Bio

Academic Appointments


Honors & Awards


  • Senior Scholar Award, Ellison Medical Foundation (2009-2013)
  • Junior Investigator Award, California Institute for Regenerative Medicine (CIRM) (2008-2013)
  • Innovation in Aging Research Award, Pfizer/American Association for Aging Research (2005-2007)
  • Klingenstein Fellow, The Esther A. & Joseph Klingenstein Fund (2005-2008)
  • Alfred P. Sloan Fellow, Sloan Foundation (2006-2008)
  • Glenn Award, The Glenn Foundation for Medical Research (2007)

Professional Education


  • B.Sc., Ecole Normale Superieure, Paris, Molecular Biology (1992)
  • Ph.D., University of Nice, France, Cell Biology (1997)
  • Postdoctoral fellow, Harvard Medical School, Neuroscience (2003)

Research & Scholarship

Current Research and Scholarly Interests


The overarching goal of our lab is to understand the genetic mechanisms of aging and longevity. Aging is a highly plastic process regulated by a combination of genetic and environmental factors.

We have a long-standing interest in the genetic pathway that connects insulin to FOXO transcription factors, a central pathway to regulate lifespan from worms to humans. We use a combination of genetic, molecular, and cellular approaches to analyze the regulation and importance of FOXO transcription factors, and more generally 'longevity genes' in mammals. We are particularly interested in the role of longevity genes in the maintenance of the pool of adult neural stem cells and intact cognitive function during aging. We also use ultra-high throughput sequencing technologies to study epigenetic changes and transcriptional networks during aging.

In parallel, our goal is to identify novel ‘longevity genes’ using short-lived animal models. Our lab uses unbiased approaches in the nematode C. elegans to identify novel pathways that control organismal longevity, particularly in response to dietary restriction. We are particularly interested in the role of chromatin modifiers in the regulation of lifespan and metabolism.

Finally, we are developing the extremely short-lived African killifish N. furzeri as a new vertebrate model for aging studies. We are taking advantage of this fish to explore the genetic architecture of longevity in vertebrates.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Males Shorten the Life Span of C. elegans Hermaphrodites via Secreted Compounds SCIENCE Maures, T. J., Booth, L. N., Benayoun, B. A., Izrayelit, Y., Schroeder, F. C., Brunet, A. 2014; 343 (6170): 541-544

    Abstract

    How an individual's longevity is affected by the opposite sex is still largely unclear. In the nematode Caenorhabditis elegans, the presence of males accelerated aging and shortened the life span of individuals of the opposite sex (hermaphrodites), including long-lived or sterile hermaphrodites. The male-induced demise could occur without mating and required only exposure of hermaphrodites to medium in which males were once present. Such communication through pheromones or other diffusible substances points to a nonindividual autonomous mode of aging regulation. The male-induced demise also occurred in other species of nematodes, suggesting an evolutionary conserved process whereby males may induce the disposal of the opposite sex to save resources for the next generation or to prevent competition from other males.

    View details for DOI 10.1126/science.1244160

    View details for Web of Science ID 000330343700047

    View details for PubMedID 24292626

  • Expansion of oligodendrocyte progenitor cells following SIRT1 inactivation in the adult brain. Nature cell biology Rafalski, V. A., Ho, P. P., Brett, J. O., Ucar, D., Dugas, J. C., Pollina, E. A., Chow, L. M., Ibrahim, A., Baker, S. J., Barres, B. A., Steinman, L., Brunet, A. 2013; 15 (6): 614-624

    Abstract

    Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor ? (PDGFR?). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFR?. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.

    View details for DOI 10.1038/ncb2735

    View details for PubMedID 23644469

  • FOXO flips the longevity SWItch. Nature cell biology Webb, A. E., Brunet, A. 2013; 15 (5): 444-446

    Abstract

    FOXO transcription factors promote longevity from worms to mammals, but the mechanisms by which FOXO extends lifespan have remained elusive. In the nematode Caenorhabditis elegans, FOXO is now shown to recruit the nucleosome remodelling complex SWI/SNF to its target genes, which is essential for FOXO to elicit stress resistance and longevity.

    View details for DOI 10.1038/ncb2749

    View details for PubMedID 23636422

  • Bridging the transgenerational gap with epigenetic memory TRENDS IN GENETICS Lim, J. P., Brunet, A. 2013; 29 (3): 176-186

    Abstract

    It is textbook knowledge that inheritance of traits is governed by genetics, and that the epigenetic modifications an organism acquires are largely reset between generations. Recently, however, transgenerational epigenetic inheritance has emerged as a rapidly growing field, providing evidence suggesting that some epigenetic changes result in persistent phenotypes across generations. Here, we survey some of the most recent examples of transgenerational epigenetic inheritance in animals, ranging from Caenorhabditis elegans to humans, and describe approaches and limitations to studying this phenomenon. We also review the current body of evidence implicating chromatin modifications and RNA molecules in mechanisms underlying this unconventional mode of inheritance and discuss its evolutionary implications.

    View details for DOI 10.1016/j.tig.2012.12.008

    View details for Web of Science ID 000316243500008

    View details for PubMedID 23410786

  • Aging and reprogramming: a two-way street CURRENT OPINION IN CELL BIOLOGY Mahmoudi, S., Brunet, A. 2012; 24 (6): 744-756

    Abstract

    Aging is accompanied by the functional decline of cells, tissues, and organs, as well as a striking increase in a wide range of diseases. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) opens new avenues for the aging field and has important applications for therapeutic treatments of age-related diseases. Here we review emerging studies on how aging and age-related pathways influence iPSC generation and property. We discuss the exciting possibility that reverting to a pluripotent stem cell stage erases several deficits associated with aging and offers new strategies for rejuvenation. Finally, we argue that reprogramming provides a unique opportunity to model aging and perhaps exceptional longevity.

    View details for DOI 10.1016/j.ceb.2012.10.004

    View details for Web of Science ID 000314743100006

    View details for PubMedID 23146768

  • Chemical Genetic Screen for AMPK alpha 2 Substrates Uncovers a Network of Proteins Involved in Mitosis MOLECULAR CELL Banko, M. R., Allen, J. J., Schaffer, B. E., Wilker, E. W., Tsou, P., White, J. L., Villen, J., Wang, B., Kim, S. R., Sakamoto, K., Gygi, S. P., Cantley, L. C., Yaffe, M. B., Shokat, K. M., Brunet, A. 2011; 44 (6): 878-892

    Abstract

    The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.

    View details for DOI 10.1016/j.molcel.2011.11.005

    View details for Web of Science ID 000298827200007

    View details for PubMedID 22137581

  • Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans NATURE Greer, E. L., Maures, T. J., Ucar, D., Hauswirth, A. G., Mancini, E., Lim, J. P., Benayoun, B. A., Shi, Y., Brunet, A. 2011; 479 (7373): 365-U204

    Abstract

    Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.

