The Guanine Nucleotide Exchanger Vav2 Interacts with c-ErbB-2 and Induces Alveolar Morphogenesis of Mammary Epithelial CellsSilvana Di Cesare1341.11.1.1561.1.27891.1.310111.2121.2.1131.2.214151.2.3161.2.417181.2.519202122231.3242252.12.226272.3282.4292.52.6302.7312.8322.9332.10342.112.123353.136605#1683738541#548578#2333.240550#1863.342433.444571#60645605#19646447#6583.548605#5123.650485#3613.751453#489523.8605#3844544.1554.2564.357584.45960614.562634.664654.76667684.869 References7071727374757677787980818283848586878889909192 Abbreviations9394 Erklärung95 Curriculum Vitae9697enTable of contentsHelpThe Guanine Nucleotide Exchanger Vav2 Interacts with c-ErbB-2 and Induces Alveolar Morphogenesis of Mammary Epithelial CellsDISSERTATION zur Erlangung des akademisches Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin von
Dipl. Biochem. Silvana Di Cesare geb. am 23.09.63 in La Plata, Argentinien

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


Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Bernhard Ronacher Prof. Dr. Harald Saumweber Prof. Dr. Walter Birchmeier Prof. Dr. Franz Theuring Tag der mündlichen Prüfung: 9. November 2001

Abstract

The ErbB receptor tyrosine kinases constitute a subfamily of four structurally related members, the EGF receptor (ErbB-1), ErbB-2, ErbB-3 and ErbB-4. ErbB receptor tyrosine kinases are critical for embryonic development of central and peripheral neural structures and heart. In addition, ErbB receptors play an important role in the postnatal development of the mammary gland. Previous studies showed that activated ErbB-2 receptor induces alveolar morphogenesis of EpH4 mammary epithelial cells that are cultured on a three-dimensional matrix (termed Matrigel). However, the downstream signaling proteins that mediate this biological activity of ErbB-2 were unknown. In this work, Vav2 was identified as a direct interaction partner of tyrosine-phosphorylated ErbB receptors using the yeast two-hybrid system. Vav2 is a member of a family of guanine nucleotide exchange factors that induce cytoskeletal rearrangements, transcriptional alterations, and have oncogenic potential when activated. To test the ability of Vav2 to mediate morphogenic signals of ErbB-2, EpH4 cells overexpressing Vav2 protein were cultured on Matrigel. Indeed, Vav2 induces alveolar morphogenesis of EpH4 cells when activated either by oncogenic mutation or tyrosine phosphorylation by ErbB-2. The morphogenic activity of Vav2 requires the Dbl homology domain, which mediates GDP/GTP exchange. Dominant-negative Vav2 specifically blocks the morphogenic signals of ErbB-2 in EpH4 cells without interfering with ErbB2-induced mitogenesis. Importantly, Vav2 is co-expressed and interacts with ErbB-2 in the mammary glands of pregnant mice. Taken together, these results point to Vav2 as a candidate to mediate ErbB-2 signals for alveolar morphogenesis in vivo, which is a relevant step in the development of the mammary gland during pregnancy.

 Zusammenfassung

Die Familie der ErbB-Rezeptor-Tyrosinkinasen besteht aus vier Mitgliedern, dem EGF-Rezeptor (ErbB-1), ErbB-2, ErbB-3 und ErbB-4. ErbB-Rezeptoren spielen eine wichtige Rolle bei der Entwicklung des Nervensystems, des Herzens und der Brustdrüsen. Ein Teil dieser Differenzierungsvorgänge läßt sich in vitro nachvollziehen: so ist zum Beispiel die Aktivierung des ErbB-2 Rezeptors ausreichend für alveoläre Morphogenese der Brustdrüsenepithelzellinie EpH4. Intrazelluläre Moleküle, die dieses ErbB2-Signal übertragen, sind allerdings noch unbekannt. Mit Hilfe eines neuen, modifizierten Hefe-2-Hybrid-Systems wurde in der vorliegenden Arbeit Vav2 als neuer Interaktionspartner von ErbB-2 identifiziert. Vav2 assoziiert mit aktiviertem ErbB-2 über eine SH2-Domäne. Die Interaktion ist direkt und ist von zwei Phosphotyrosinen in ErbB-2 abhängig. Vav2 kann den GDP/GTP-Austausch bei GTPasen der Rho-Familie vermitteln. Dadurch kann der Umbau des Zytoskeletts und Veränderungen der Transkription sowie Zelltransformation induziert werden. In einem dreidimensionalen Zellkultursystem kann aktiviertes Vav2 in EpH4 Zellen die Bildung von alveolären Zellaggregaten induzieren. In diesen Alveolen umgibt eine Schicht polarisierter milchproduzierender Zellen ein zentrales Lumen. Diese Vav2-vermittelte Morphogenese ist abhängig von der katalytischen GDP/GTP-Austausch Aktivität von Vav2. Katalytisch-inaktives Vav2 kann die morphogenetische Aktivität von ErbB-2 in EpH4-Zellen verhindern, ohne die mitogene Aktivität von ErbB-2 zu beeinflussen. Vav2 ist mit ErbB-2 coexprimiert und interagiert mit dem Rezeptor in Brustdrüsenzellen schwangerer Mäuse. Diese Untersuchungen deuten darauf hin, dass Vav2 eine wichtige Funktion bei der durch ErbB-2 induzierten alveolären Morphogenese der Brustdrüse spielt.

