DOCSLIB.ORG

The Role of PI3K/AKT/Mtor Signalling in Pluripotency and Cell Fate

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Role of PI3K/AKT/Mtor Signalling in Pluripotency and Cell Fate © 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 3050-3060 doi:10.1242/dev.137075 REVIEW Proliferation, survival and metabolism: the role of PI3K/AKT/ mTOR signalling in pluripotency and cell fate determination Jason S. L. Yu and Wei Cui* ABSTRACT The embryonic lethality that results from disruption of PI3K/ Phosphatidylinositide 3 kinases (PI3Ks) and their downstream AKT/mTOR signalling hinders the interrogation of this pathway mediators AKT and mammalian target of rapamycin (mTOR) during differentiation through traditional developmental models. constitute the core components of the PI3K/AKT/mTOR signalling Derivation of pluripotent stem cells (PSCs), including embryonic cascade, regulating cell proliferation, survival and metabolism. stem cells (ESCs) and induced PSCs (iPSCs), has mitigated this Although these functions are well-defined in the context of problem to some extent. These cells can undergo self-renewal tumorigenesis, recent studies – in particular those using pluripotent indefinitely, and can also give rise to all the cells of the adult stem cells – have highlighted the importance of this pathway to organism, providing researchers with a unique cell-based model to development and cellular differentiation. Here, we review the recent interrogate cellular differentiation. This, coupled with increasingly in vitro and in vivo evidence for the role PI3K/AKT/mTOR signalling defined culture conditions and better tools for genetic manipulation, plays in the control of pluripotency and differentiation, with a particular has allowed researchers to overcome the technically challenging focus on the molecular mechanisms underlying these functions. interrogation of the molecular basis for crosstalk interactions. As a result, recent studies have begun to delineate the contribution of KEY WORDS: PI3K, AKT, mTOR, Pluripotency, Proliferation and PI3K/AKT/mTOR signalling in preserving the ability of PSCs to differentiation, Metabolism self-renew and differentiate (Paling et al., 2004; Zhou et al., 2009; Singh et al., 2012; Yu et al., 2015), and it has become apparent that Introduction this signalling pathway is crucial for embryonic development and is During development, cellular differentiation is a crucial process by indispensable for the self-renewal of PSCs. In this Review, we which all major tissues of the adult organism are formed. This is discuss the functions of this pathway during embryonic development governed by the spatial and temporal activation of signalling and in cell fate determination of PSCs, with a particular focus on the pathways, classically demonstrated through gain- and loss-of- putative molecular mechanisms underlying these functions. function studies performed in developmental models, such as zebrafish, Xenopus and mice. Genetic manipulation of signalling PI3K/AKT/mTOR signalling components induces defects in either specific tissues or PI3Ks are a unique family of intracellular lipid kinases that developmental stages, thus implying that distinct pathways specify phosphorylate the 3′-hydroxyl group of the inositol ring of particular lineages. However, it is increasingly evident that this is a phosphatidylinositides (PtdIns) and are divided into three classes. gross oversimplification, given that signalling pathways do not operate The most studied is class I PI3K (Fig. 1), which stimulates the in isolation, but participate in a myriad of crosstalk interactions that conversion of membrane-bound phosphatidylinositol-(4,5)- cumulatively determine cell fate. Therefore, even pathways that do not bisphosphate [PtdIns(4,5)P2; PIP2] to phosphatidylinositol-(3,4,5)- appear to specify a lineage