Functional investigation of the class II tumor suppressor gene H-REV107-1

Dissertation
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von

Irina Nazarenko

geb. am 24. Januar 1975 in Kustanay, Kasachstan

Präsident der Humboldt-Universität zu Berlin:
Prof. Dr. Jürgen Mlynek

Dekan: Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Michael Linscheid

Gutachter:
1. Prof. Dr. Thomas Börner
2. Prof. Dr. Reinhold Schäfer
3. Prof. Dr. Dr. Christian Hagemeier

Tag der Einreichung:11.06.03
Tag der mündlichen Prüfung:16.09.03

Zusammenfassung

Das Klasse II Tumorsuppressor-Gen H-REV107-1, ist in normalen Geweben ubiquitär exprimiert, zeigt jedoch häufig Expressionsverluste, vorzugsweise in Tumoren des Brustgewebes, des Ovars und der Lunge. Das H-REV107-1 Protein wirkt in vitro und in vivo als Wachstumssuppressor. Um den Mechanismus der H-REV107-1 bedingten Wachstumshemmung zu verstehen, haben wir mit Hilfe des LexA-basierten Hefe-2-Hybrid Systems interagierende Proteine identifiziert. Diese Suche wurde mit einem H-REV107-1 Deletionskonstrukt durchgeführt, dem die Membran-bindende Domäne fehlte. Diese Analyse lieferte eine Vielzahl von potentiellen Interaktionspartnern, darunter der Retinsäure Rezeptor RARG, das Calcium-bindende Proteine S100A6, der Translations-Elongationsfaktor ETF und das weitgehend unbekannte Protein p14.5 Die Bindungen des H-REV107-1 Proteins an die beiden potentiellen Kandidaten, den Transkriptionsfactor PC4 und die regulatorische Untereinheit der Protein Phosphatase 2A (PR65), wurden genauer untersucht.

Wir haben dabei einen Proteinkomplex aus H-REV107-1, PC4 und STAT1 (Signal Transducer and Activator of Transcription 1) identifiziert, der vermutlich eine Rolle in der IFNγ - abhängigen Wachstumshemmung in Ovarialkarzinom Zellen spielt. Da sich die Expression des H-REV107-1 Gens durch IFNγ über den Transkriptionsfaktor IRF-1 stimulieren läßt, und in verschiedenen Zelllinien sowohl zur Hemmung des Wachstums, als auch zur Apoptose führt, vermuteten wir verschiedene Mechanismen der Wachstumshemmung durch den IFNγ-Signalweg und H-REV107-1.

Weitere Analysen der H-REV107-1 – vermittelten Apoptose zeigten, daß die Interaktion zwischen H-REV107-1 und PR65 eine wichtige Rolle in diesem Prozeß spielt. Um die Proteindomäne zu identifizieren, welche für die direkte Wechselwirkung von H-REV107-1 mit PR65 verantwortlich ist, wurden H-REV107-1 Mutanten generiert und mittels Co-Immunpräzipitation getestet. Die Prolin-reiche Sequenz am N-Terminus des H-REV107-1 Proteins konnte als verantwortliche Domäne für die Interaktion bestimmt werden.

Die funktionelle Analyse dieser Interaktion zeigte die Hemmung der Protein Phosphatase 2A (PP2A) Aktivität in Ovarialkarzinom Zellen durch H-REV107-1. Der Einsatz der Mutanten im Phosphatase-Aktivitätstest zeigte, daß die selbe Domäne, die die Interaktion vermittelt, auch für die Hemmung der Phosphatase 2A verantwortlich ist. Diese Fakten deuteten auf eine wichtige Rolle der Phosphatase 2A in Ovarialkarzinom Zellen hin, weil sowohl die Verwendung des PP2A Inhibitors (Okadainsäure), als auch die Transfektion der Zellen mit einem H-REV107-1 - Expressionsplasmid zur Apoptose führten. Damit konnten wir zeigen, daß PP2A für das Überleben der Ovarialkarzinomzellen notwendig ist, und die Reaktivierung des H-REV107-1 Proteins durch IFNγ zur Hemmung der Phosphatase und damit zur Apoptose führt.