    View details for DOI 10.1038/nature10572

    View details for Web of Science ID 000297059700038

    View details for PubMedID 22012258

  • Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging CELL REPORTS Liu, L., Cheung, T. H., Charville, G. W., Hurgo, B. M., Leavitt, T., Shih, J., Brunet, A., Rando, T. A. 2013; 4 (1): 189-204

    Abstract

    The ability to maintain quiescence is critical for the long-term maintenance of a functional stem cell pool. To date, the epigenetic and transcriptional characteristics of quiescent stem cells and how they change with age remain largely unknown. In this study, we explore the chromatin features of adult skeletal muscle stem cells, or satellite cells (SCs), which reside predominantly in a quiescent state in fully developed limb muscles of both young and aged mice. Using a ChIP-seq approach to obtain global epigenetic profiles of quiescent SCs (QSCs), we show that QSCs possess a permissive chromatin state in which few genes are epigenetically repressed by Polycomb group (PcG)-mediated histone 3 lysine 27 trimethylation (H3K27me3), and a large number of genes encoding regulators that specify nonmyogenic lineages are demarcated by bivalent domains at their transcription start sites (TSSs). By comparing epigenetic profiles of QSCs from young and old mice, we also provide direct evidence that, with age, epigenetic changes accumulate and may lead to a functional decline in quiescent stem cells. These findings highlight the importance of chromatin mapping in understanding unique features of stem cell identity and stem cell aging.

    View details for DOI 10.1016/j.celrep.2013.05.043

    View details for Web of Science ID 000321901900018

    View details for PubMedID 23810552

  • Energy metabolism and energy-sensing pathways in mammalian embryonic and adult stem cell fate JOURNAL OF CELL SCIENCE Rafalski, V. A., Mancini, E., Brunet, A. 2012; 125 (23): 5597-5608

    Abstract

    Metabolism is influenced by age, food intake, and conditions such as diabetes and obesity. How do physiological or pathological metabolic changes influence stem cells, which are crucial for tissue homeostasis? This Commentary reviews recent evidence that stem cells have different metabolic demands than differentiated cells, and that the molecular mechanisms that control stem cell self-renewal and differentiation are functionally connected to the metabolic state of the cell and the surrounding stem cell niche. Furthermore, we present how energy-sensing signaling molecules and metabolism regulators are implicated in the regulation of stem cell self-renewal and differentiation. Finally, we discuss the emerging literature on the metabolism of induced pluripotent stem cells and how manipulating metabolic pathways might aid cellular reprogramming. Determining how energy metabolism regulates stem cell fate should shed light on the decline in tissue regeneration that occurs during aging and facilitate the development of therapies for degenerative or metabolic diseases.

    View details for DOI 10.1242/jcs.114827

    View details for Web of Science ID 000315164200002

    View details for PubMedID 23420198

  • Methylation by Set9 modulates FoxO3 stability and transcriptional activity AGING-US Calnan, D. R., Webb, A. E., White, J. L., Stowe, T. R., Goswami, T., Shi, X., Espejo, A., Bedford, M. T., Gozani, O., Gygi, S. P., Brunet, A. 2012; 4 (7): 462-479

    Abstract

    The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

    View details for Web of Science ID 000307474000004

    View details for PubMedID 22820736

  • Aging and the control of the insulin-FOXO signaling pathway M S-MEDECINE SCIENCES Brunet, A. 2012; 28 (3): 316-320

    Abstract

    Aging is a complex process that is accompanied by the onset of a series of age-related diseases, including Alzheimer's disease. Aging is controlled by a combination of genetic and environmental factors. Among the genes that regulate aging, the insulin-FOXO signaling pathway plays a central role, as this pathway regulates lifespan in multiple species, such as worms, flies, and mice. In humans, exceptional longevity - being a centenarian - is also associated with genetic variation in this insulin-FOXO pathway. Recent evidence indicates that the FOXO family of transcription factors plays a key role in the self-renewal of adult and embryonic stem cells, which could contribute to tissue regeneration. Understanding the mechanisms underlying aging should help better prevent and treat age-dependent diseases.

    View details for DOI 10.1051/medsci/2012283021

    View details for Web of Science ID 000303365100021

    View details for PubMedID 22480657

  • Epigenetic memory of longevity in Caenorhabditis elegans. Worm Benayoun, B. A., Brunet, A. 2012; 1 (1): 77-81

    Abstract

    A recent study by Greer et al. in the nematode C. elegans has shown transgenerational epigenetic inheritance of longevity in the descendants of worms deficient for subunits of a complex responsible for histone H3 lysine 4 trimethylation (H3K4me3). In this commentary, we discuss the implications of this epigenetic memory of longevity and the potential mechanisms underlying this phenomenon. The transgenerational inheritance of longevity could result from heritable depletion of H3K4me3 at particular aging-regulating gene loci that would only be progressively replenished. The epigenetic memory of longevity could also be explained by the transgenerational transmission of other molecules, for example other proteins or non-coding RNAs. The discovery of an epigenetic memory of longevity in worms raises the intriguing possibility that environmental cues modulating longevity in ancestors might affect subsequent generations in a non-Mendelian manner. Another remaining intriguing question is whether transgenerational inheritance of longevity also exists in other species, including mammals.

    View details for DOI 10.4161/worm.19157

    View details for PubMedID 24058828

  • Histone methylation makes its mark on longevity TRENDS IN CELL BIOLOGY Han, S., Brunet, A. 2012; 22 (1): 42-49

    Abstract

    How long organisms live is not entirely written in their genes. Recent findings reveal that epigenetic factors that regulate histone methylation, a type of chromatin modification, can affect lifespan. The reversible nature of chromatin modifications suggests that therapeutic targeting of chromatin regulators could be used to extend lifespan and healthspan. This review describes the epigenetic regulation of lifespan in diverse model organisms, focusing on the role and mode of action of chromatin regulators that affect two epigenetic marks, trimethylated lysine 4 of histone H3 (H3K4me3) and trimethylated lysine 27 of histone H3 (H3K27me3), in longevity.

    View details for DOI 10.1016/j.tcb.2011.11.001

    View details for Web of Science ID 000299450400005

    View details for PubMedID 22177962

  • Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging. G3 (Bethesda, Md.) Valenzano, D. R., Sharp, S., Brunet, A. 2011; 1 (7): 531-538

    Abstract

    The African killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in captivity. N. furzeri comprises several wild-derived strains with striking differences in longevity ranging from 3 to 9 months, which makes it a powerful vertebrate model for aging research. The short life cycle of N. furzeri should also facilitate studies on adult traits that are specific to vertebrates. Although progress has been made to generate a genetic linkage map and to start sequencing the genome of N. furzeri, tools to genetically manipulate this species of fish have not yet been developed. Here, we report the first establishment of transgenesis in N. furzeri. We use the Tol2 transposase system to generate transgenic N. furzeri that express green fluorescent protein driven by the Xenopus cytoskeletal actin promoter or the zebrafish heat-shock protein 70 promoter. We successfully generate stable transgenic lines of N. furzeri with germline transmission of integrated transgene. The development of transgenesis in N. furzeri provides a powerful tool to investigate the mechanisms underlying aging and longevity in a short-lived vertebrate model. Transgenesis in this fish will also facilitate the study of other phenotypes, including adult tissue regeneration and cognitive behavior.