independently may act to augment or trisphosphate [PtdIns(3,4,5)P3; PIP3] (Vanhaesebroeck et al., 2010; inhibit pathways that are essential for lineage specification. However, Thorpe et al., 2015). PIP3 acts as a secondary messenger, facilitating the inherent complexity and redundant nature of signalling makes the the recruitment and activation of kinases that possess the pleckstrin molecular dissection of such crosstalk events difficult. homology (PH) domain, such as PI3K-dependent kinase-1 (PDK1). PI3K/AKT/mTOR signalling contributes to a variety of processes The signalling duration of PIP3 is subject to regulation by that are critical in mediating many aspects of cellular function, phosphatase and tensin homolog (PTEN), which acts to oppose including nutrient uptake, anabolic reactions, cell growth and PI3K activity. The serine/threonine kinase AKT, also known as survival. The core components of the pathway, namely protein kinase B (PKB), possesses a PH domain, and is recruited to phosphatidylinositide 3-kinases (PI3Ks), AKT and mammalian the plasma membrane along with PDK1. Phosphorylation of amino target of rapamycin (mTOR, also known as mechanistic TOR), have acid residues T308 and S473 by PDK1 and mTORC2, respectively, been shown to be frequently hyperactivated in the majority of is essential for full AKT activation. Following activation, AKT can cancers and have therefore been the focus of many studies in this phosphorylate many target proteins, most notably glycogen field (Vanhaesebroeck et al., 2010; Thorpe et al., 2015). By contrast, synthase kinase 3 (GSK3), tuberous sclerosis 2 (TSC2), caspase 9 the importance of this pathway in development is understated, even and PRAS40 (AKT1S1), which explains its relatively wide though loss any of the core components often results in embryonic spectrum of downstream effects in promoting cell proliferation, lethality (Bi et al., 1999; Peng et al., 2003; Murakami et al., 2004). differentiation, apoptosis, angiogenesis and metabolism. mTOR is an evolutionarily conserved serine/threonine protein Institute of Reproductive and Developmental Biology, Department of Surgery and kinase that belongs to the PI3K-related kinase family (PIKK). The Cancer, Imperial College London, Du Cane Road, London W12 0NN, UK. accessibility of the mTOR active site is governed in part by mTOR- associated proteins that form two distinct complexes: mTOR *Author for correspondence ([email protected]) complex 1 (mTORC1) and 2 (mTORC2). These complexes differ J.S.L.Y., 0000-0001-5203-3603; W.C., 0000-0003-2019-380X in their composition, mode of activation and rapamycin sensitivity DEVELOPMENT 3050 REVIEW Development (2016) 143, 3050-3060 doi:10.1242/dev.137075 PIP2 PIP3 PIP2 Box 1. Regulation of mTOR complexes P P RTK P P Cell membrane P P P mTORC1 PTEN Activation of mTORC1 is regulated by multiple intracellular and P P PI3K extracellular cues, involving both RAG and RHEB (Ras homolog PDK1 T308 mLST8 enriched in brain) guanosine triphosphatases (GTPases). RAG S473 P P GTPase exists as a heterodimer of RAGA/B (RRAGA/B) with RAGC/D PKB/AKT mTORT DEPTOR mSIN1 (RRAGC/D) and is anchored to the lysosome by the Ragulator complex RICTOR P (Bar-Peled et al., 2012). RAG GTPase is activated by the presence of GSK3 mTORC2 P nutrients (e.g. amino acids) through the combined action of the amino TSC2 TSC1 acid transporter SLC38A9 (Rebsamen et al., 2015; Wang et al., 2015) P P mLST8 SGK1 PKC and the Ragulator complex as a guanine nucleotide exchange factor (GEF), along with other proteins, which recruit mTORC1 to the lysosome mTOR DEPTOR RHEB surface where it encounters RHEB. The activation of RHEB is negatively PRAS40 RAPTOR controlled by tuberous sclerosis 1 and 2 (TSC1/2). Notably, many mTORC1 upstream signalling inputs regulate mTORC1 via phosphorylation of TSC1/2. For example, growth factor-induced PI3K/AKT