Eigene Schlagworte: H-REV107-1, PP2A, Apoptose, IFNγ, Tumorsuppressor

Abstract

The H-REV107-1 class II tumor suppressor gene is ubiquitously expressed in normal tissues and down-regulated in human breast, ovarian and lung tumours. H-REV107-1 has the capacity to suppress growth of tumour cells in vitro and in vivo. To understand the mechanism of H-REV107-1 mediated growth suppression I performed a search for H-REV107-1 interacting proteins using a LexA-based yeast two-hybrid system. I screened a human kidney cDNA library with a truncated form of the H-REV107-1 as a bait. This analysis revealed numerous potential interaction partners. Among them the retinoic acid receptor gamma (RARG), the calcium-binding protein S100A6, the translation termination factor ETF1, and the potential translational inhibitor protein P14.5.

The interaction of H-REV107-1 with the transcriptional co-activator PC4 and with the regulatory subunit A of protein phosphatase 2A (PR65) was analysed in detail. H-REV107-1 binds ectopically expressed and endogenous PC4. In addition, a multiprotein complex between H-REV107-1, PC4 and the signal transducer and activator of transcription 1 (STAT1) was demonstrated. This complex is likely to be involved in IFNγ-mediated growth suppression of ovarian carcinoma cells. Endogenous H-REV107-1 can be induced after application of IFNγ through the IRF-1 transcription factor. This up-regulation of H-REV107-1 leads either to growth suppression via a G1 arrest or to apoptosis depending on the cell line, suggesting different mechanisms of IFNγ-, and H-REV107-1- mediated growth suppression.

Further investigation of the mechanism of H-REV107-1-dependent apoptosis revealed an important role of the interaction between H-REV107-1 and the PR65 protein. The use of several H-REV107-1 mutant proteins generated after disruption of the highly conserved domains identified the proline-rich N-terminal domain responsible for the interaction with PR65 in Co-immunoprecipitation studies. Functional investigation of the H-REV107-1 – PR65 interaction demonstrated that wild-type H-REV107-1 is able to inhibit PP2A activity, however a mutant protein lacking the N-terminal domain was devoid of this function. We sought to identify the functional relevance of the PP2A activity in ovarian carcinoma cells with normally have suppressed the H-REV107-1 gene. Treatment of OVCAR-3 cells with the PP2A inhibitor Okadaic acid and transient transfection of the cells with wild-type H-REV107-1 resulted in the activation of caspase-9, suggesting a role for PP2A in the survival of ovarian carcinoma cells. We suggest, that the down-regulation of H-REV107-1 in ovarian carcinomas is a prerequisite for the PP2A-dependent activation of yet unknown signalling pathways mediating tumour cell survival. Reactivation of H-REV107-1 gene expression via IFNγ leads to the inhibition of PP2A activity and tumour cell death.