    View details for DOI 10.1534/g3.111.001271

    View details for PubMedID 22384364

  • The H3K27 demethylase UTX-1 regulates C. elegans lifespan in a germline-independent, insulin-dependent manner AGING CELL Maures, T. J., Greer, E. L., Hauswirth, A. G., Brunet, A. 2011; 10 (6): 980-990

    Abstract

    Aging is accompanied by alterations in epigenetic marks that control chromatin states, including histone acetylation and methylation. Enzymes that reversibly affect histone marks associated with active chromatin have recently been found to regulate aging in Caenorhabditis elegans. However, relatively little is known about the importance for aging of histone marks associated with repressed chromatin. Here, we use a targeted RNAi screen in C. elegans to identify four histone demethylases that significantly regulate worm lifespan, UTX-1, RBR-2, LSD-1, and T26A5.5. Interestingly, UTX-1 belongs to a conserved family of histone demethylases specific for lysine 27 of histone H3 (H3K27me3), a mark associated with repressed chromatin. Both utx-1 knockdown and heterozygous mutation of utx-1 extend lifespan and increase the global levels of the H3K27me3 mark in worms. The H3K27me3 mark significantly drops in somatic cells during the normal aging process. UTX-1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin-FoxO signaling pathway. These findings identify the H3K27me3 histone demethylase UTX-1 as a novel regulator of worm lifespan in somatic cells.

    View details for DOI 10.1111/j.1474-9726.2011.00738.x

    View details for Web of Science ID 000297003800007

    View details for PubMedID 21834846

  • The pro-longevity gene FoxO3 is a direct target of the p53 tumor suppressor ONCOGENE Renault, V. M., Thekkat, P. U., Hoang, K. L., WHITE, J. L., Brady, C. A., Broz, D. K., Venturelli, O. S., Johnson, T. M., Oskoui, P. R., Xuan, Z., Santo, E. E., Zhang, M. Q., Vogel, H., Attardi, L. D., Brunet, A. 2011; 30 (29): 3207-3221

    Abstract

    FoxO transcription factors have a conserved role in longevity, and act as tissue-specific tumor suppressors in mammals. Several nodes of interaction have been identified between FoxO transcription factors and p53, a major tumor suppressor in humans and mice. However, the extent and importance of the functional interaction between FoxO and p53 have not been fully explored. Here, we show that p53 regulates the expression of FoxO3, one of the four mammalian FoxO genes, in response to DNA damaging agents in both mouse embryonic fibroblasts and thymocytes. We find that p53 transactivates FoxO3 in cells by binding to a site in the second intron of the FoxO3 gene, a genomic region recently found to be associated with extreme longevity in humans. While FoxO3 is not necessary for p53-dependent cell cycle arrest, FoxO3 appears to modulate p53-dependent apoptosis. We also find that FoxO3 loss does not interact with p53 loss for tumor development in vivo, although the tumor spectrum of p53-deficient mice appears to be affected by FoxO3 loss. Our findings indicate that FoxO3 is a p53 target gene, and suggest that FoxO3 and p53 are part of a regulatory transcriptional network that may have an important role during aging and cancer.

    View details for DOI 10.1038/onc.2011.35

    View details for Web of Science ID 000293006800001

    View details for PubMedID 21423206

  • Epigenetic regulation of aging stem cells ONCOGENE Pollina, E. A., Brunet, A. 2011; 30 (28): 3105-3126

    Abstract

    The function of adult tissue-specific stem cells declines with age, which may contribute to the physiological decline in tissue homeostasis and the increased risk of neoplasm during aging. Old stem cells can be 'rejuvenated' by environmental stimuli in some cases, raising the possibility that a subset of age-dependent stem cell changes is regulated by reversible mechanisms. Epigenetic regulators are good candidates for such mechanisms, as they provide a versatile checkpoint to mediate plastic changes in gene expression and have recently been found to control organismal longevity. Here, we review the importance of chromatin regulation in adult stem cell compartments. We particularly focus on the roles of chromatin-modifying complexes and transcription factors that directly impact chromatin in aging stem cells. Understanding the regulation of chromatin states in adult stem cells is likely to have important implications for identifying avenues to maintain the homeostatic balance between sustained function and neoplastic transformation of aging stem cells.

    View details for DOI 10.1038/onc.2011.45

    View details for Web of Science ID 000292726300001

    View details for PubMedID 21441951

  • MicroRNA programs in normal and aberrant stem and progenitor cells GENOME RESEARCH Arnold, C. P., Tan, R., Zhou, B., Yue, S., Schaffert, S., Biggs, J. R., Doyonnas, R., Lo, M., Perry, J. M., Renault, V. M., Sacco, A., Somervaille, T., Viatour, P., Brunet, A., Cleary, M. L., Li, L., Sage, J., Zhang, D., Blau, H. M., Chen, C., Chen, C. 2011; 21 (5): 798-810

    Abstract

    Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ?22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

    View details for DOI 10.1101/gr.111385.110

    View details for Web of Science ID 000290088000018

    View details for PubMedID 21451113

  • A CRTCal Link between Energy and Life Span CELL METABOLISM Brunet, A. 2011; 13 (4): 358-360

    Abstract

    Cutting down calories prolongs life, but how this works remains largely unknown. A recent study in Nature (Mair et al., 2011) shows that life span extension triggered by the energy-sensing protein kinase AMPK is mediated by an evolutionarily conserved transcriptional circuit involving CRTC-1 and CREB.

    View details for DOI 10.1016/j.cmet.2011.03.012

    View details for Web of Science ID 000289381300006

    View details for PubMedID 21459320

  • Energy metabolism in adult neural stem cell fate PROGRESS IN NEUROBIOLOGY Rafalski, V. A., Brunet, A. 2011; 93 (2): 182-203

    Abstract

    The adult mammalian brain contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes and are thought to be involved in certain forms of memory, behavior, and brain injury repair. Neural stem cell properties, such as self-renewal and multipotency, are modulated by both cell-intrinsic and cell-extrinsic factors. Emerging evidence suggests that energy metabolism is an important regulator of neural stem cell function. Molecules and signaling pathways that sense and influence energy metabolism, including insulin/insulin-like growth factor I (IGF-1)-FoxO and insulin/IGF-1-mTOR signaling, AMP-activated protein kinase (AMPK), SIRT1, and hypoxia-inducible factors, are now implicated in neural stem cell biology. Furthermore, these signaling modules are likely to cooperate with other pathways involved in stem cell maintenance and differentiation. This review summarizes the current understanding of how cellular and systemic energy metabolism regulate neural stem cell fate. The known consequences of dietary restriction, exercise, aging, and pathologies with deregulated energy metabolism for neural stem cells and their differentiated progeny will also be discussed. A better understanding of how neural stem cells are influenced by changes in energy availability will help unravel the complex nature of neural stem cell biology in both the normal and diseased state.

    View details for DOI 10.1016/j.pneurobio.2010.10.007

    View details for Web of Science ID 000287950400003

    View details for PubMedID 21056618

  • The MicroRNA Cluster miR-106b similar to 25 Regulates Adult Neural Stem/Progenitor Cell Proliferation and Neuronal Differentiation AGING-US Brett, J. O., Renault, V. M., Rafalski, V. A., Webb, A. E., Brunet, A. 2011; 3 (2): 108-124

    Abstract

    In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.