inactivates P P TSC2 by phosphorylation of S939/T1462, resulting in the activation of S6K1/2 P 4E-BP1 ULK1 RHEB and therefore mTORC1. Conversely, energy deficiency-activated P adenosine monophosphate protein kinase (AMPK; PRKA) triggers the S6 EIF4E phosphorylation of TSC2-T1227/S1387, enhancing TSC1/2 activity and inhibiting mTORC1. Recently, arginine has been shown to activate Fig. 1. Schematic of the PI3K/AKT/mTOR signalling cascade and its main mTORC1 via a RAG GTPase-independent mechanism (Carroll et al., downstream effectors. Upon growth factor stimulation (yellow diamond), 2016). active PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2)to mTORC2 phosphatidylinositol (3,4,5)-triphosphate (PIP3), which then recruits AKT and In contrast to mTORC1, the factors and regulative processes driving permits phosphorylation of T308 and S473 through PDK1 and mTORC2, mTORC2 activation remain relatively understudied. So far, only PI3K respectively. Activated AKT inhibits GSK3 and TSC2 via phosphorylation, stimulation has been shown to promote mTORC2 activation in inactive TSC1/2 is unable to bind RAS homolog enriched in brain (RHEB), association with the mSIN1 PH domain and the ribosome (Liu et al., which subsequently enables its activation of mTORC1 at the surface of 2015; Zinzalla et al., 2011). The exact molecular details await further lysosome, initiating its effect on many downstream proteins, including S6K, elucidation. 4E-BP1 and ULK1. Active mTORC2 can regulate several AGC kinases, such as SGK1 and PKC, in addition to AKT. ‘P’ in a green circle indicates activating phosphorylation, in a red circle inhibitory phosphorylation and in a brown circle particularly in the context of embryonic development. Nonetheless, phosphorylation that is not induced by this signalling cascade. genetic manipulation of key components by way of gene knockout has ascertained their importance in driving organismal growth and (Yang et al., 2013; Liu et al., 2015). Although mTOR in both tissue
Load more

Recommended publications
  • Accumulation of Dephosphorylated 4EBP After Mtor Inhibition with Rapamycin Is Sufficient to Disrupt Paracrine Transformation By
    Oncogene (2014) 33, 2405–2412 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc SHORT COMMUNICATION Accumulation of dephosphorylated 4EBP after mTOR inhibition with rapamycin is sufficient to disrupt paracrine transformation by the KSHV vGPCR oncogene D Martin, Q Nguyen, A Molinolo and JS Gutkind Dysregulation of the PI3K/Akt/mTOR pathway is one of the most frequent events in human cancer. However, the clinical benefits of PI3K/Akt/mTOR inhibitors have not yet achieved their predicted potential in many of the most prevalent human cancers. Of interest, treatment of Kaposi’s sarcoma (KS) patients with rapamycin provided the first evidence of the antineoplastic activity of mTOR inhibitors in humans, becoming the standard of care for KS arising in renal transplant patients. Thus, the study of KS may provide a unique opportunity to dissect the contribution of specific mTOR downstream targets to cancer development. The KS- associated herpesvirus (KSHV) is the etiological agent for KS, and the KSHV-encoded oncogene viral-G protein-coupled receptor (vGPCR) promotes the potent activation of the PI3K-Akt-mTOR pathway by both direct and paracrine mechanisms. We focused on a direct target of mTOR, EIF4EBP1/2/3 (4EBP), which inhibits the translation of eukaryotic initiation factor 4E (eiF4E)-bound mRNAs. 4EBP phosphorylation by mTOR relieves its inhibitory activity, hence resulting in increased eiF4E-dependent mRNA translation. We developed a paracrine transformation model, recapitulating the cellular composition of KS lesions, in which vGPCR-expressing cells promote the rapid proliferation of endothelial cells, thus expressing KSHV-latent genes by the release of growth factors.