Keywords: H-REV107-1, PP2A, Apoptosis, IFNγ, Tumor Suppressor

Table of contents

• 1.  Introduction
  • 1.1. Multi-Step Progression of Tumors
    • 1.1.1. Oncogenes
    • 1.1.2. Tumor Suppressor Genes
      • 1.1.2.1. The Class I Tumor Suppressor Genes
      • 1.1.2.2. The Class II Tumor Suppressor Genes
    • 1.1.3. Mechanisms of Gene Silencing
      • 1.1.3.1. DNA Methylation and Deacetylation
      • 1.1.3.2. Inhibition of Positive Regulators of Transcription
      • 1.1.3.3. Inhibition of Expression by Activation of Oncogenic Signaling
  • 1.2. H-REV107-1 is a Member of the NlpC/P60 Protein Superfamily
    • 1.2.1. The NlpC/P60 Protein Superfamily
    • 1.2.2.  The LRAT-Like Protein Family
  • 1.3.  Purpose of this Work
• 2.  Materials and Methods
  • 2.1. Materials
    • 2.1.1. Chemicals
    • 2.1.2. Kits
    • 2.1.3.  Enzymes
    • 2.1.4.  Antibodies
    • 2.1.5.  Fluorophore-Labelled Antibodies
    • 2.1.6. cDNA Library
    • 2.1.7. Mammalian Cell Lines
    • 2.1.8.  E. coli Strains
    • 2.1.9.  Yeast Strains (OriGene Technologies, Inc., MD, USA)
    • 2.1.10. Plasmids and Expression Constructs
    • 2.1.11. Oligonucleotides (MWG-Biotech, Ebersberg, Germany)
  • 2.2.  Methods
    • 2.2.1.  Yeast Two-Hybrid System
      • 2.2.1.1. Yeast Expression Vectors and General Procedure
      • 2.2.1.2. Yeast Strain Storage and Culturing
        • 2.2.1.2.1. Storage
        • 2.2.1.2.2. Culturing
      • 2.2.1.3.  Yeast Transformation
        • 2.2.1.3.1. Preparation of Fresh Competent Yeast
        • 2.2.1.3.2. Transformation
      • 2.2.1.4. β-Galactosidase Assay
        • 2.2.1.4.1. In Vivo Plate Assay Using X-gal – Containing Medium
        • 2.2.1.4.2.  Colony-Lift Filter Assay
      • 2.2.1.5. Secondary Test of Positives Colonies
      • 2.2.1.6. Mating Test
      • 2.2.1.7. Yeast Plasmid Isolation
      • 2.2.1.8. Preparation of Electrocompetent E. coli KC8
      • 2.2.1.9. Transformation of the Electrocompetent E. coli KC8 with Yeast Plasmids
      • 2.2.1.10. Yeast Protein Isolation
    • 2.2.2.  Bacterial Culture
      • 2.2.2.1.  Routine Culturing and Storage Conditions
      • 2.2.2.2.  Transformation
      • 2.2.2.3. Mini-Preparation of Plasmid DNA
      • 2.2.2.4. Large-Scale Preparation of Plasmid DNA
      • 2.2.2.5. Measurement of DNA Concentration
    • 2.2.3. Enzymatic Manipulation and Analysis of DNA
      • 2.2.3.1. Digestion of DNA with Restriction Endonucleases
      • 2.2.3.2.  DNA Ligation
      • 2.2.3.3. Vector Dephosphorylation
      • 2.2.3.4.  Polymerase Chain Reaction (PCR)
      • 2.2.3.5.  Purification of PCR-Amplified Fragments of DNA
      • 2.2.3.6. Site-Directed Mutagenesis
      • 2.2.3.7. Sequencing
      • 2.2.3.8. Electrophoretic Separation of DNA
      • 2.2.3.9. Elution of DNA Fragments from a Gel
    • 2.2.4.  Culturing of Mammalian Cells
      • 2.2.4.1.  Routine Culturing
      • 2.2.4.2. Freezing and Thawing Procedure
      • 2.2.4.3. Cell Treatments
      • 2.2.4.4.  Transfection of Mammalian Cells
      • 2.2.4.5. Colonie Formation Assay
    • 2.2.5. Apoptosis Assays
      • 2.2.5.1. DAPI-Staining of Apoptotic Nuclei
      • 2.2.5.2. Flow Cytometric Analysis of Annexin V
    • 2.2.6. Analysis of Proteins
      • 2.2.6.1. Protein Isolation from Mammalian Cells
      • 2.2.6.2.  Subcellular Fractionation
