceedings of the National Academy of Sciences.

[45]  P. Coffer,et al.  Stressed marrow: FoxOs stem tumour growth , 2007, Nature Cell Biology.

[46]  M. Mumby.  The 3D structure of protein phosphatase 2A: new insights into a ubiquitous regulator of cell signaling. , 2007, ACS chemical biology.

[47]  Yonghong Xiao,et al.  FoxOs Are Lineage-Restricted Redundant Tumor Suppressors and Regulate Endothelial Cell Homeostasis , 2007, Cell.

[48]  G. Gores,et al.  Transcriptional Regulation of Bim by FoxO3A Mediates Hepatocyte Lipoapoptosis* , 2007, Journal of Biological Chemistry.

[49]  K. Arden.  FoxOs in Tumor Suppression and Stem Cell Maintenance , 2007, Cell.