    [Show full text]
  • Adaptive Capabilities of the Pi3k/Akt/Mtor Pathway in Acute Myeloid Leukemia Revealed by the Use of Selective Inhibitors
    Alma Mater Studiorum – Università di Bologna SCIENZE BIOMEDICHE: PROGETTO 5 “SCIENZE MORFOLOGICHE UMANE E MOLECOLARI” Ciclo XXV SSD : BIO 16, SETTORE CONCORSUALE :05H1 ADAPTIVE CAPABILITIES OF THE PI3K/AKT/MTOR PATHWAY IN ACUTE MYELOID LEUKEMIA REVEALED BY THE USE OF SELECTIVE INHIBITORS Presentata da Jessika Bertacchini Relatori Prof.ssa Lucia Manzoli Prof.ssa Sandra Marmiroli Coordinatore Chiar. mo Prof. Lucio Cocco TABLE OF CONTENTS o ABSTRACT o INTRODUCTION o ACUTE MYELOID LEUKEMIA o PROGNOSIS AND GENETICS o DEREGULATED SIGNAL TRANSDUCTION PATHWAYS IN ACUTE MYELOID LEUKEMIA o PI3K/AKT/MTOR SIGNAL TRANSDUCTION PATHWAY IN ACUTE MYELOID LEUKEMIA o PI3K o AKT/PKB o Mtor o NEGATIVE REGULATION OF PI3K/AKT/mTOR PATHWAY o PI3K/AKT/mTOR PATHWAY AND SURVIVAL o PI3K/AKT/mTOR PATHWAY AND CELL CYCLE o PI3K/AKT/mTOR PATHWAY AND METABOLISM o PI3K/AKT/mTOR INHIBITORS o PI3K INHIBITORS o AKT INHIBITORS o mTOR INHIBITORS o TYROSINE KINASE RECEPTOR o TYROSINE KINASE RECEPTOR IN ACUTE MYELOID LEUKEMIA o AIMS o MATERIALS AND METHODS o PATIENTS DEMOGRAPHICS o CELL CULTURE AND DRUG TREATMENTS o ARRAY ASSEMBLY o RESULTS o RESULTS o DISCUSSION o BIBLIOGRA 1.ABSTRACT The objective of the study was to investigate the sensitivity of primary blasts from AML patients to PI3K/Akt/mTor inhibitors through reverse-phase protein microarray. Reverse-phase microarray assays using phosphospecific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. Mapping of deregulated kinases and protein signaling networks within tumors can provide a means to stratify patients with shared biological characteristics to the most optimal treatment, and identify drug targets. In particular, the PI3K/AKT/mTOR signaling pathways are frequently activated in blast cells from patients with acute myelogenous leukemia (AML), a neoplastic disorder characterized by the accumulation of genetically altered myelogenous cells displaying deregulated intracellular signalling pathways and aggressive clinical behavior with poor prognosis.
    [Show full text]
  • Comparative Mapping of Bovine Chromosome 27 with Human Chromosome 8 Near a Dairy Form QTL in Cattle
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Agriculture: Agricultural Publications from USDA-ARS / UNL Faculty Research Service, Lincoln, Nebraska 2006 Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle E. E. Connor USDA-ARS M. S. Ashwell North Carolina State University at Raleigh R. Schnabel University of Missouri-Columbia J. L. Williams Roslin Institute (Edinburgh) Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Part of the Agricultural Science Commons Connor, E. E.; Ashwell, M. S.; Schnabel, R.; and Williams, J. L., "Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle" (2006). Publications from USDA-ARS / UNL Faculty. 698. https://digitalcommons.unl.edu/usdaarsfacpub/698 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Original Article Cytogenet Genome Res 112:98–102 (2006) DOI: 10.1159/000087519 Comparative mapping of bovine chromosome 27 with human chromosome 8 near a dairy form QTL in cattle E.E. Connor,a M.S. Ashwell,a,b R. Schnabel,c J.L. Williamsd a Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD; b North Carolina State University, Department of Animal Science, Raleigh, NC; c University of Missouri-Columbia, Animal Sciences Unit, Columbia, MO (USA); d Roslin Institute (Edinburgh), Roslin, Midlothian, Scotland (UK) Manuscript received 30 December 2004; accepted in revised form for publication by T.