    View details for Web of Science ID 000288170400008

    View details for PubMedID 21386132

  • Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans NATURE Greer, E. L., Maures, T. J., Hauswirth, A. G., Green, E. M., Leeman, D. S., Maro, G. S., Han, S., Banko, M. R., Gozani, O., Brunet, A. 2010; 466 (7304): 383-U137

    Abstract

    The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

    View details for DOI 10.1038/nature09195

    View details for Web of Science ID 000279867100052

    View details for PubMedID 20555324

  • Mapping Loci Associated With Tail Color and Sex Determination in the Short-Lived Fish Nothobranchius furzeri GENETICS Valenzano, D. R., Kirschner, J., Kamber, R. A., Zhang, E., Weber, D., Cellerino, A., Englert, C., Platzer, M., Reichwald, K., Brunet, A. 2009; 183 (4): 1385-1395

    Abstract

    The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in captivity, with a median life span of 9-11 weeks for the shortest-lived strain. Natural populations of N. furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome involved in sex determination and tail color by genotyping microsatellite markers in the F(2) progeny of a cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and markedly reduced recombination in the male sex-determining region. Our results demonstrate that both naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the genetic architecture of traits that characterize this group of vertebrates, including short life span and adaptation to extreme environmental conditions.

    View details for DOI 10.1534/genetics.109.108670

    View details for Web of Science ID 000272435000016

    View details for PubMedID 19786620

  • FoxO3 Regulates Neural Stem Cell Homeostasis CELL STEM CELL Renault, V. M., Rafalski, V. A., Morgan, A. A., Salih, D. A., Brett, J. O., Webb, A. E., Villeda, S. A., Thekkat, P. U., Guillerey, C., Denko, N. C., Palmer, T. D., Bufte, A. J., Brunet, A. 2009; 5 (5): 527-539

    Abstract

    In the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3(-/-) mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3(-/-) mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species.

    View details for DOI 10.1016/j.stem.2009.09.014

    View details for Web of Science ID 000272019500014

    View details for PubMedID 19896443

  • CANCER When restriction is good NATURE Brunet, A. 2009; 458 (7239): 713-714

    View details for DOI 10.1038/458713a

    View details for Web of Science ID 000265193600026

    View details for PubMedID 19360073

  • Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C-elegans AGING CELL Greer, E. L., Brunet, A. 2009; 8 (2): 113-127

    Abstract

    Dietary restriction (DR) has the remarkable ability to extend lifespan and healthspan. A variety of DR regimens have been described in species ranging from yeast to mammals. However, whether different DR regimens extend lifespan via universal, distinct, or overlapping pathways is still an open question. Here we examine the genetic pathways that mediate longevity by different DR regimens in Caenorhabditis elegans. We have previously shown that the low-energy sensing AMP-activated protein kinase AMPK/aak-2 and the Forkhead transcription factor FoxO/daf-16 are necessary for longevity induced by a DR regimen that we developed (sDR). Here we find that AMPK and FoxO are necessary for longevity induced by another DR regimen, but are dispensable for the lifespan extension induced by two different DR methods. Intriguingly, AMPK is also necessary for the lifespan extension elicited by resveratrol, a natural polyphenol that mimics some aspects of DR. Conversely, we test if genes previously reported to mediate longevity by a variety of DR methods are necessary for sDR-induced longevity. Although clk-1, a gene involved in ubiquinone biosynthesis, is also required for sDR-induced lifespan extension, we find that four other genes (sir-2.1, FoxA/pha-4, skn-1, and hsf-1) are all dispensable for longevity induced by sDR. Consistent with the observation that different DR methods extend lifespan by mostly independent genetic mechanisms, we find that the effects on lifespan of two different DR regimens are additive. Understanding the genetic network by which different DR regimens extend lifespan has important implications for harnessing the full benefits of DR on lifespan and healthspan.

    View details for DOI 10.1111/j.1474-9726.2009.00459.x

    View details for Web of Science ID 000264607200004

    View details for PubMedID 19239417

  • AMP-activated Protein Kinase and FoxO Transcription Factors in Dietary Restriction-induced Longevity INTERNATIONAL SYMPOSIUM ON OLFACTION AND TASTE Greer, E. L., Banko, M. R., Brunet, A. 2009; 1170: 688-692

    Abstract

    Aging is regulated by modifications in single genes and by simple changes in the environment. The signaling pathway connecting insulin to FoxO transcription factors integrates environmental stimuli to regulate lifespan. FoxO transcription factors are directly phosphorylated in response to insulin/growth factor signaling by the protein kinase Akt, thereby causing their sequestration in the cytoplasm. In the absence of insulin/growth factors, FoxO factors translocate to the nucleus where they trigger a range of cellular responses, including resistance to oxidative stress--a phenotype highly coupled with lifespan extension. Our recent results indicate that FoxO transcription factors are also regulated in response to nutrient deprivation by the AMP-activated protein kinase (AMPK) pathway. The energy-sensing AMPK directly phosphorylates FoxO transcription factors at six regulatory sites. AMPK phosphorylation enhances FoxO transcriptional activity, leading to the expression of specific target genes involved in stress resistance and changes in energy metabolism. The AMPK-FoxO pathway plays a crucial role in the ability of a dietary restriction regimen to extend lifespan in Caenorhabditis elegans. Understanding the intricate signaling networks that translate environmental conditions like dietary restriction into changes in gene expression that extend lifespan will be of critical importance to identify ways to delay the onset of aging and age-dependent diseases.

    View details for DOI 10.1111/j.1749-6632.2009.04019.x

    View details for Web of Science ID 000270495700118

    View details for PubMedID 19686213

  • The FoxO code ONCOGENE Calnan, D. R., Brunet, A. 2008; 27 (16): 2276-2288

    Abstract

    The FoxO family of Forkhead transcription factors plays an important role in longevity and tumor suppression by upregulating target genes involved in stress resistance, metabolism, cell cycle arrest and apoptosis. FoxO transcription factors translate a variety of environmental stimuli, including insulin, growth factors, nutrients and oxidative stress, into specific gene-expression programs. These environmental stimuli control FoxO activity primarily by regulating their subcellular localization, but also by affecting their protein levels, DNA-binding properties and transcriptional activity. The precise regulation of FoxO transcription factors is enacted by an intricate combination of post-translational modifications (PTMs), including phosphorylation, acetylation and ubiquitination, and binding protein partners. An intriguing possibility is that FoxO PTMs may act as a 'molecular FoxO code' read by selective protein partners to rapidly regulate gene-expression programs. The effective control of FoxO activity in response to environmental stimuli is likely to be critical to prevent aging and age-dependent diseases, including cancer, neurodegenerative diseases and diabetes.

    View details for DOI 10.1038/onc.2008.21

    View details for Web of Science ID 000254782700003

    View details for PubMedID 18391970

  • FoxO transcription factors in the maintenance of cellular homeostasis during aging CURRENT OPINION IN CELL BIOLOGY Salih, D. A., Brunet, A. 2008; 20 (2): 126-136

    Abstract

    The FoxO family of Forkhead transcription factors functions at the interface of tumor suppression, energy metabolism, and organismal longevity. FoxO factors are key downstream targets of insulin, growth factor, nutrient, and oxidative stress stimuli that coordinate a wide range of cellular outputs. FoxO-dependent cellular responses include gluconeogenesis, neuropeptide secretion, atrophy, autophagy, apoptosis, cell cycle arrest, and stress resistance. This review will discuss the roles of the mammalian FoxO family in a variety of cell types, from stem cells to mature cells, in the context of the whole organism. Given the overwhelming evidence that the FoxO factors promote longevity in invertebrates, this review will also discuss the potential role of the FoxO factors in the aging of mammalian organisms.

