DOCSLIB.ORG

Interaction of Calcineurin with a Domain of Thetranscription Factor NFAT1 That Controls Nuclear Import

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interaction of Calcineurin with a Domain of Thetranscription Factor NFAT1 That Controls Nuclear Import Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8907-8912, August 1996 Biochemistry Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import (protein phosphatase/nuclear localization sequence/immunosuppression/T cell activation/signal transduction) CHUN Luo*t#, KAREN T.-Y. SHAW*t§, ANURADHA RAGHAVANT, JOSE ARAMBURU*, FRANcisco GARCIA COZAR*, BRiAN A. PERRINOII, PATRICK G. HOGAN1, AND ANJANA RAO*,** *Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute and Department of Pathology, and IDepartment of Neurobiology, Harvard Medical School, Boston, MA 02115; and IlThe Vollum Institute, Oregon Health Sciences University, Portland, OR 97201 Communicated by Stephen C. Harrison, Harvard University, Cambridge, MA, May 8, 1996 (received for review February 7, 1996) ABSTRACT The nuclear import of the nuclear factor of hallmarks of activation is dependent on calcineurin, since each activated T cells (NFAT)-family transcription factors is ini- is blocked by CsA or FK506 (30). Here we present evidence tiated by the protein phosphatase calcineurin. Here we iden- suggesting that the close control of NFAT1 activation by the tify a regulatory region of NFAT1, N terminal to the DNA- calcium/calcineurin pathway reflects a protein-protein inter- binding domain, that controls nuclear import of NFAT1. The action that targets calcineurin to NFAT1. regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated sub- cellular localization identical to that of full-length NFAT1. MATERIALS AND METHODS The corresponding region of NFATc likewise binds cal- cDNA Constructs. The cDNAs encoding murine NFAT1, cineurin, suggesting that the efficient activation ofNFAT1 and human NFAT1(1-415), murine NFAT1(399-927), and mu- NFATc by calcineurin reflects a specific targeting of the rine NFAT1(398-694) were subcloned into pEFTAG, a de- phosphatase to these proteins. The presence in other NFAT- rivative of the expression vector pEF-BOS (31) that has been family transcription factors of several sequence motifs from modified to encode an influenza hemagglutinin (HA) peptide the regulatory region of NFAT1, including its probable nu- at the N terminus of the expressed proteins. Details of the clear localization sequence, indicates that a conserved protein subcloning steps are available from the authors. Murine domain may control nuclear import of all NFAT proteins. NFAT1(398-694) is the DNA-binding domain that has been expressed in bacteria as the hexahistidine-tagged protein NFAT1 (nuclear factor of activated T cells 1) (previously NFATpXS(1-297) (11). termed NFATp) (1-3) is a member of the NFAT family of Bacterial expression plasmids for the glutathione S- transcription factors, proteins that play a key role in the transferase (GST) fusion proteins GST-NFAT1(1-415) and regulation of cytokine gene transcription during the immune GST-NFATc(1-418) were made by subcloning the corre- response (4-6). Other members of the NFAT family include sponding regions of human NFAT1 and NFATc cDNAs into NFATc (7), NFATx/NFAT4 (8-10), and NFAT3 (9). NFAT- pGEX2T. family proteins show strong sequence conservation in their Site-Directed Mutagenesis. Single-stranded plasmid DNA DNA-binding domains (8-12), and moderate sequence con- containing uracil (32) was prepared from a pLGP3 vector (33) servation in a second region located immediately N terminal with the murine NFAT1 cDNA insert. Site-directed mutagen- to the DNA-binding domain (54). esis (32) using the oligonucleotides 5'-TCACCCGGTGC- A central feature of the regulation of NFAT-family proteins CGCTGCAGCTCATTCGTGCGCA-3' and 5 '-GTCAT- is their pronounced sensitivity to the immunosuppressive drugs CAACGGAGCTGCAGCCACTAGTCAGCCACAGCA-3' cyclosporin A (CsA) and FK506 (13, 14). These drugs, which was carried out to replace the codons for KRR or KRKR, are potent inhibitors of cytokine gene