    [Show full text]
  • Human 4E-BP1 / EIF4EBP1 Protein (His Tag)
    Human 4E-BP1 / EIF4EBP1 Protein (His Tag) Catalog Number: 10022-H07E General Information SDS-PAGE: Gene Name Synonym: 4E-BP1; 4EBP1; BP-1; PHAS-I Protein Construction: A DNA sequence encoding the human 4EBP1 (NP_004086.1) (Ser 2-Ile 118) with a N-terminal polyhistidine tag was expressed. Source: Human Expression Host: E. coli QC Testing Purity: > 90 % as determined by SDS-PAGE Endotoxin: Protein Description Please contact us for more information. The translational suppressor eIF4E binding protein-1, 4E-BP1 functions as Stability: a key regulator in cellular growth, differentiation, apoptosis and survival. The Eif4ebp1 gene, encoding 4E-BP1, is a direct target of a transcription Samples are stable for up to twelve months from date of receipt at -70 ℃ factor activating transcription factor-4 (ATF4), a master regulator of gene expression in stress responses. 4E-BP1 is characterized by its capacity to Predicted N terminal: Met bind specifically to eIF4E and inhibit its interaction with eIF4G. Molecular Mass: Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap- dependent translation, in cell stress. Binding of eIF4E to eIF4G is inhibited The secreted recombinant human 4EBP1 comprises 124 amino acids with in a competitive manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases a predicted molecular mass of 13.4 kDa. It migrates as an approximately the affinity of this protein for eIF4E, thus favouring the binding of eIF4G 19 kDa band in SDS-PAGE under reducing conditions. and enhancing translation. 4E-BP1 is important for beta-cell survival under endoplasmic reticulum (ER) stress. 4E-BP1 mediates the regulation of Formulation: protein translation by hormones, growth factors and other stimuli that signal through the MAP kinase and mTORC1 pathways.
    [Show full text]
  • Relevance of Translation Initiation in Diffuse Glioma Biology and Its
    cells Review Relevance of Translation Initiation in Diffuse Glioma Biology and its Therapeutic Potential Digregorio Marina 1, Lombard Arnaud 1,2, Lumapat Paul Noel 1, Scholtes Felix 1,2, Rogister Bernard 1,3 and Coppieters Natacha 1,* 1 Laboratory of Nervous System Disorders and Therapy, GIGA-Neurosciences Research Centre, University of Liège, 4000 Liège, Belgium; [email protected] (D.M.); [email protected] (L.A.); [email protected] (L.P.N.); [email protected] (S.F.); [email protected] (R.B.) 2 Department of Neurosurgery, CHU of Liège, 4000 Liège, Belgium 3 Department of Neurology, CHU of Liège, 4000 Liège, Belgium * Correspondence: [email protected] Received: 18 October 2019; Accepted: 26 November 2019; Published: 29 November 2019 Abstract: Cancer cells are continually exposed to environmental stressors forcing them to adapt their protein production to survive. The translational machinery can be recruited by malignant cells to synthesize proteins required to promote their survival, even in times of high physiological and pathological stress. This phenomenon has been described in several cancers including in gliomas. Abnormal regulation of translation has encouraged the development of new therapeutics targeting the protein synthesis pathway. This approach could be meaningful for glioma given the fact that the median survival following diagnosis of the highest grade of glioma remains short despite current therapy. The identification of new targets for the development of novel therapeutics is therefore needed in order to improve this devastating overall survival rate. This review discusses current literature on translation in gliomas with a focus on the initiation step covering both the cap-dependent and cap-independent modes of initiation.
