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Integrin-Mediated Cell Adhesion

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Nina Dierdorf

an der

Universität Konstanz des Fachbereichs Biologie

Konstanz, Juni 2015

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Tag der mündlichen Prüfung: 24.09.2015

Erstgutachter: Prof. Dr. Christof Hauck

Zweitgutachter: Prof. Dr. Daniel Legler

Drittgutachter: Prof. Dr. Steffen Backert

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Meinen Eltern

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An dieser Stelle möchte ich mich ganz herzlich bei einigen Menschen bedanken, die mich während meiner Promotionszeit begleitet haben.

Mein besonderer Dank gilt Prof. Christof Hauck, der mir in meiner gesamten Promotionsphase mit seinem fachlichen Knowhow und seiner freundlichen Art stets zur Seite stand, es schaffte mich in den richtigen Momenten zu motivieren und mir Mut zu machen. Lieber Christof, vielen Dank für die lehrreichen und schönen Jahre in deinem Labor!

Des Weiteren bedanke ich mich ganz herzlich bei Prof. Daniel Legler für die freundliche Übernahme des Zweitgutachtens und Prof. Alexander Bürkle für den Prüfungsvorsitz.

Ich möchte mich bei allen Mitgliedern der AG Hauck für diese erlebnisreiche und schöne Zeit bedanken. Das gute Miteinander in unserer Gruppe wird mir immer mit Freude in Erinnerung bleiben. Besonders bedanke ich mich bei unserer Sekretärin Anne für ihr organisatorisches Talent und ihre herzliche Art, bei unseren tollen Laborengeln Susi, Petra und Claudia für ihre Unterstützung und ihr Engagement, sowie bei meiner Masterstudentin Sarah für ihre Mithilfe an der Phosphatasen-Front.

Lexi möchte ich für die schöne gemeinsame Urlaubszeit danken, die mich immer erfolgreich allen Stress hat vergessen lassen und die ich sehr genossen habe. Bei Alexa bedanke ich mich für ihre unverwechselbare Art, durch die immer alles fröhlicher und besser wurde.

Des Weiteren bedanke ich mich bei Chris, Julia und Arnaud und meinen ehemaligen Kollegen Naja, Alex B., Maike, Timo und Nori für ihre Aufrichtigkeit und Freundschaft, für ihre Hilfsbereitschaft, ihren Rat in fachlichen Fragen, den Spaß und das Lachen im und außerhalb des Labors.

Bei meinem Konstanzer Familienersatz Ina, Dusi, Alex, Kerstin und Ragi möchte ich mich für ihre Unterstützung, ihr Verständnis und die tolle gemeinsame Zeit bedanken.

Zu guter Letzt gilt mein Dank meiner Familie und besonders meinen wundervollen Eltern. Ich sage euch viel zu selten, wie dankbar ich bin, euch an meiner Seite zu haben!

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Inhaltsverzeichnis

Summary ... 1

Zusammenfassung ... 3

1. Introduction ... 5

1.1. The Integrin Family ... 5

1.1.1. Integrins and Diseases ... 6

1.1.2. Integrin Structure and Signaling ... 9

1.1.3. Integrin Activators and Inactivators ... 13

1.1.4. Site-Specific Phosphorylation of Integrins and Their Regulators ... 17

1.2. Phosphatases ... 19

1.2.1. The Serine/Threonine Phosphatase PPM1F ... 22

1.3. Aims of the Work ... 26

2. Results ... 28

2.1. An ShRNA-Based Screening Approach Identified PPM1F as a Negative Regulator of Integrin-Mediated Cell Adhesion... 28

2.2. PPM1F Negatively Regulates β1 Integrin Activity and Affects Integrin-Mediated Cell Migration in Primary Fibroblasts ... 32

2.3. Loss of PPM1F Results in Redistribution and Clustering of Talin- Positive Focal Adhesions ... 33

2.4. PPM1F Co-Localizes with the Inactive β1 Integrin and Its Negative Regulator FilaminA Along Actin Stress Fibers... 35