transcription in acti- respectively, and plasmids carrying the mutant sequences were vated T cells, B cells, mast cells, and NK cells (15-18), act by identified by DNA sequencing. EcoNI-BglII and MluI-HindIII binding to intracellular immunophilins and inhibiting the fragments, carrying respectively the region encoding the KRR activity of the calmodulin-dependent phosphatase calcineurin to AAA substitution and the region encoding the KRKR to (19, 20). Inhibition of calcineurin with the immunosuppressive AAAT substitution, were used to replace the corresponding drugs prevents the appearance of NFAT DNA-binding activity fragments of pBluescript-mNFAT1-C (C.L., unpublished in the cell nucleus when T cells and other immune system cells work). TheAatII-SalI fragments of the resulting plasmids were are stimulated (1, 18, 21-26), and blocks NFAT-dependent subcloned into pEFTAG for expression in T cells. The se- reporter gene transcription (21, 23, 25). Conversely, overex- quence of the entire fragment subcloned from the pLGP3 pression of calcineurin or expression of constitutively active vector was verified by DNA sequencing by the dideoxy chain- calcineurin partially replaces the calcium requirement for termination method (34). NEAT-dependent transcriptional activation in T cells (17, 27-29). Immunocytochemical Localization of Recombinant Pro- Detailed analysis of one NFAT-family protein, NFAT1, has teins. Plasmids were introduced into Jurkat cells by electro- provided more insight into the molecular mechanisms by which poration, and into clone 7W2 cells by the DEAE-dextran calcineurin regulates NFAT activity. NFAT1 is present in the method. Two days later cells in medium at 37°C were treated cytoplasm of resting T cells and translocates into the nucleus in response to stimulation with ionomycin, antigen, or anti- Abbreviations: NFAT, nuclear factor of activated T cells; CsA, cyclo- CD3 (30, 55). Early events in the activation of NFAT1 are its sporin A; HA, hemagglutinin; GST, glutathione S-transferase; NLS, rapid dephosphorylation, an increase in its affinity for NFAT nuclear localization sequence. sites in DNA, and its nuclear import (30). Each of these three tC.L. and K.T.-Y.S. contributed equally to this work. IPresent address: Center for Blood Research and Harvard Medical School, Boston, MA 02115. The publication costs of this article were defrayed in part by page charge §Present address: Department of Physiology and Cell Biology, Uni- payment. This article must therefore be hereby marked "advertisement" in versity of Nevada School of Medicine, Reno, NV 89557. accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 8907 Downloaded by guest on September 25, 2021 8908 Biochemistry: Luo et al. Proc. Natl. Acad. Sci. USA 93 (1996) as indicated, then fixed and stained with the anti-HA tag N terminal to the DNA-binding domain, human NFAT1(1- antibody (12CA5; Boehringer Mannheim) and Cy-3-labeled 415); the region from the beginning of the DNA-binding donkey anti-mouse IgG (Jackson ImmunoResearch). Tran- domain to the C terminus, murine NFAT1(399-927) (here sient expression of the tagged protein was detectable in 1-5% denoted NFAT1AN); and the DNA-binding domain itself, of human Jurkat T cells and in 0.2-2% of murine clone 7W2 murine NFAT1(397-694) [here denoted NFAT1(DBD)]. cells. At least three independent experiments were analyzed Each recombinant protein was tagged at its N terminus with an for each recombinant protein. influenza HA peptide. In Vitro Dephosphorylation. Unstimulated Ar-5 T cells were HA-tagged recombinant NFAT1 shows correctly regulated lysed in RIPA buffer (30) supplemented with protease inhib- intracellular localization in human Jurkat T cells. The protein itors and phosphatase inhibitors, and NFAT1 was immuno- is restricted to the cytoplasm in unstimulated cells, it translo- precipitated. Immunoprecipitates were resuspended in cal- cates to the cell nucleus on stimulation with ionomycin, and its cineurin assay buffer (50 mM Hepes, pH 7.4/2 mM MnCl2/0.5 translocation is completely prevented by pretreatment with mM EDTA/15 mM 