    [Show full text]
  • DATASHEET Eukaryotic Translation Initiation Factor 4E-Binding
    DATASHEET Abbexa Ltd, Innovation Centre, Cambridge Science Park, Cambridge, CB4 0EY, UK Telephone: +44 (0) 1223 755950 - Fax: +44 (0) 1223 755951 - E-Mail: [email protected] Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 (4EBP1) Antibody Catalogue No.:abx015717 Western blot analysis using 4E-BP1 antibody against truncated 4E-BP1 recombinant protein (1) and A431 cell lysate (2). Immunohistochemical analysis of paraffin-embedded human pancreas carcinoma (A), esophagus carcinoma tissue (B) and ovary tumor tissue (C), showing cytoplasmic and membrane localization using 4E-BP1 antibody with DAB staining. 4E-BP1 (eukaryotic translation Initiation Factor 4E Binding Protein 1),also called ELF4EBP1/BP-1/PHAS-I,which is located on chromosome 8p12, with 118-amino acid protein (about 13kDa).Binding of eIF4EBP1 to eIF4E is reversible and is dependent on the phosphorylation status of eIF4EBP1.Non phosphorylated eIF4EBP1 will bind strongly to eIF4E while (24kDa), the phosphorylated form will not.Akt, TOR, MAP kinase, S6 kinase, and Cdc2 are known kinases capable of inactivating eIF4EBP1 binding to eIF4E by phosphorylating either threonines 35, 45, 69 or serine 64.Although, not all phosphorylation events equally block the eIF4EBP1-eIF4E interaction. Target: 4EBP1 Reactivity: Human Host: Mouse Clonality: Monoclonal Tested Applications: ELISA, WB, IHC Recommended dilutions: ELISA: 1/10000, WB: 1/500 - 1/2000, IHC: 1/200 - 1/1000. Optimal dilutions/concentrations should be determined by the end user. Immunogen: Purified recombinant fragment of 4E-BP1 expressed in E. coli. Purification: Unpurified Ascites. Registered in England: 8475531 - VAT Reg No: GB 167 9240 79 Page 1 DATASHEET Abbexa Ltd, Innovation Centre, Cambridge Science Park, Cambridge, CB4 0EY, UK Telephone: +44 (0) 1223 755950 - Fax: +44 (0) 1223 755951 - E-Mail: [email protected] Isotype: IgG1 Conjugation: Unconjugated Storage: Aliquot and store at -20 °C.
    [Show full text]
  • 4E-BP1 Antibody [9E12D9] Cat
    4E-BP1 Antibody [9E12D9] Cat. No.: 32-101 4E-BP1 Antibody [9E12D9] Specifications HOST SPECIES: Mouse SPECIES REACTIVITY: Human IMMUNOGEN: Ni-NTA purified truncated recombinant 4EBP1 expressed in E. Coli strain BL21 (DE3). TESTED APPLICATIONS: ELISA, WB Western Blot:1:500 - 1:1,000. APPLICATIONS: ELISA:Propose dilution 1:10,000. Determining optimal working dilutions by titration test. Properties CLONALITY: Monoclonal ISOTYPE: IgG1 CONJUGATE: Unconjugated BUFFER: Ascitic fluid containing 0.03% sodium azide. 4E-BP1 monoclonal antibody can be stored at -20˚C, stable for one year. As with all STORAGE CONDITIONS: antibodies care should be taken to avoid repeated freeze thaw cycles. Antibodies should not be exposed to prolonged high temperatures. September 25, 2021 1 https://www.prosci-inc.com/4e-bp1-antibody-9e12d9-32-101.html Additional Info OFFICIAL SYMBOL: EIF4EBP1 Phosphorylated heat- and acid-stable protein regulated by insulin 1, 4E-BP1, BP-1, 4EBP1, ALTERNATE NAMES: PHAS-I ACCESSION NO.: Q13541 PROTEIN GI NO.: 34921508 GENE ID: 1978 USER NOTE: Optimal dilutions for each application to be determined by the researcher. Background and References 4E-BP1(eukaryotic translation Initiation Factor 4E Binding Protein 1),also called ELF4EBP1/BP-1/PHAS-I ,which is located on chromosome 8p12, with 118-amino acid protein (about 13 kDa). Binding of eIF4EBP1 to eIF4E is reversible and is dependent on the phosphorylation status of eIF4EBP1. Non phosphorylated eIF4EBP1 will bind strongly to BACKGROUND: eIF4E while(24 kDa), the phosphorylated form will not. Akt, TOR, MAP kinase, S6 kinase, and Cdc2 are known kinases capable of inactivating eIF4EBP1 binding to eIF4E by phosphorylating either threonines 35, 45, 69 or serine 64.