2.5. PPM1F Dephosphorylates T788/T789 within β1 Integrin Tail ... 37

2.6. Dephosphorylation of β1 IntegrinT788/T789 Affects Binding of the Integrin Regulators FilaminA and Talin ... 41

2.7. PPM1F Knockout Leads to Developmental Defects and Embryonic Lethality at E10.5 in Mice ... 45

2.8. PPM1F Knockout Embryos Exhibit an Abnormal Forebrain Structure ... 49

2.9. PPM1F Knockout Fibroblasts Display Enhanced β1 Integrin

Activity and β1 Integrin Tail Phosphorylation ... 51

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Inhaltsverzeichnis

2.10. Re-Expression of Wildtype but not the Inactive PPM1F in

Knockout Fibroblasts Restores Cell Migration Potential ... 52

2.11. PPM1F+/- Mice Exhibit Reduced PPM1F Brain Expression and Display Increased Activity in a 1 h Open Field Test ... 54

3. Discussion and Outlook ... 57

3.1. PPM1F: The Molecular Switch to Inactivate Integrins ... 57

3.2. PPM1F and Its Role in Cell Migration ... 64

3.3. Manipulating PPM1F Phosphatase for Therapeutic Benefits ... 67

3.4. PPM1F and Its Role in Brain Development ... 69

3.5. Heterozygous PPM1F+/- Mice: A Novel Model to Study ADHD? .. 71

4. Material ... 75

4.1. Eukaryotic Cells ... 75

4.2. Media for Eukaryotic Cells... 77

4.3. Prokaryotic Cells ... 77

4.4. Media for Prokaryotic Cells ... 78

4.5. Antibiotics ... 78

4.6. Antibodies ... 78

4.6.1. Primary Antibodies ... 78

4.6.2. Secondary Antibodies ... 80

4.7. Dyes and Toxins ... 81

4.8. Enzymes and Proteins ... 81

4.9. Plasmids ... 82

4.10. Phospho-Peptides ... 83

4.11. Oligonucleotides ... 84

4.12. Buffers and Solutions ... 88

4.13. Chemicals ... 92

4.14. Kits ... 92

4.15. Laboratory Equipment and Consumables ... 92

4.16. Software ... 93

5. Methods ... 94

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5.1. Standard Laboratory Work ... 94

5.2. Work with Eukaryotic Cells ... 94

5.2.1. Cell Culture, Transfection of Cells and Cell Lysis ... 94

5.2.2. Isolation of Genomic DNA from Eukaryotic Cells ... 95

5.2.3. Lentivirus Production and Generation of Stable Cell Lines ... 95

5.2.4. Cell Adhesion Assay ... 95

5.2.5. Re-Plating Assay ... 96

5.2.6. Integrin Activity Assay (Adhering Cells) ... 96

5.2.7. Integrin Activity Assay (Suspending Cells) ... 97

5.2.8. Immunofluorescence Staining for FACS Analysis ... 97

5.2.9. Immunofluorescence Staining for Microscopic Preparations ... 98

5.2.10.Single Cell Tracking... 98

5.3. Work with Mice ... 99

5.3.1. Mice and Mice Maintenance ... 99

5.3.2. Isolation of Genomic DNA from Tail Biopsies ... 99

5.3.3. Genotyping ... 99

5.3.4. Generation of PPM1F Knockout Fibroblasts ... 100

5.3.5. LacZ Staining of Frozen Tissue Sections ... 101

5.3.6. Fluorescent Immunohistochemistry Staining of Frozen Mouse Embryonic Tissue Sections ... 101

5.3.7. Whole-Mount Histochemical Detection of β-Galactosidase Activity . 102 5.3.8. Paraffin Embedding, Sectioning and Mounting of Whole-Mount Stained Embryos ... 103