2-mercaptoethanol) with 0.1 mg/ml BSA, CsA (Fig. 1). In a minority of cells, nuclear translocation in 20 ,uM leupeptin, and 10 ,ug/ml aprotinin, and incubated at response to ionomycin is only partial, a finding that may reflect 30°C for 10 min with 100 nM calcineurin (35) and 500 nM the high levels of expression of the recombinant protein in purified calmodulin. SDS lysates were prepared as described individual cells. However, because recombinant protein is (30). The samples were examined by Western blotting with never seen in the nucleus of unstimulated cells or of cells anti-NFAT1 antiserum (anti-67.1) (30, 36). treated with CsA, the incomplete translocation in a few cells Expression constructs encoding HA-tagged NFAT1 or does not affect the interpretation of the experiments. NFAT1(1-415) were introduced into COS cells using DEAE- Recombinant NFAT1(1-415) is regulated in the same way dextran combined with adenovirus (37). After 48 h, cytosolic as the full-length protein, and its translocation is similarly extracts were prepared in lysis buffer [20 mM tris HCl, pH sensitive to CsA (Fig. 1). In contrast, the truncated proteins 7.4/30 mM NaCl/5 mM EDTA/10 mM iodoacetamide/2 mM NFAT1AN and NFAT1(DBD), which lack the N-terminal phenylmethylsulfonyl fluoride (PMSF)/10 ,ug/ml aprotinin/25 region of NFAT1, are not restricted to the cytoplasm in ,uM leupeptin/5% glycerol/0.05% Nonidet P-40]. Cytosolic unstimulated cells and show no redistribution in response
Load more

Recommended publications
  • Myoferlin Regulation by NFAT in Muscle Injury, Regeneration and Repair
    Research Article 2413 Myoferlin regulation by NFAT in muscle injury, regeneration and repair Alexis R. Demonbreun1,2, Karen A. Lapidos2,3, Konstantina Heretis2, Samantha Levin2, Rodney Dale1, Peter Pytel4, Eric C. Svensson1,3 and Elizabeth M. McNally1,2,3,* 1Committee on Developmental Biology, 2Department of Medicine, 3Department of Molecular Genetics and Cell Biology, and 4Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6088, Chicago, IL 60637, USA *Author for correspondence ([email protected]) Accepted 9 April 2010 Journal of Cell Science 123, 2413-2422 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.065375 Summary Ferlin proteins mediate membrane-fusion events in response to Ca2+. Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease.
    [Show full text]
  • Cytoplasmic-Nuclear Localization of NFAT Responsiveness By
    The Journal of Immunology Active Protein Kinase B Regulates TCR Responsiveness by Modulating Cytoplasmic-Nuclear Localization of NFAT and NF-␬B Proteins1 Amiya K. Patra, Shin-Young Na, and Ursula Bommhardt2 T cell activation leads to the induction of the transcription factors of the NFAT and NF-␬B families, important regulators of T cell activation and function. In this study we demonstrate that TCR/CD3-stimulated T cells from mice expressing a constitutively active form of protein kinase B (myr PKB␣) lack significant nuclear accumulation/shuttling of NFATc1 and NFATp as well as NF-␬〉p65 and RelB proteins. Notably, despite this deficit in nuclear NFAT and NF-␬B proteins, myr PKB T cells show lower activation threshold for proliferation, enhanced cell cycle progression and increased production of Th1 and Th2 cytokines similar to signals provided by CD28 costimulation. The enhanced T cell response correlates with increased expression of cyclins D3 and B1 and cytokine-induced Src homology 2 protein, and inactivation of the forkhead transcription factor FKHR. In addition, coimmunoprecipitation studies indicate a direct regulation of NFATc1 by active PKB. Together, our results demonstrate that the positive regulatory role of myr PKB on TCR responsiveness, subsequent cell division, and effector function is linked to a negative regulatory mechanism on the nuclear accumulation/shuttling of NFAT and NF-␬〉 proteins. The Journal of Immunology, 2004, 172: 4812–4820. D4 T cell activation, expansion, and differentiation re- by the immunosuppressants cyclosporin A (CsA)3 or FK506 pre- quire recognition of specific Ag presented by MHC class vents activation and nuclear entry of NFAT.