    [Show full text]
  • Activation of Diverse Signalling Pathways by Oncogenic PIK3CA Mutations
    ARTICLE Received 14 Feb 2014 | Accepted 12 Aug 2014 | Published 23 Sep 2014 DOI: 10.1038/ncomms5961 Activation of diverse signalling pathways by oncogenic PIK3CA mutations Xinyan Wu1, Santosh Renuse2,3, Nandini A. Sahasrabuddhe2,4, Muhammad Saddiq Zahari1, Raghothama Chaerkady1, Min-Sik Kim1, Raja S. Nirujogi2, Morassa Mohseni1, Praveen Kumar2,4, Rajesh Raju2, Jun Zhong1, Jian Yang5, Johnathan Neiswinger6, Jun-Seop Jeong6, Robert Newman6, Maureen A. Powers7, Babu Lal Somani2, Edward Gabrielson8, Saraswati Sukumar9, Vered Stearns9, Jiang Qian10, Heng Zhu6, Bert Vogelstein5, Ben Ho Park9 & Akhilesh Pandey1,8,9 The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets. 1 McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 527, Baltimore, Maryland 21205, USA.
    [Show full text]
  • The Human Gene Connectome As a Map of Short Cuts for Morbid Allele Discovery
    The human gene connectome as a map of short cuts for morbid allele discovery Yuval Itana,1, Shen-Ying Zhanga,b, Guillaume Vogta,b, Avinash Abhyankara, Melina Hermana, Patrick Nitschkec, Dror Friedd, Lluis Quintana-Murcie, Laurent Abela,b, and Jean-Laurent Casanovaa,b,f aSt. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY 10065; bLaboratory of Human Genetics of Infectious Diseases, Necker Branch, Paris Descartes University, Institut National de la Santé et de la Recherche Médicale U980, Necker Medical School, 75015 Paris, France; cPlateforme Bioinformatique, Université Paris Descartes, 75116 Paris, France; dDepartment of Computer Science, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; eUnit of Human Evolutionary Genetics, Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, Institut Pasteur, F-75015 Paris, France; and fPediatric Immunology-Hematology Unit, Necker Hospital for Sick Children, 75015 Paris, France Edited* by Bruce Beutler, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 15, 2013 (received for review October 19, 2012) High-throughput genomic data reveal thousands of gene variants to detect a single mutated gene, with the other polymorphic genes per patient, and it is often difficult to determine which of these being of less interest. This goes some way to explaining why, variants underlies disease in a given individual. However, at the despite the abundance of NGS data, the discovery of disease- population level, there may be some degree of phenotypic homo- causing alleles from such data remains somewhat limited. geneity, with alterations of specific physiological pathways under- We developed the human gene connectome (HGC) to over- come this problem.
    [Show full text]
  • MTORC1 Regulates Cardiac Function and Myocyte Survival Through 4E-BP1 Inhibition in Mice
    Amendment history: Corrigendum (October 2010) MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice Denghong Zhang, … , Nahum Sonenberg, Gianluigi Condorelli J Clin Invest. 2010;120(8):2805-2816. https://doi.org/10.1172/JCI43008. Research Article Cardiology Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor- ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory […] Find the latest version: https://jci.me/43008/pdf Research article MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice Denghong Zhang,1 Riccardo Contu,2 Michael V.G.