5.3.9. Fixation, Paraffin Embedding and Sectioning of E10.5 Embryos... 104

5.3.10.Behavioral Testing: 1-h Open Field Test ... 104

5.4. Molecular Biological Methods ... 105

5.4.1. Generation of DNA Constructs ... 105

5.4.2. SOEing (Synthesis by Overlap Extension) PCR for the Generation of a PPM1F Rescue Mutant ... 107

5.4.3. Site Directed Mutagenesis ... 108

5.4.4. ShRNA Construction and Cloning ... 108

5.5. Protein Biochemical Methods ... 109

5.5.1. Expression and Phosphate-Free Purification of GST-Tagged PPM1F and PPM1FD360A in HEK293T Cells ... 109

5.5.2. In vitro Kinase Assay ... 109

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Inhaltsverzeichnis

5.5.3. In vitro Phosphatase Assay ... 110

5.5.4. Protein Microarray ... 110

5.5.5. Generation of a Polyclonal Antibody Directed Against mPPM1F ... 111

6. References ... 113

7. Appendix ... 125

7.1. Publications ... 125

7.1.1. Publication Part of This Thesis ... 125

7.1.2. External Publications ... 125

7.2. Declaration of Contributions ... 125

7.3. List of Figures ... 126

7.4. List of Tables ... 127

7.5. Abbreviations ... 128

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Summary

Integrin-mediated cell adhesion is a fundamental process that is critical for the formation and maintenance of multicellular organisms. Integrins are widely expressed transmembrane receptors, indispensable for a multitude of physiological events such as embryogenesis, maintenance of the tissue integrity, survival or immune response. These cellular processes are a result of a complex and precisely controlled interplay of numerous integrin-associated proteins including protein and lipid kinases, phosphatases, small G-proteins and adaptor proteins. To allow cells to sense and respond to their variable microenvironment, integrins have developed a responsive receptor activation mechanism that is characterized by large conformational changes which have been termed integrin activation.

In this study, we identified the serine/threonine phosphatase PPM1F in an shRNA-based screening approach to negatively affect integrin-mediated cell adhesion. Knockdown of PPM1F in different cell types led to increased cell adhesion due to enhanced β1 integrin activity. Consequently, integrin-dependent cellular functions such as cell migration were significantly impaired in PPM1F knockout fibroblasts. Re-expression of the wildtype phosphatase, but not the enzymatic dead version, could rescue cell migration capability.

We further found PPM1F to co-localize with the inactive β1 integrin and the integrin inactivating protein FilaminA along the actin cytoskeleton and additionally to affect the subcellular distribution of the integrin activator Talin. We discovered that PPM1F directly dephosphorylates the highly conserved threonine residues Thr788/Thr789 within the cytoplasmic tail of β1 integrin and thereby modulates the binding properties of the integrin regulators Talin and FilaminA. While Talin seems to prefer binding to the phosphorylated threonine residues, FilaminA association is negatively affected by phosphorylation. Thereby, PPM1F activity constitutes a molecular switch that orchestrates protein association with β1 integrin tails. To address the relevance of PPM1F function in an intact organism, we employed mice exhibiting a disruption of the PPM1F gene. Mating of heterozygous PPM1F+/- mice did not result in homozygous offspring and the genotypic ratio suggested embryonic lethality. Interestingly, we could identify homozygous knockout embryos at embryonic day E10.5 but not at later time

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Summary

points. We further observed that the complete loss of PPM1F resulted in brain developmental defects. PPM1F deficient embryos exhibited an abnormal forebrain structure characterized by a disturbed organization and orientation of neural progenitor cells within the ventricular zone of the telencephalon. Additionally, knockout embryos displayed a defective delimitation of the pia mater and the neuroepithelial cells. We further received the first indication that PPM1F might also support proper brain functions in adult mice. Notably, heterozygous PPM1F knockout mice showed reduced PPM1F brain expression levels accompanied by elevated activity and reduced anxiety- related behavior in a 1h open field test. Taking together, the data presented in this thesis identify PPM1F as an essential protein phosphatase, which controls a phospho-switch to regulate integrin activity and integrin-mediated processes. Furthermore, PPM1F activity is particular important during mammalian brain development and its absence cannot be compensated by other phosphatases. Modulation of PPM1F expression or activity might strike a new path to manipulate integrin function in cells and tissues.

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Zusammenfassung

Die Integrin-vermittelte Zelladhäsion ist ein elementarer Prozess, der essentiell für die Entstehung und Aufrechterhaltung von multizellulären Organismen ist. Integrine sind ubiquitär exprimierte Transmembranrezeptoren, welche unabdingbar für eine Reihe von physiologischen Ereignissen wie die Embryogenese, die Aufrechterhaltung der Gewebeintegrität, das Überleben oder die Immunantwort sind. Diese zellulären Prozesse sind das Ergebnis eines komplexen und fein regulierten Zusammenspiels von verschiedensten Integrin-assoziierten Proteinen wie zum Beispiel Protein- und Lipidkinasen, Phosphatasen, kleinen G-Proteinen und Adapterproteinen. Damit Zellen ihre Umgebung wahrnehmen und auf sie reagieren können, werden Integrine in ihrer Bindungsfähigkeit an Liganden reguliert, ein Prozess, welcher als Integrin Aktivierung bezeichnet wird und durch umfangreiche Konformationsänderungen der Integrin Untereinheiten gekennzeichnet ist.