    View details for DOI 10.1016/j.ceb.2008.02.005

    View details for Web of Science ID 000255546800003

    View details for PubMedID 18394876

  • Signaling networks in aging JOURNAL OF CELL SCIENCE Greer, E. L., Brunet, A. 2008; 121 (4): 407-412

    View details for DOI 10.1242/jcs.021519

    View details for Web of Science ID 000253425800001

    View details for PubMedID 18256383

  • FOXO transcription factors in ageing and cancer ACTA PHYSIOLOGICA Greer, E. L., Brunet, A. 2008; 192 (1): 19-28

    Abstract

    Ageing is associated with an increased onset of cancer. Understanding the molecular mechanisms that underlie the age dependency of cancer will have important implications for preventing and treating this pathology. The signalling pathway connecting insulin and FOXO transcription factors provides the most compelling example for a conserved genetic pathway at the interface between ageing and cancer. FOXO transcription factors (FOXO) promote longevity and tumour suppression. FOXO transcription factors are directly phosphorylated in response to insulin/growth factor signalling by the protein kinase Akt, thereby causing their sequestration in the cytoplasm. In the absence of insulin/growth factors, FOXO factors translocate to the nucleus where they trigger a range of cellular responses, including resistance to oxidative stress, a phenotype highly coupled with lifespan extension. FOXO factors integrate stress stimuli via phosphorylation, acetylation and mono-ubiquitination of a series of regulatory sites. Understanding how FOXO proteins integrate environmental conditions to control specific gene expression programmes will be pivotal in identifying ways to slow the onset of cancer in ageing individuals.

    View details for DOI 10.1111/j.1748-1716.2007.01780.x

    View details for Web of Science ID 000251667000004

    View details for PubMedID 18171426

  • Aging and cancer: killing two birds with one worm NATURE GENETICS Brunet, A. 2007; 39 (11): 1306-1307

    View details for Web of Science ID 000250575900006

    View details for PubMedID 17968345

  • The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor JOURNAL OF BIOLOGICAL CHEMISTRY Greer, E. L., Oskoui, P. R., Banko, M. R., Maniar, J. M., Gygi, M. P., Gygi, S. P., Brunet, A. 2007; 282 (41): 30107-30119

    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.

    View details for DOI 10.1074/jbc.M705325200

    View details for Web of Science ID 000249981200041

    View details for PubMedID 17711846

  • An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C-elegans CURRENT BIOLOGY Greer, E. L., Dowlatshahi, D., Banko, M. R., Villen, J., Hoang, K., Blanchard, D., Gygi, S. P., Brunet, A. 2007; 17 (19): 1646-1656

    Abstract

    Dietary restriction (DR) is the most effective environmental intervention to extend lifespan in a wide range of species. However, the molecular mechanisms underlying the benefits of DR on longevity are still poorly characterized. AMP-activated protein kinase (AMPK) is activated by a decrease in energy levels, raising the possibility that AMPK might mediate lifespan extension by DR.By using a novel DR assay that we developed and validated in C. elegans, we find that AMPK is required for this DR method to extend lifespan and delay age-dependent decline. We find that AMPK exerts its effects in part via the FOXO transcription factor DAF-16. FOXO/DAF-16 is necessary for the beneficial effects of this DR method on lifespan. Expression of an active version of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent manner. Lastly, we find that AMPK activates FOXO/DAF-16-dependent transcription and phosphorylates FOXO/DAF-16 at previously unidentified sites, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK.Our study shows that an energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans.

    View details for DOI 10.1016/j.cub.2007.08.047

    View details for Web of Science ID 000250125200023

    View details for PubMedID 17900900

  • Ageing - From stem to stern NATURE Brunet, A., Rando, T. A. 2007; 449 (7160): 288-?

    View details for DOI 10.1038/449288a

    View details for Web of Science ID 000249557300030

    View details for PubMedID 17882206

  • FOXO transcription factors CURRENT BIOLOGY Carter, M. E., Brunet, A. 2007; 17 (4): R113-R114

    View details for Web of Science ID 000244463100006

    View details for PubMedID 17307039

  • FOXO transcription factors at the interface between longevity and tumor suppression ONCOGENE Greer, E. L., Brunet, A. 2005; 24 (50): 7410-7425

    Abstract

    A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.

    View details for DOI 10.1038/sj.onc.1209086

    View details for Web of Science ID 000233201900004

    View details for PubMedID 16288288

  • Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase SCIENCE Brunet, A., Sweeney, L. B., Sturgill, J. F., Chua, K. F., Greer, P. L., Lin, Y. X., Tran, H., Ross, S. E., Mostoslavsky, R., Cohen, H. Y., Hu, L. S., Cheng, H. L., Jedrychowski, M. P., Gygi, S. P., Sinclair, D. A., Alt, F. W., Greenberg, M. E. 2004; 303 (5666): 2011-2015

    Abstract

    The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

    View details for DOI 10.1126/science.1094637

    View details for Web of Science ID 000220429800040

    View details for PubMedID 14976264

  • PEA-15 binding to ERK1/2 MAPKs is required for its modulation of integrin activation JOURNAL OF BIOLOGICAL CHEMISTRY Chou, F. L., Hill, J. M., Hsieh, J. C., Pouyssegur, J., Brunet, A., Glading, A., Uberall, F., Ramos, J. W., Werner, M. H., Ginsberg, M. H. 2003; 278 (52): 52587-52597

    Abstract

    Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the alphaG helix and within the mitogen-activated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.

    View details for DOI 10.1074/jbc.M309322200

    View details for Web of Science ID 000187480700077

    View details for PubMedID 14506247

  • The many forks in FOXO's road. Science's STKE : signal transduction knowledge environment Tran, H., Brunet, A., Griffith, E. C., Greenberg, M. E. 2003; 2003 (172): RE5-?

    Abstract

    The FOXO family of transcription factors constitute an evolutionarily conserved subgroup within the larger family known as winged helix or Forkhead transcriptional regulators. Building upon work in the nematode, researchers have uncovered a role for these proteins in a diverse set of cellular responses that include glucose metabolism, stress response, cell cycle regulation, and apoptosis. At the organismal level, FOXO transcription factors are believed to function in various pathological processes ranging from cancer and diabetes to organismal aging. A number of studies have also shed light on the signaling pathways that regulate FOXO activity in response to external stimuli and have identified multiple FOXO target genes that mediate this varied set of biological responses.

    View details for PubMedID 12621150

  • DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein SCIENCE Tran, H., Brunet, A., Grenier, J. M., Datta, S. R., Fornace, A. J., DiStefano, P. S., Chiang, L. W., Greenberg, M. E. 2002; 296 (5567): 530-534

    Abstract

    The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. We show that mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appeared to be a direct target of FOXO3a that mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span.

    View details for Web of Science ID 000175179400046

    View details for PubMedID 11964479

  • 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport JOURNAL OF CELL BIOLOGY Brunet, A., Kanai, F., Stehn, J., Xu, J., Sarbassova, D., Frangioni, J. V., Dalal, S. N., DeCaprio, J. A., Greenberg, M. E., Yaffe, M. B. 2002; 156 (5): 817-828

    Abstract

    14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal alpha-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.