    [Show full text]
  • The Role of Hif-1Alpha in Epigenetic Regulation of Transcription
    From Department of Cell & Molecular Biology (CMB) Karolinska Institutet, Stockholm, Sweden THE ROLE OF HIF-1ALPHA IN EPIGENETIC REGULATION OF TRANSCRIPTION Nikola Vojnovic Stockholm 2018 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB 2018 © Nikola Vojnovic, 2018, ISBN 978-91-7831-274-0 The role of HIF-1alpha in epigenetic regulation of transcription THESIS FOR DOCTORAL DEGREE (Ph.D.) By Nikola Vojnovic Principal Supervisor: Opponent: Randall S Johnson Sven Påhlman Karolinska Institutet Lund Universitet Department of CMB Department of Translation cancer research Co-supervisor(s): Examination Board: Katarina Gradin Ann-Kristin Östlund Farrants Karolinska Institutet Stockholm University Department of CMB Department of Molecular Bioscience Wenner-Gren Susanne Schiliso Karolinska Institutet Department of MTC Martin Rottenberg Karolinska Institutet Department of MTC “The significant problems we have cannot be solved at the same level of thinking with which we created them”. - Albert Einstein ABSTRACT The oxygen level inside cells, determine the amount of HIF protein. By directly being involved in HIF protein turnover rates, through a mechanism, by which, oxygen is utilized as a co-factor, for the PHD enzymes, regulating HIF protein stability. This allows for rapid stabilization of the HIFs and subsequent gene activation during low oxygen tensions inside cells. I investigated the role of HIF specific epigenetic effects, in cancer cell line models, as well as primary mouse CD8+ T-lymphocytes. The data in Paper I illustrates how HIF has the ability to modulate chromatin through a HIF-1α dependent chromatin remodeling event, in hypoxia responsive gene promoters.
    [Show full text]
  • Dephosphorylation of the Nuclear Factor of Activated T Cells (NFAT) Transcription Factor Is Regulated by an RNA-Protein Scaffold Complex
    Correction BIOCHEMISTRY Correction for “Dephosphorylation of the nuclear factor of ac- B. Sacks, and Anjana Rao, which appeared in issue 28, July 12, tivated T cells (NFAT) transcription factor is regulated by an 2011, of Proc Natl Acad Sci USA (108:11381–11386; first pub- RNA-protein scaffold complex,” by Sonia Sharma, Gregory M. lished June 27, 2011; 10.1073/pnas.1019711108). Findlay, Hozefa S. Bandukwala, Shalini Oberdoerffer, Beate The authors note that, due to a printer’s error, Fig. 1 appeared Baust, Zhigang Li, Valentina Schmidt, Patrick G. Hogan, David incorrectly. The corrected figure and its legend appear below. A C 670 kDa 158 kDa 670 KDa 158 KDa 52 54 56 58 60 62 64 66 68 V IB: NFAT1 Fraction: 5052 54 56 5860 62 64 66 68 70 72 74 76 250 IB: IQGAP1 IB: NFAT1 150 100 IB: IQGAP2 5052 54 56 5860 62 64 66 68 70 72 74 76 250 IB: CaM IB: IQGAP1 150 IB: CK1 ε 100 IB: CnA B 100 bp Fr. 54-60 Fr. 74-80 Fr. 87-93 Load H2O NFAT1: +-- High Low protein protein Fig. 1. NFAT1 coelutes with IQGAP and NRON in resting T cell lysates by size-exclusion chromatography. Hypotonic lysates from (A and B) HA-NFAT1 Jurkat T cells or (C) primary murine CD8+ T cells were fractionated on a Superdex 200 size-exclusion column. (A and C) Individual fractions were analyzed by SDS-PAGE and Western blotting for NFAT1, IQGAP1, IQGAP2, calmodulin (CaM), casein kinase epsilon (CK1ε) and calcineurin A (CnA). Column void volume (V)isin- dicated.