In dieser Studie haben wir mittels eines shRNA-basierten Screens die Serin/Threonin Phosphatase PPM1F identifiziert, welche einen negativen Einfluss auf die Integrin- vermittelte Zelladhäsion hat. Der Knockdown von PPM1F führte zu einer Zunahme der Zelladhäsion in verschiedenen Zelltypen und wurde außerdem von erhöhter Integrinaktivität begleitet. Infolgedessen wurde auch die Integrin-abhängige Zellmigration in PPM1F dezimierten Fibroblasten stark beeinträchtigt. Die Reexpression der aktiven, nicht aber der enzymatisch inaktiven Phosphatase, konnte die Bewegungsfähigkeit der Zellen wieder vollständig herstellen. Des Weiteren konnten wir feststellen, dass PPM1F gemeinsam mit dem inaktiven β1 Integrin und dem Integrin- inaktivierenden Protein FilaminA entlang des Aktin Zytoskeletts lokalisiert und zusätzlich die subzelluläre Verteilung des Integrin-aktivierenden Proteins Talin beeinflusst. Wir haben herausgefunden, dass PPM1F direkt die hoch konservierten Threoninreste Thr788/Thr789 in der zytoplasmatischen Domäne von β1 Integrin dephosphoryliert und dadurch die Bindungseigenschaften der Integrin Regulatoren Talin und FilaminA beeinflusst. Während Talin die Bindung an die phosphorylierten Threoninreste zu bevorzugen scheint, wird die Assoziation von FilaminA durch die Phosphorylierung beeinträchtigt. Dies lässt den Schluss zu, dass PPM1F wie ein molekularer Schalter wirkt, welcher die Bindung von Proteinen an die zytoplasmatische

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Zusammenfassung

Domäne von β1 Integrin reguliert. Zusätzlich haben wir uns gefragt, wie relevant die Funktion von PPM1F in einem intakten Organismus und im Kontext anderer Serin/Threonin Phosphatasen ist. Um diese Frage zu beantworten, haben wir Mäuse mit einer Genetrap-Mutation erworben. Durch Kreuzungen von heterozygoten PPM1F Tieren haben wir herausgefunden, dass es keinen homozygoten Nachwuchs -weder nach dem Abstillen noch perinatal- gibt. Auch das genotypische Verhältnis von Wildtyp zu heterozygoten Tieren weist darauf hin, dass der komplette Verlust von PPM1F zum Abbruch der Embryonalentwicklung führt. An Embryonaltag E10.5 konnten homozygote Knockout Embryos in utero identifiziert werden, nicht aber zu späteren Zeitpunkten der Embryonalentwicklung. Des Weiteren haben wir festgestellt, dass der komplette Verlust von PPM1F zur Schädigungen in der embryonalen Hirnentwicklung führt. PPM1F defiziente Embryonen wiesen eine abnormale Vorderhirn Struktur auf, die durch eine gestörte Orientierung der neuralen Vorläuferzellen innerhalb der ventrikulären Zone des Telencephalons sowie durch eine fehlerhafte Trennung der Pia mater von den neuroepithelialen Zellen gekennzeichnet ist. Zusätzlich haben wir erste Anhaltspunkte dafür erhalten, dass PPM1F möglicherweise auch in die korrekte Hirnfunktion in adulten Mäusen involviert ist. Heterozygote knockout Tiere wiesen eine stark reduzierte PPM1F Expression im zentralen Nervensystem auf, was zusätzlich durch Verhaltensunterschiede, wie eine erhöhte Aktivität und ein reduziertes Angstauftreten, begleitet wurde. Zusammenfassend weisen diese Daten darauf hin, dass PPM1F eine essentielle Phosphatase ist, welche die Integrinaktivität negativ reguliert und somit Integrin-abhängige Prozesse beeinflusst. Des Weiteren spielt PPM1F eine wichtige Rolle während der Säugerhirnentwicklung und ihr Fehlen kann nicht durch die Expression anderer Phosphatasen kompensiert werden. Eine gezielte Veränderung in der PPM1F Expression oder Aktivität könnte in Zukunft dazu genutzt werden, um in Integrin- gesteuerte Prozesse einzugreifen.