    View details for DOI 10.1083/jcb.200112059

    View details for Web of Science ID 000176426300007

    View details for PubMedID 11864996

  • Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1 MOLECULAR BIOLOGY OF THE CELL Shin, I., Bakin, A. V., Rodeck, U., Brunet, A., ARTEAGA, C. L. 2001; 12 (11): 3328-3339

    Abstract

    The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

    View details for Web of Science ID 000172357200003

    View details for PubMedID 11694570

  • Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway CURRENT OPINION IN NEUROBIOLOGY Brunet, A., Datta, S. R., Greenberg, M. E. 2001; 11 (3): 297-305

    Abstract

    The PI3K-Akt signaling pathway plays a critical role in mediating survival signals in a wide range of neuronal cell types. The recent identification of a number of substrates for the serine/threonine kinase Akt suggests that it blocks cell death by both impinging on the cytoplasmic cell death machinery and by regulating the expression of genes involved in cell death and survival. In addition, recent experiments suggest that Akt may also use metabolic pathways to regulate cell survival.

    View details for Web of Science ID 000169286200005

    View details for PubMedID 11399427

  • Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a) MOLECULAR AND CELLULAR BIOLOGY Brunet, A., Park, J., Tran, H., Hu, L. S., HEMMINGS, B. A., Greenberg, M. E. 2001; 21 (3): 952-965

    Abstract

    Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

    View details for Web of Science ID 000166353700025

    View details for PubMedID 11154281

  • Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3 JOURNAL OF BIOLOGICAL CHEMISTRY Nichols, A., Camps, M., Gillieron, C., Chabert, C., Brunet, A., Wilsbacher, J., Cobb, M., Pouyssegur, J., Shaw, J. P., Arkinstall, S. 2000; 275 (32): 24613-24621

    Abstract

    Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

    View details for Web of Science ID 000088683300053

    View details for PubMedID 10811804

  • Cellular survival: a play in three Akts GENES & DEVELOPMENT Datta, S. R., Brunet, A., Greenberg, M. E. 1999; 13 (22): 2905-2927

    View details for Web of Science ID 000084048600001

    View details for PubMedID 10579998

  • Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms SCIENCE Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., Greenberg, M. E. 1999; 286 (5443): 1358-1362

    Abstract

    A mechanism by which the Ras-mitogen-activated protein kinase (MAPK) signaling pathway mediates growth factor-dependent cell survival was characterized. The MAPK-activated kinases, the Rsks, catalyzed the phosphorylation of the pro-apoptotic protein BAD at serine 112 both in vitro and in vivo. The Rsk-induced phosphorylation of BAD at serine 112 suppressed BAD-mediated apoptosis in neurons. Rsks also are known to phosphorylate the transcription factor CREB (cAMP response element-binding protein) at serine 133. Activated CREB promoted cell survival, and inhibition of CREB phosphorylation at serine 133 triggered apoptosis. These findings suggest that the MAPK signaling pathway promotes cell survival by a dual mechanism comprising the posttranslational modification and inactivation of a component of the cell death machinery and the increased transcription of pro-survival genes.

    View details for Web of Science ID 000083675500042

    View details for PubMedID 10558990

  • Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor CELL Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., ANDERSON, M. J., Arden, K. C., Blenis, J., Greenberg, M. E. 1999; 96 (6): 857-868

    Abstract

    Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.

    View details for Web of Science ID 000079300100012

    View details for PubMedID 10102273

  • Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry EMBO JOURNAL Brunet, A., Roux, D., Lenormand, P., Dowd, S., Keyse, S., Pouyssegur, J. 1999; 18 (3): 664-674

    Abstract

    Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry.

    View details for Web of Science ID 000078597500017

    View details for PubMedID 9927426

  • Growth factor-induced p42/p44 MAPK nuclear translocation and retention requires both MAPK activation and neosynthesis of nuclear anchoring proteins JOURNAL OF CELL BIOLOGY Lenormand, P., Brondello, J. M., Brunet, A., Pouyssegur, J. 1998; 142 (3): 625-633

    Abstract

    Mitogen-activated protein kinases (p42/p44 MAPK, also called Erk2 and Erk1) are key mediators of signal transduction from the cell surface to the nucleus. We have previously shown that the activation of p42/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear translocation of these kinases. However, the means by which p42 and p44 MAPK translocate into the nucleus after cytoplasmic activation is still not understood and cannot simply be deduced from their protein sequences. In this study, we have demonstrated that activation of the p42/ p44 MAPK pathway was necessary and sufficient for triggering nuclear translocation of p42 and p44 MAPK. First, addition of the MEK inhibitor PD 98059, which blocks activation of the p42/p44 MAPK pathway, impedes the nuclear accumulation, whereas direct activation of the p42/p44 MAPK pathway by the chimera DeltaRaf-1:ER is sufficient to promote nuclear accumulation of p42/p44 MAPK. In addition, we have shown that this nuclear accumulation of p42/p44 MAPK required the neosynthesis of short-lived proteins. Indeed, inhibitors of protein synthesis abrogate nuclear accumulation in response to serum and accelerate p42/p44 MAPK nuclear efflux under conditions of persistent p42/p44 MAPK activation. In contrast, inhibition of targeted proteolysis by the proteasome synergistically potentiated p42/p44 MAPK nuclear localization by nonmitogenic agonists and markedly prolonged nuclear localization of p42/p44 MAPK after mitogenic stimulation. We therefore conclude that the MAPK nuclear translocation requires both activation of the p42/p44 MAPK module and neosynthesis of short-lived proteins that we postulate to be nuclear anchors.

    View details for Web of Science ID 000075415700003

    View details for PubMedID 9700154

  • Signal transduction pathways from the membrane to the nucleus: variations on common themes BULLETIN DU CANCER Brunet, A. 1998; 85 (6): 527-537

    Abstract

    During their life, cells are exposed to a wide variety of extracellular stimuli and have to develop appropriate biological responses. Signal transduction from the plasma membrane, which is in contact with the extracellular environment, to the nucleus, where gene expression is achieved, thus represents a fundamental process for the development and maintenance of life in organisms. Signalling pathways are extremely diverse and range from direct strategies, such as the steroid hormone receptor and JAK/STAT (signal transducers and activators of transcription) pathways, to multi-step strategies, such as the NF-kappa B (nuclear factor kappa B), PKA (protein kinase A) and Ras/MAPK (mitogen-activated protein kinase) pathways. In order to modulate gene expression, all these pathways must ultimately achieve nuclear localization. The mechanisms by which these varied signalling components cross the nuclear envelope are equally as diverse. However, despite the variety of the means used, cells have adopted several common themes for signal transduction, particularly interaction between proteins as a mean to transport the signal and phosphorylation as a post-translational modification carrying information. Finally, all signalling pathways have been conserved throughout evolution, inghlighting their advantage for cells. In mammals, proteins that participate in signal transmission represent a frequent target for mutations leading to tumor development. Unraveling signalling pathways thus represents an important step in the fight against cancer.

    View details for Web of Science ID 000074999500006

    View details for PubMedID 9752280

  • Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation JOURNAL OF BIOLOGICAL CHEMISTRY Englaro, W., Bertolotto, C., Busca, R., Brunet, A., Pages, G., Ortonne, J. P., Ballotti, R. 1998; 273 (16): 9966-9970

    Abstract

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.