    [Show full text]
  • Isoform-Selective NFAT Inhibitor: Potential Usefulness and Development
    International Journal of Molecular Sciences Review Isoform-Selective NFAT Inhibitor: Potential Usefulness and Development Noriko Kitamura 1 and Osamu Kaminuma 1,2,* 1 Laboratory of Allergy and Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan; [email protected] 2 Department of Disease Model, Research Institute of Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan * Correspondence: [email protected]; Tel.: +81-82-257-5819 Abstract: Nuclear factor of activated T cells (NFAT), which is the pharmacological target of immuno- suppressants cyclosporine and tacrolimus, has been shown to play an important role not only in T cells (immune system), from which their name is derived, but also in many biological events. There- fore, functional and/or structural abnormalities of NFAT are linked to the pathogenesis of diseases in various organs. The NFAT protein family consists of five isoforms, and each isoform performs diverse functions and has unique expression patterns in the target tissues. This diversity has made it difficult to obtain ideal pharmacological output for immunosuppressants that inhibit the activity of almost all NFAT family members, causing serious and wide-ranging side effects. Moreover, it remains unclear whether isoform-selective NFAT regulation can be achieved by targeting the struc- tural differences among NFAT isoforms and whether this strategy can lead to the development of better drugs than the existing ones. This review summarizes the role of the NFAT family members in biological events, including the development of various diseases, as well as the usefulness of and Citation: Kitamura, N.; problems associated with NFAT-targeting therapies, including those dependent on current immuno- Kaminuma, O.
    [Show full text]
  • UCLA Electronic Theses and Dissertations
    UCLA UCLA Electronic Theses and Dissertations Title CHRONIC ALCOHOL EXPOSURE INCREASES GLYCOLYSIS AND CANCER STEMNESS OF OSCC THROUGH NFAT ACTIVATION Permalink https://escholarship.org/uc/item/3kt8968d Author Nguyen, Anthony Publication Date 2021 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA LOS ANGELES CHRONIC ALCOHOL EXPOSURE INCREASES GLYCOLYSIS AND CANCER STEMNESS OF OSCC THROUGH NFAT ACTIVATION A thesis submitted in partial satisfaction of the requirement for the degree Master of Science in Oral Biology by Anthony Nguyen 2021 © Copyright by Anthony Nguyen 2021 ABSTRACT OF THE THESIS CHRONIC ALCOHOL EXPOSURE INCREASES GLYCOLYSIS AND CANCER STEMNESS OF OSCC THROUGH NFAT ACTIVATION by Anthony Nguyen Master of Science in Oral Biology University of California, Los Angeles, 2021 Professor Ki-Hyuk Shin, Co-Chair Professor Yong Kim, Co-Chair Chronic alcohol consumption is associated with cancer development and progression. Previous reports have demonstrated that cancer cell metabolism is heavily involved in the development of cancer, including a side population know as cancer stem cells (CSCs). Therefore, it is generally accepted targeting cancer cell metabolism may provide a new and effective method for the treatment of tumors. There are emerging reports of the effects of alcohol on glucose metabolism, however the effects of alcohol and cancer metabolism and its underlying mechanisms are not fully understood in cancers including OSCC. In this study, we investigated the local effect of alcohol on cancer cell metabolism and its underlying mechanism by which alcohol promotes oral cancer progression. We report chronic alcohol promotes ii malignant growth of OSCC.
    [Show full text]
  • The Multifaceted Output of C-Jun Biological Activity: Focus at the Junction of CD8 T Cell Activation and Exhaustion
    cells Review The Multifaceted Output of c-Jun Biological Activity: Focus at the Junction of CD8 T Cell Activation and Exhaustion Athanasios G. Papavassiliou 1 and Anna Maria Musti 2,* 1 Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece; [email protected] 2 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy * Correspondence: [email protected]; Tel.: +39-3337543732 Received: 22 September 2020; Accepted: 11 November 2020; Published: 13 November 2020 Abstract: c-Jun is a major component of the dimeric transcription factor activator protein-1 (AP-1), a paradigm for transcriptional response to extracellular signaling, whose components are basic-Leucine Zipper (bZIP) transcription factors of the Jun, Fos, activating transcription factor (ATF), ATF-like (BATF) and Jun dimerization protein 2 (JDP2) gene families. Extracellular signals regulate c-Jun/AP-1 activity at multiple levels, including transcriptional and posttranscriptional regulation of c-Jun expression and transactivity, in turn, establishing the magnitude and the duration of c-Jun/AP-1 activation. Another important level of c-Jun/AP-1 regulation is due to the capability of Jun family members to bind DNA as a heterodimer with every other member of the AP-1 family, and to interact with other classes of transcription factors, thereby acquiring the potential to integrate diverse extrinsic and intrinsic signals into combinatorial regulation of gene expression. Here, we review how these features of c-Jun/AP-1 regulation underlie the multifaceted output of c-Jun biological activity, eliciting quite distinct cellular responses, such as neoplastic transformation, differentiation and apoptosis, in different cell types.