      • 2.2.6.3. Determination of Protein Concentration
      • 2.2.6.4. One-Dimensional SDS Gel Electrophoresis (PAGE)
      • 2.2.6.5. Western Blot Analysis
    • 2.2.7.  Protein interaction analysis
      • 2.2.7.1. Glutathione-S-Transferase Fusion System
        • 2.2.7.1.1. Production of GST-Fusion Protein in Bacteria
        • 2.2.7.1.2. GST Pull-Down Assay
    • 2.2.8. Co-Immunoprecipitation
    • 2.2.9. Immunofluorescence Analysis and Confocal Microscopy
    • 2.2.10. Phosphatase Assay
• 3.  Results
  • 3.1. Identification of Proteins Interacting with H-REV107-1
    • 3.1.1. Screening of a Human Kidney cDNA Library to Identify Potential Interacting Partners of the H-REV107-1 Protein
    • 3.1.2.  Sequencing Analysis of Clones Encoding Putative Interaction Partners of the H-REV107-1 Protein
    • 3.1.3. Verification of Specificity of Interactions Using the Mating Test
    • 3.1.4.  Examination of Protein Expression in Yeast
    • 3.1.5.  Generation of the H-REV107-1V5 and ΔCH-REV107-1HA Expression Vectors.
  • 3.2.  PC4
    • 3.2.1. H-REV107-1 Interacts with PC4 in COS-7 Cells
    • 3.2.2.  Examination of the Intracellular Localisation of the Ectopically Expressed H-REV107-1 and PC4 Proteins
    • 3.2.3. H-REV107-1 Interacts with Endogenous PC4 in COS-7 Cells
    • 3.2.4. H-REV107-1, PC4, and STAT1 Form a Protein Complex related to IFNγ-signaling
  • 3.3. PR65
    • 3.3.1. H-REV107-1 Interacts with PR65 in COS-7 Cells
    • 3.3.2.  H-REV107-1 and PR65 are Co-Localised in COS-7 Cells
    • 3.3.3.  H-REV107-1 Interacts with PR65 in a Cell-Free System
    • 3.3.4. Homodimer Formation of H-REV107-1
    • 3.3.5.  Determination of the H-REV107-1 Domains Responsible for Interaction with PR65 and Homodimer Formation
      • 3.3.5.1. Generation of the H-REV107-1 Mutant Proteins
      • 3.3.5.2. The N-terminal Domain of the H-REV107-1 Protein is Required for the Interaction with PR65 and for Homodimer Formation
  • 3.4. Investigation of a Role of the H-REV107-1 - PR65 Interaction in Apoptosis
    • 3.4.1. H-REV107-1 Does not Induces Apoptosis in Rat Fibroblasts FE-8
    • 3.4.2. The ΔC107-ΔN Interaction Deficient Mutant Fails to Induce Apoptosis in Human Ovarian Carcinoma Cell Lines A27/80 and OVCAR-3
    • 3.4.3. Cellular Re-Distribution of the Endogenous PR65 Protein Correlates with the H-REV107-1 Induced Apoptosis in OVCAR-3 Cells
    • 3.4.4. H-REV107-1 Inhibits PP2A Activity in vitro
    • 3.4.5. Okadaic Acid Induces Apoptosis in OVCAR-3 Cells
    • 3.4.6. PP2A Inhibition in OVCAR-3 Cells Leads to the Activation of Procaspase-9
  • 3.5. Confirmation of Interaction between H-REV107-1 and RARG, S100A6, ETF1, and P14.5
    • 3.5.1. RARG
    • 3.5.2. S100A6, ETF1, and P14.5
      • 3.5.2.1. S100A6
      • 3.5.2.2. ETF1
      • 3.5.2.3.  P14.5
• 4.  Discussion
  • 4.1. Yeast Two–Hybrid System
  • 4.2. H-REV107-1 is a Target of IRF-1 and Modulates IFNγ - Dependent Inhibition of Cellular Growth by Different Mechanisms
  • 4.3. H-REV107-1 Participates in the Cross-Talk between Retinoic Acid and IFNγ-Dependent Pathways
  • 4.4. H-REV107-1 – Mediated Cell Death through Inhibition of PP2A Activity
  • 4.5. Possible Participation of H-REV107-1 in Calcium Metabolism
• List of Abbreviations
• References
• Danksagung
• Selbständigkeitserklärung