Table of contents

  • 1  Introduction

    • 1.1 Development of the Mammary Gland

      • 1.1.1 Regulation by Systemic Hormones

      • 1.1.2 Local Effects of Multiple Growth Factors

      • 1.1.3 The Role of Extracellular Matrix

    • 1.2 The ErbB Family of Receptor Tyrosine Kinases

      • 1.2.1 ErbB Ligands Contain an EGF-like Domain

      • 1.2.2 Generation of Signal Diversity

      • 1.2.3 Intracellular Effectors of ErbB Receptors

      • 1.2.4 ErbB Signaling in Embryonic Development

      • 1.2.5 ErbB Receptors in Mammary Gland Development

    • 1.3 Vav Family of Guanine Nucleotide Exchange Factors

  • 2  Materials and Methods

    • 2.1 Amplification of the Hollenberg cDNA Yeast Expression Library

    • 2.2 Yeast Plasmids and Strains

    • 2.3 Yeast Two-Hybrid Screen

    • 2.4 Verification of Interacting Clones

    • 2.5 Mutagenesis of the Yeast TprMet-ErbB2 Baits

    • 2.6  Mammalian Expression Plasmids

    • 2.7 Site-Directed Mutagenesis

    • 2.8 Mammalian Cell Lines

    • 2.9 Immunoprecipitation and Western Blotting

    • 2.10 Matrigel Assays

    • 2.11 Light and Electron Microscopy

    • 2.12 In Situ Hybridization Analysis

  • 3  Results

    • 3.1 Yeast Two-Hybrid Screen with ErbB-2 Bait Proteins

    • 3.2 Distinct Phosphotyrosine Residues of ErbB-2 Bind to Various Interaction Partners

    • 3.3 Vav2 Interacts with Receptor Tyrosine Kinases of the ErbB Subfamily

    • 3.4 Vav2 Induces Alveolar Morphogenesis of EpH4 Mammary Epithelial Cells

    • 3.5 Vav2 and ErbB-2 Can Both Directly and Indirectly Associate in Mammalian Cells

    • 3.6 Vav2 and ErbB-2 Are Associated in Mammary Epithelium During Pregnancy

    • 3.7 The Dbl-Homology Domain of Vav2 Is Required for Its Morphogenic Activity in EpH4 Cells

    • 3.8 Catalytically Inactive Vav2 Blocks Neuregulin-Mediated Morphogenesis of EpH4 Cells

  • 4  Discussion

    • 4.1 Modified Yeast Two-Hybrid System: A Powerful Tool to Search for Phosphotyrosine-Interacting Proteins

    • 4.2 New Insights into the Intracellular Signaling Pathways of ErbB-2

    • 4.3 Vav2 Couples to Receptor Tyrosine Kinases via Its SH2 Domain

    • 4.4 Vav2 Mediates ErbB-2 Signals for Alveolar Morphogenesis of EpH4 Mammary Epithelial Cells

    • 4.5  The Morphogenic Activity of Vav2 on Mammary Epithelial Cells May Involve Changes in Actin Cytoskeleton

    • 4.6 Vav2 Is a Specific Effector of Neuregulin Signals for Alveolar Morphogenesis

    • 4.7 Possible Molecular Mechanisms Involving Vav2 as Critical Effector of Alveolar Morphogenesis

    • 4.8 Conclusions

  • References

  • Abbreviations

  • Erklärung

  • Curriculum Vitae

Tables

  • Table 1. Interaction partners of ErbB-2 isolated in independent yeast two-hybrid screens using different baits.

  • Table 2. Characterization of specific binding sites of ErbB-2 for its interaction partners.

  • Table 3. Interaction of the various ErbB-2 partners with different receptor tyrosine kinases.

Images

  • Figure 1. Structure of the chimeric ErbB-2 baits used in yeast two-hybrid screens.

  • Figure 2. Interaction partners of ErbB-2 in the yeast system are SH2 domains.

  • Figure 3. The SH2 domains of substrates isolated in yeast two-hybrid screens interact with phosphotyrosine residues of the ErbB-2 C-terminal tail.

  • Figure 4. Vav2 directly interacts with phosphotyrosines Y1 and Y3 of c-ErbB-2.

  • Figure 5. Constitutively active Vav2 induces alveolar morphogenesis of EpH4 mammary epithelial cells.

  • Figure 6. Constitutively active Vav2 induces polarization of alveolar EpH4 mammary epithelial cells.

  • Figure 7. Tyrosine phosphorylation of full-size Vav2 by ErbB-2 activates its morphogenic potential.

  • Figure 8. Analysis of the interaction between Vav2 and ErbB-2 in mammalian cells and in mammary tissue.

  • Figure 9. Vav2 and ErbB-2 are co-expressed in mammary alveolar epithelium during pregnancy.

  • Figure 10. The Dbl homology domain of Vav2 is essential to promote alveolar morphogenesis of EpH4 mammary epithelial cells.

  • Figure 11. Catalytically inactive Vav2 harbouring a mutation in the Dbl homology domain blocks the morphogenic signals of neuregulin in EpH4 mammary epithelial cells.

  • Figure 12. Catalytically inactive Vav2 does not interfere with branching morphogenesis of EpH4 mammary epithelial cells.