    [Show full text]
  • Grail Controls Th2 Cell Development by Targeting STAT6 for Degradation
    ARTICLE Received 9 Apr 2014 | Accepted 17 Jul 2014 | Published 22 Aug 2014 DOI: 10.1038/ncomms5732 Grail controls Th2 cell development by targeting STAT6 for degradation Anupama Sahoo1, Andrei Alekseev1, Lidiya Obertas1 & Roza Nurieva1 T helper (Th)-2 cells are the major players in allergic asthma; however, the mechanisms that control Th2-mediated inflammation are poorly understood. Here we find that enhanced expression of Grail, an E3 ubiquitin ligase, in Th2 cells depends on interleukin (IL)-4-signalling components, signal transducer and activator of transcription 6 (Stat6) and Gata3, that bind to and transactivate the Grail promoter. Grail deficiency in T cells leads to increased expression of Th2 effector cytokines in vitro and in vivo and Grail-deficient mice are more susceptible to allergic asthma. Mechanistically, the enhanced effector function of Grail-deficient Th2 cells is mediated by increased expression of Stat6 and IL-4 receptor a-chain. Grail interacts with Stat6 and targets it for ubiquitination and degradation. Thus, our results indicate that Grail plays a critical role in controlling Th2 development through a negative feedback loop. 1 Department of Immunology, M. D. Anderson Cancer Center, Houston, Texas 77030, USA. Correspondence and requests for materials should be addressed to R.N. (email: [email protected]). NATURE COMMUNICATIONS | 5:4732 | DOI: 10.1038/ncomms5732 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5732 D4 þ T helper (Th) cells are critical components in conditions and analysed for Grail mRNA expression. Consistent adaptive immunity. After activation, CD4 þ Th cells with a previous study, which showed upregulation of Grail in Th2 Cdifferentiate into various effector subsets characterized cells during chronic schistosomiasis23, Grail mRNA level was by expression of lineage-specific transcriptional factors and significantly increased in Th2 cells compared with other T-helper cytokines that determine their effector functions1.
    [Show full text]
  • Regulates NFAT-Mediated Activation GERALD P
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9545-9552, September 1996 Cell Bio[ogy This contribution is part ofthe special series ofInaugural Articles by members of the National Academy of Sciences ekcted on April 25, 1995. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation GERALD P. LINETrE*, YING LI*, KEVIN RoTH, AND STANLEY J. KORSMEYERt Howard Hughes Medical Institute and Division of Molecular Oncology, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Stanley J. Korsmeyer, July 17, 1996 ABSTRACT BCL-2-deficient T cells demonstrate accel- well as a proliferation program (15). Mice deficient in a cell erated cell cycle progression and increased apoptosis follow- cycle regulator, the retinoblastoma gene product (Rb), dem- ing activation. Increasing the levels of BCL-2 retarded the Go onstrate abnormal mitotic figures as well as death of embry- -- S transition, sustained the levels of cyclin-dependent onic neurons (16, 17). In parallel, the overexpression of E2F, kinase inhibitor p27KiPl, and repressed postactivation death. which mimics Rb deficiency, can also result in apoptosis (18, Proximal signal transduction events and immediate early gene 19). From this perspective, there would appear to be a capacity transcription were unaffected. However, the transcription and to identify aberrations in proliferation that would trigger the synthesis of interleukin 2 and other delayed early cytokines apoptotic elimination of dangerous cells. were markedly attenuated by BCL-2. In contrast, a cysteine We chose to approach this complex issue from the opposite protease inhibitor that also blocks apoptosis had no substan- perspective, asking whether regulators of cell death impact the tial affect upon cytokine production.