Tables

• Table 1 Human proteins belonging to the LRAT-like family of proteins
• Table 2 Generated yeast strains and respective selective media
• Table 3 List of genes encoding putative interacting partners of the H-REV107-1 protein
• Table 4 List of known false positives found in yeast two-hybrid screenings
• Table 5 List of the genes encoding potential H-REV107-1 interacting partners chosen for the mating test
• Table 6 Cell cycle analysis of FE-8 cells harbouring tet-inducible H-rev107

Images

• Fig. 1 Schematic presentation of the NlpC/P60 protein superfamily
• Fig. 2 Circular permutation of the NlpC/P60 conservative domain
• Fig. 3 Aminoacid sequence alignment of the nine human proteins belong to the LRAT-like protein family
• Fig. 4 Schematic diagram of the LexA Two-Hybrid System (BD Biosciences, Clontech, CA, USA)
• Fig. 5 Screening of a AD fusion library for proteins that interact with H-REV107-1 (BD Biosciences, Clontech, CA, USA)
• Fig. 6 Yeast Two Hybrid screen for H-REV107-1 interacting proteins, and strategy for their validation
• Fig. 7 Interaction between H-REV107-1 and potential binding proteins found in the yeast two-hybrid screening is verified using the mating assay
• Fig. 8 Expression of the S100A6, ETF1, PC4, and P14.5 proteins is confirmed in yeast
• Fig. 9 The H-REV107-1 HA and V5 fusion proteins
• Fig. 10 The PC4-V5 and ΔCH-REV107-1HA proteins interact with each other in COS-7 cells
• Fig. 11 The H-REV107-1 protein is distributed through the cytoplasm in COS-7 cells, whereas the PC4-V5 protein is localised preferably in the nucleus
• Fig. 12 The H-REV107-1 protein is distributed through the nuclear and cytoplasmic fractions of the transiently transfected COS-7 cells
• Fig. 13 H-REV107-1 interacts with the endogenous PC4 protein
• Fig. 14 Up-regulation of H-REV107-1 expression in OVCAR-3 cells after IFNγ-induction
• Fig. 15 Induction of the H-REV107-1 expression upon IFNγ - treatment leads to cell death
• Fig. 16 STAT1 and P21WAF1 expression after IFNγ - induction in OVCAR-3 and A27/80 cells.
• Fig. 17 STAT1 and PC4 proteins interact with H-REV107-1
• Fig. 18 Co-immunoprecipitation of ΔCH-REV107-1HA and PR65-V5 in COS-7 cells
• Fig. 19 The H-REV107-1 and PR65-V5 proteins ectopically expressed in COS-7 cells are co-localised in the cytoplasm
• Fig. 20 H-REV107-1 interacts with PR65 in a cell-free conditions
• Fig. 21 The H-REV107-1 protein forms a homodimer in COS-7 cells
• Fig. 22 H-REV107-1 mutants generated for search of the domains responsible for interaction
• Fig. 23 The ΔC107-ΔN mutant does not form homodimers
• Fig. 24 The ΔN107 mutant fails to interact with PR65
• Fig. 25 H-rev107 expression after doxycycline induction in FE-8 cells
• Fig. 26 Reduction of colony formation in OVCAR-3 and A27/80 cells
• Fig. 27 Induction of apoptosis in OVCAR-3 cells after H-REV107-1 over-expression is correlated with a re-distribution of the PR65 protein.
• Fig. 27 Induction of apoptosis in OVCAR-3 cells after H-REV107-1 over-expression is correlated with a re-distribution of the PR65 protein.
• Fig. 28 The H-REV107-1 protein inhibits the catalytic activity of PP2A in vitro
• Fig. 29 Control Western blot analysis of the precipitated protein complexes
• Fig. 30 Induction of apoptosis by OA treatment in OVCAR-3 cells
• Fig. 31 H-REV107-1 overexpression and OA treatment of OVCAR-3 cells activate procaspases –9 and –3
• Fig. 32 TTNPB and DR5 are essential for the interaction between RARG and H-REV107-
• Fig. 33 Calcyclin (S100A6) interacts with H-REV107-1 in COS-7 cells
• Fig. 34 H-REV107-1 interacts weakly with ETF1 in COS-7 cells
• Fig. 35 H-REV107-1 fails to bind P14.5 in COS-7 cells
• Fig. 36 Prediction of protein phosphorylation sites of the H-REV107-1 protein
• Fig. 37 Hypothetical scheme of H-REV107-1 participation in the IFNγ-signaling in OVCAR-3 and A27/80 cells
• Fig. 38 Schematic presentation of the mechanism of H-REV107-1 – mediated cell death