    View details for Web of Science ID 000073128800087

    View details for PubMedID 9545341

  • Involvement of extracellular signal-regulated kinase module in HIV-mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors JOURNAL OF IMMUNOLOGY Briant, L., Robert-Hebmann, V., Sivan, V., Brunet, A., Pouyssegur, J., Devaux, C. 1998; 160 (4): 1875-1885

    Abstract

    Although the molecular mechanisms by which the HIV-1 triggers either T cell activation, anergy, or apoptosis remain poorly understood, it is well established that the interaction of HIV-1 envelope glycoproteins with cell surface CD4 delivers signals to the target cell, resulting in activation of transcription factors such as NF-kappa B and AP-1. In this study, we report the first evidence indicating that kinases MEK-1 (MAP kinase/Erk kinase) and ERK-1 (extracellular signal-regulated kinase) act as intermediates in the cascade of events that regulate NF-kappa B and AP-1 activation upon HIV-1 binding to cell surface CD4. We found that CEM cells transfected with dominant negative forms of MEK-1 or ERK-1 do not display NF-kappa B activation after HIV-1 binding to CD4. In contrast, NF-kappa B activation was observed in these cells after PMA stimulation. Although the different cell lines studied expressed similar amounts of CD4 and p56(lck), HIV-1 replication and HIV-1-induced apoptosis were slightly delayed in cells expressing dominant negative forms of MEK-1 or ERK-1 compared with parental CEM cells and cells expressing a constitutively active mutant form of MEK-1 or wild-type ERK-1. In light of recently published data, we propose that a positive signal initiated following oligomerization of CD4 by the virus is likely to involve a recruitment of active forms of p56(lck), Raf-1, MEK-1, and ERK-1, before AP-1 and NF-kappa B activation.

    View details for Web of Science ID 000073704800042

    View details for PubMedID 9469449

  • The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44(MAPK) cascade JOURNAL OF BIOLOGICAL CHEMISTRY Brondello, J. M., Brunet, A., Pouyssegur, J., McKenzie, F. R. 1997; 272 (2): 1368-1376

    Abstract

    Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and MKP-2 are two members of a recently described family of dual specificity phosphatases that are capable of dephosphorylating p42/p44MAPK. Overexpression of MKP-1 or MKP-2 inhibits MAP kinase-dependent intracellular signaling events and fibroblast proliferation. By using specific antibodies that recognize endogenous MKP-1 and MKP-2 in CCL39 cells, we show that MKP-1 and MKP-2 are not expressed in quiescent cells, but are rapidly induced following serum addition, with protein detectable as early as 30 min (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 and MKP-2 is sustained, with protein detectable up to 14 h after serum addition. Induction of MKP-1 and, to a lesser extent, MKP-2 temporally correlates with p42/p44MAPK inactivation. To analyze the contribution of the MAP kinase cascade to MKP-1 and MKP-2 induction, we examined CCL39 cells transformed with either v-ras or a constitutively active direct upstream activator of MAP kinase, mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and MKP-2 are constitutively expressed, with MKP-2 being prevalent. In addition, in CCL39 cells expressing an estradiol-inducible deltaRaf-1::ER chimera, activation of Raf alone is sufficient to induce MKP-1 and MKP-2. The role of the MAP kinase cascade in MKP induction was highlighted by the MKK-1 inhibitor PD 098059, which blunted both the activation of p42/p44MAPK and the induction of MKP-1 and MKP-2. However, the MAP kinase cascade is not absolutely required for the induction of MKP-1, as this phosphatase, but not MKP-2, was induced to detectable levels by agents that stimulate protein kinases A and C. Thus, activation of the p42/p44MAPK cascade promotes the induction of MKP-1 and MKP-2, which may then attenuate p42/p44MAPK-dependent events in an inhibitory feedback loop.

    View details for Web of Science ID A1997WC04800097

    View details for PubMedID 8995446

  • Mammalian MAP kinase modules: how to transduce specific signals ESSAYS IN BIOCHEMISTRY, VOL 32, 1997 Brunet, A., Pouyssegur, J. 1997; 32: 1-16

    Abstract

    MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events. They are used to transduce a variety of information in organisms as diverse as yeasts, worms, flies or mammals. MAPK modules integrate signals coming from membrane receptor activation and, by the ability of MAPK to translocate into the nucleus and phosphorylate nuclear targets such as transcription factors, they relay extracellular signals into a genomic response. Since several MAPK modules transducing different information are expressed in the same cell, in yeast or in mammals, the question arises as to how fidelity is maintained between the distinct MAPK modules of a single cell. Two levels of specificity have been documented: the molecular selectivity of each enzyme for its substrate, which is particularly evident for the MKK-MAPK couple, permits specificity within one particular module; exogenous proteins, such as the yeast Ste5 protein, may serve as 'chaperone' proteins to tether all the members of a module and restrict signal transduction to this module. In mammalian cells, the MAPK modules are not strictly independent and one pathway may interfere with another. It remains to be determined whether this interference is of physiological relevance.

    View details for Web of Science ID 000072180100001

    View details for PubMedID 9493007

  • Cyclin D1 expression is regulated positively by the p42/p44(MAPK) and negatively by the p38/HOG(MAPK) pathway JOURNAL OF BIOLOGICAL CHEMISTRY Lavoie, J. N., LALLEMAIN, G., Brunet, A., Muller, R., Pouyssegur, J. 1996; 271 (34): 20608-20616

    Abstract

    We have previously shown that the persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts (Pagès, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouysségur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323) and postulated that MAPKs control the activation of G1 cyclin-dependent complexes. We examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression were compared in CCL39 cells, using a co-transfection procedure. We found that inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibited expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increased cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) revealed that in absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade showed an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3) together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression was observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhanced cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities.

    View details for Web of Science ID A1996VD33700059

    View details for PubMedID 8702807

  • Identification of MAP kinase domains by redirecting stress signals into growth factor responses SCIENCE Brunet, A., Pouyssegur, J. 1996; 272 (5268): 1652-1655

    Abstract

    Mitogen-activated protein kinase (MAPK) cascades, termed MAPK modules, channel extracellular signals into specific cellular responses. Chimeric molecules were constructed between p38 and p44 MAPKs, which transduce stress and growth factor signals, respectively. A discrete region of 40 residues located in the amono-terminal p38MAPK lobe directed the specificity of response to extracellular signals, whereas the p44MAPK chimera, expressed in vivo, redirected stress signals into early mitogenic responses, demonstrating the functional independence of these domains.