    [Show full text]
  • Molecular Orchestration of Differentiation and Function of Regulatory T Cells
    Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Molecular orchestration of differentiation and function of regulatory T cells Li-Fan Lu2 and Alexander Rudensky1 Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA During the last decade, a unique mechanism of negative CD4 T cells recognize MHC class II molecules bound to regulation of immune responses and inflammation by peptides generated in the endosomes and lysosomes, a dedicated population of so-called regulatory T cells although some important exceptions to this rule exist (Treg) has become a focus of intensive investigation. (Trombetta and Mellman 2005). Through the discovery of transcription factor Foxp3 as Sequence analyses of peptides displayed by MHC a central molecular determinant of differentiation and molecules revealed that the vast majority of peptides function of Treg cells, the complex biology of these cells, bound to MHC class I and class II molecules is derived including maintenance of immunological tolerance to from self-proteins. Furthermore, TCR have an intrinsic ‘‘self’’ and regulation of immune responses to pathogens, bias toward recognition of MHC molecules, and ‘‘tick- commensals, and tumors, has become amenable to mech- ling’’ of TCR through certain low-affinity interactions anistic studies. In this review, we discuss the molecular with self-peptide–MHC complexes displayed by thymic aspects of Treg cell lineage commitment, maintenance, cortical epithelial cells is prerequisite for thymic T-cell and function. precursors to pass a critical differentiation checkpoint dubbed positive selection. In the periphery, analogous The cornerstone of the adaptive immune system is the interactions facilitate the long-term maintenance of random generation of antigen-specific receptors in the mature CD4 and CD8 T cells and further shape TCR process of somatic gene rearrangement.
    [Show full text]
  • Fos-Jun Interactions That Mediate Transcription Regulatory Speci®City
    Oncogene (2001) 20, 2438 ± 2452 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Close encounters of many kinds: Fos-Jun interactions that mediate transcription regulatory speci®city Yurii Chinenov1 and Tom K Kerppola*,1 1Howard Hughes Medical Institute, University of Michigan Medical School Ann Arbor, Michigan, MI 48109-0650, USA Fos and Jun family proteins regulate the expression of a Wang et al., 1992). However, only a few of the genes myriad of genes in a variety of tissues and cell types. that mediate the essential functions of speci®c Fos and This functional versatility emerges from their interac- Jun family members have been identi®ed (Bakin and tions with related bZIP proteins and with structurally Curran, 1999; Fu et al., 2000) unrelated transcription factors. These interactions at Fos and Jun family proteins function as dimeric composite regulatory elements produce nucleoprotein transcription factors that bind to AP-1 regulatory complexes with high sequence-speci®city and regulatory elements in the promoter and enhancer regions of selectivity. Several general principles including binding numerous mammalian genes (Curran and Franza, cooperativity and conformational adaptability have 1988). Jun proteins form both homodimers and emerged from studies of regulatory complexes containing heterodimers with Fos proteins, whereas Fos proteins Fos-Jun family proteins. The structural properties of do not form homodimers and require heterodimeriza- Fos-Jun family proteins including opposite orientations tion to bind DNA. The DNA-binding and dimerization of heterodimer binding and the ability to bend DNA can domains among dierent family members are highly contribute to the assembly and functions of such conserved and dierent members of the Fos and Jun complexes.
    [Show full text]
  • Structure of the DNA-Binding Domains from NFAT, Fos and Jun Bound
    articles Structure of the DNA-binding domainsfromNFAT,Fosand Jun bound specifically to DNA 8 Lin Chen*, J. N. Mark Glover*†, Patrick G. Hogan‡, Anjana Rao‡ & Stephen C. Harrison*† * Department of Molecular and Cellular Biology and † Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA ‡ Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA ........................................................................................................................................................................................................................................................ The nuclear factor of activated Tcells (NFAT) and the AP-1 heterodimer, Fos–Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-A˚ -resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs. Engagement of the T-cell antigen receptor (TCR) by presented unrelated structures might make, and to work out the mechanism antigen induces a cascade of transcriptional responses1,2. The for this part of the convergent, multipathway T-cell response, we synthesis of interleukin (IL)-2, an important early event, depends have crystallized a quaternary complex containing the bZIP ele- on a regulatory region that extends about 300 base pairs upstream of ments of Fos and Jun, the RHR of human NFAT1 (ref. 12), and a the transcriptional start site, and several signalling pathways, from DNA fragment that has the sequence of the distal antigen-receptor the TCR and from co-stimulatory receptors, converge on this response element (ARRE2) from the murine IL-2 promoter23.
    [Show full text]