    [Show full text]
  • UNDERSTANDING the ROLE of EIF4A in GENE REGULATION in HEALTH and DISEASE THOMAS EDWARD WEBB B.Sc
    UNDERSTANDING THE ROLE OF EIF4A IN GENE REGULATION IN HEALTH AND DISEASE THOMAS EDWARD WEBB B.Sc. (Hons, Warwick) M.Sc. (Oxon) Thesis submitted to The University of Nottingham for the degree of Doctor of Philosophy (July 2012) 1 Abstract eIF4A Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase responsible for unwinding the secondary structure of mRNAs. In humans, eIF4A exists as three separate paralogs: eIF4AI and eIF4AII possess a high degree of homology while eIF4AIII is distinct. Knockdown of eIF4AII had no effect on the expression of a reporter construct containing a structured RNA hairpin. Knockdown of eIF4AI and treatment with hippuristanol (an eIF4A inhibitor) caused a dramatic reduction in the hairpin-mediated gene. This reporter system was developed as part of this project to act as a screen for eIF4A activity along with an in vitro screening approach. PDCD4 The activity of eIF4A is suppressed in vivo by the tumour suppressor PDCD4. The fact that loss of PDCD4 function increases the severity of DNA damage is probably attributable its eIF4A-suppressive activity. Alzheimer's Disease Based on previous microarray data, it was supposed that eIF4A inhibition may be therapeutically beneficial in the treatment of Alzheimer's disease. As part of this project, it was demonstrated that eIF4A suppression significantly reduced the expression of reporter genes preceded by the 5’ UTRs of genes predicted to play harmful roles in Alzheimer’s disease. The expression of reporter genes preceded by the 5’ UTR sequences of genes predicted to be beneficial in Alzheimer's were not affected by this suppression.
    [Show full text]
  • Genomic and Expression Analysis of the 8P11–12 Amplicon in Human Breast Cancer Cell Lines
    [CANCER RESEARCH 64, 40–47, January 1, 2004] Genomic and Expression Analysis of the 8p11–12 Amplicon in Human Breast Cancer Cell Lines Michael E. Ray,1 Zeng Quan Yang,1 Donna Albertson,4 Celina G. Kleer,3 Joseph G. Washburn,3 Jill A. Macoska,2 and Stephen P. Ethier1 1Departments of Radiation Oncology and 2Urology, 3Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan, and 4Cancer Research Institute, University of California San Francisco, San Francisco, California ABSTRACT In our panel of 11 breast cancer cell lines, 3 lines (SUM-44, SUM-52, and SUM-225) have an amplification in the 8p11-p12 Gene amplification is an important mechanism of oncogene activation region (8, 14, 15). However, unlike FGFR2, we have been unable to in breast and other cancers. Characterization of amplified regions of the find direct evidence that FGFR1 is the driving oncogene in these cell genome in breast cancer has led to the identification of important onco- genes including erbB-2/HER-2, C-MYC, and fibroblast growth factor re- lines. We have undertaken a detailed genomic and expression analysis ceptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10–15% of of the 8p11-p12 amplicon in these three cell lines, using array com- human breast cancers. The putative oncogene FGFR1 localizes to this parative genomic hybridization (CGH), Southern blot analysis, North- region; however, we show evidence that FGFR inhibition fails to slow ern blot analysis, and chromosome 8-specific cDNA microarrays. growth of three breast cancer cell lines with 8p11-p12 amplification. We These experiments have identified new candidate oncogenes and have present a detailed analysis of this amplicon in three human breast cancer provided support for the hypothesis that genes other than FGFR1 may cell lines using comparative genomic hybridization, traditional Southern play a causal role in breast cancer progression in tumors with ampli- and Northern analysis, and chromosome 8 cDNA microarray expression fication in the 8p11–12 region.
    [Show full text]