    View details for Web of Science ID A1996UR09300047

    View details for PubMedID 8658140

  • THE MOUSE P44 MITOGEN-ACTIVATED PROTEIN-KINASE (EXTRACELLULAR SIGNAL-REGULATED KINASE-1) GENE - GENOMIC ORGANIZATION AND STRUCTURE OF THE 5'-FLANKING REGULATORY REGION JOURNAL OF BIOLOGICAL CHEMISTRY Pages, G., Stanley, E. R., Le Gall, M., Brunet, A., Pouyssegur, J. 1995; 270 (45): 26986-26992

    Abstract

    Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1995TE58300046

    View details for PubMedID 7592946

  • B-RAF PROTEIN ISOFORMS INTERACT WITH AND PHOSPHORYLATE MEK-1 ON SERINE RESIDUE-218 AND RESIDUE-222 ONCOGENE Papin, C., Eychene, A., Brunet, A., Pages, G., Pouyssegur, J., Calothy, G., Barnier, J. V. 1995; 10 (8): 1647-1651

    Abstract

    The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

    View details for Web of Science ID A1995QU68100022

    View details for PubMedID 7731720

  • [MAP kinase module: role in the control of cell proliferation]. Comptes rendus des séances de la Société de biologie et de ses filiales Brunet, A., Brondello, J. M., L'Allemain, G., Lenormand, P., McKenzie, F., Pagès, G., Pouysségur, J. 1995; 189 (1): 43-57

    Abstract

    A kinase cascade highly conserved throughout evolution, Raf/MAP kinase kinase kinase (MAPKKK)-->MAP kinase kinase (MAPKK)-->MAP kinase (MAPK)-->ribosomal S6 kinase (p90 RSK), is thought to play a crucial role in signal transduction from the membrane to the nucleus. In mammalian cells, this cascade is connected both to tyrosine kinase receptors and G protein-coupled receptors. Although the mode of activation at the receptor level differs, all mitogens activate the ubiquitously expressed isoforms of MAPK, p42 and p44. We have cloned, epitope tagged and expressed in fibroblasts, the Hamster MAPKK and p44 MAPK in order to analyze their time-course of activation, their subcellular localization, their regulatory phosphorylation sites and their role in cell cycle entry. We have demonstrated that MAPK activation was rapid, biphasic and persistent. The sustained phase of activation is only obtained with potent mitogenic agents, correlating with their ability to elicit cell cycle entry. Activation of MAPKK is also rapid and persistent but does not distinguish between mitogenic and non mitogenic factors, indicating that a distinction occurs at the MAPK level, probably by the action of specific phosphatases such as MAPK phosphatase MKP-1. Both isoforms of MAPK are translocated into the nucleus upon growth factor addition whereas the upstream activators (MAPKKK, Raf and MAPKK) remain cytoplasmic. MAPK translocation, together with the ability of MAPK to phosphorylate transcription factors, indicates that MAPK might constitute a relay between cytoplasmic and nuclear events. Finally we show that interfering with the MAP kinase cascade, by expressing either MAPK antisense, a MAPK dominant negative mutant or the MAPK specific phosphatase, MKP-1, suppresses the growth factor induced G0 to G1 transition. In addition, permanently activated versions of MAPKK reduce growth factor requirement, allow autonomous cell growth and induce tumor formation in nude mice. We therefore conclude that MAP kinase activation is both necessary and sufficient to trigger cell cycle entry.

    View details for PubMedID 7648366

  • CONSTITUTIVELY ACTIVE MUTANTS OF MAP KINASE KINASE (MEK1) INDUCE GROWTH FACTOR-RELAXATION AND ONCOGENICITY WHEN EXPRESSED IN FIBROBLASTS ONCOGENE Brunet, A., Pages, G., Pouyssegur, J. 1994; 9 (11): 3379-3387

    Abstract

    The Mitogen Activated Protein Kinase (MAPK) module operates downstream of Ras to convey cell surface signals to the nucleus via the nuclear translocation of p42/p44 MAPKs. We have previously established that MAPK activation is obligatory and must persist in the G1 phase to allow resting fibroblasts to exit from G0 (Pagès et al., Proc. Natl. Acad. Sci.1993, 90, 8319-8323). It remained to be established whether MAPK activation was sufficient to trigger cell proliferation. To this aim, we generated and expressed in Chinese hamster lung fibroblasts, constitutively active mutants of hamster MAP kinase kinase (MAPKK). Three mutants: S218D, S222D and S218D/S222D in which we substituted the Raf1/MAPKKK-dependent regulatory phosphorylation sites by aspartic acid residues, displayed increased basal activity when expressed in fibroblasts. Two of them, S218D and S218D/S222D which have a basal activity higher than serum-stimulated wild type-MAPKK (respectively 2- and 5-fold), induced activation of p42 MAPK in growth factor-deprived cells. Interestingly, only these two mutants led to a growth factor-independent state as judged by early gene transcription (activation of the fos promoter), increased sensitivity to growth factors for reinitiation of DNA synthesis, autonomous cell cycling and rapid tumor formation in nude mice. Therefore we conclude that the downstream elements of the growth factor signalling cascade, MAPKK-MAPK, are both necessary and sufficient to promote growth factor signals and autonomous cell cycling in fibroblasts.

    View details for Web of Science ID A1994PM65800035

    View details for PubMedID 7936666

  • CONSTITUTIVE MUTANT AND PUTATIVE REGULATORY SERINE PHOSPHORYLATION SITE OF MAMMALIAN MAP KINASE KINASE (MEK1) EMBO JOURNAL Pages, G., Brunet, A., LALLEMAIN, G., Pouyssegur, J. 1994; 13 (13): 3003-3010

    Abstract

    In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.

    View details for Web of Science ID A1994NW77100007

    View details for PubMedID 8039496

  • GROWTH FACTOR-STIMULATED MAP KINASE INDUCES RAPID RETROPHOSPHORYLATION AND INHIBITION OF MAP KINASE KINASE (MEK1) FEBS LETTERS Brunet, A., Pages, G., Pouyssegur, J. 1994; 346 (2-3): 299-303

    Abstract

    The MAP kinase module (Raf/MAPKKK-MAPKK-MAPK) has been shown to be sequentially activated after mitogenic stimulation. Here we demonstrate, by site directed mutagenesis, that MAPK is able to retrophosphorylate its own activator, MAPKK, on two threonine residues Thr-292 and Thr-386 in vitro, and that these sites are also phosphorylated in vivo. A comparison of the kinetics of serum-mediated activation of a wild-type MAPKK and of a mutant unable to undergo phosphorylation by MAPK suggests that this retrophosphorylation may be involved in a negative feedback control of the cascade in vivo.

    View details for Web of Science ID A1994NR56900034

    View details for PubMedID 8013650

  • GROWTH-FACTORS INDUCE NUCLEAR TRANSLOCATION OF MAP KINASES (P42(MAPK) AND P44(MAPK)) BUT NOT OF THEIR ACTIVATOR MAP KINASE KINASE (P45(MAPKK)) IN FIBROBLASTS JOURNAL OF CELL BIOLOGY Lenormand, P., Sardet, C., Pages, G., LALLEMAIN, G., Brunet, A., Pouyssegur, J. 1993; 122 (5): 1079-1088

    Abstract

    Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.

    View details for Web of Science ID A1993LU31000010

    View details for PubMedID 8394845

  • MAP KINASE CASCADE - AN ESSENTIAL SIGNALING ROUTE THAT CONTROLS CELL-PROLIFERATION NEGATIVE REGULATION OF HEMATOPOIESIS Pouyssegur, J., Brunet, A., CHAMBARD, J. C., LALLEMAIN, G., Lenormand, P., Pages, G. 1993; 229: 55-63
  • MAP KINASE CASCADE AND THE CONTROL OF CELL-PROLIFERATION MOLECULAR ONCOLOGY AND CLINICAL APPLICATIONS Pages, G., Brunet, A., CHAMBARD, J. C., LALLEMAIN, G., Lenormand, P., Pouyssegur, J. 1993: 153-164

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