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1. Introduction

1.1. The Integrin Family

Cells recognize and respond to their micro environment through a multitude of transmembrane proteins such as the well characterized and intensively studied integrin family. Integrins are widely expressed cell adhesion receptors that are found on the surface of all metazoan cells, indicating that this family evolved relatively early in the history of multicellular animals (Nichols et al. 2006; Rokas 2008). Only some years ago it was discovered, that also unicellular relatives of Metazoa such as the filasterean Capsaspora owczarzaki express integrins and that this key genes, formerly been stated as crucial for metazoan origins, have risen much earlier (Sebe-Pedros et al. 2010; Suga et al. 2013). Furthermore, homologous sequences of the component domains of integrin α and β subunits have even been identified in prokaryotes (Whittaker and Hynes 2002).

Since these evolutionary conserved receptors are key players during cell adhesion and migration, it is obvious that integrins are essential for the regulation of fundamental physiological events like embryogenesis, maintenance of the tissue integrity, angiogenesis, or immune response (Calderwood 2004; Harburger and Calderwood 2009). All integrins are highly glycosylated, non-covalently linked, heterodimeric transmembrane receptors composed of an α and a β subunit, mediating cell-cell as well as cell-matrix interactions. In the mammalian genome 18 α and 8 β subunit genes are encoded and to date 24 different heterodimers have been identified at the protein level (Hynes 2002; Humphries et al. 2006), exhibiting overlapping substrate specificity and cell type-specific expression patterns (Fig. 1).

Figure 1. The integrin family. The mammalian integrin subunits and their distinct αβ associations are depicted. 18 α subunits can associate with 8 β subunits to form 24 defined heterodimers, which can further be classified into different subfamilies based on ligand specificity, evolutionary relationships (coloring of α subunits) and expression pattern (with regard to β2 and β7 restricted to leukocytes) (Barczyk, Carracedo et al. 2010).

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1. Introduction

Integrin molecules act like a molecular bridge, ensuring the bidirectional connection of the actin cytoskeleton (Martin et al. 2002) to the extracellular matrix (ECM).

Engagement of integrins by their ligands such as the ECM proteins collagen or laminin induce their clustering into focal adhesions, followed by the recruitment of numerous cytoplasmic proteins that indirectly link the intracellular domains of these receptors to the actin cytoskeleton. This linkage is not only essential for the generation of contractile forces between the cell and the substrate during cell migration but also for the transfer of signals into the cell and thereby integrating cells within their micro environment.

1.1.1. Integrins and Diseases

Integrins are dynamic and precisely regulated cell adhesion molecules that mediate the transfer of information across the membrane. These receptors play a crucial role in regulating matrix remodeling, differentiation, cell migration or survival (Harburger and Calderwood 2009). Hence, it is perspicuous that alterations in integrin structure and function can result in pathological conditions in multicellular organisms.

In line with their role in early embryonic development, mutations were rarely observed in the genes encoding for the α4, α5, αv, β1 or β8 integrin. However, various mutations within integrins with more tissue-specific expression have been connected to human diseases such as the autosomal recessive inherited disorder leukocyte adhesion deficiency (LAD). LAD was first recognized in the 1970s and is characterized by recurrent bacterial infections, impaired wound healing, as well as abnormalities in numerous of adhesion-dependent functions of granulocytes, monocytes, and lymphoid cells (Anderson and Springer 1987). LAD type I (LAD-I) has been linked to mutations in the β2 integrin, leading to a non-functional protein or to reduced or missing cell surface expression. β2 integrin is restricted to the hematopoietic system and part of all four leukocyte integrin heterodimers like the αLβ2 (also known as LFA-1 or CD11a/CD18), αMβ2 (alias MAC-1 or CD11b/CD18), αXβ2 (also known as CD11c/CD18) and αDβ2 (alias CD11d/CD18). Mice expressing a hypomorphic allele of the β2 integrin show an impaired inflammatory response to chemically induced peritonitis and have persistent skin inflammation due to inappropriate neutrophil activation but show no evidence of

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resemble LAD-I (Scharffetter-Kochanek et al. 1998) and have deficiencies in T cell activation and extravasation (Grabbe et al. 2002).

It has also been shown that mutations in the gene of ITGA2B and ITGB3, which encode for the two subunits of the platelet integrin αIIbβ3 lead to the bleeding disorder Glanzmann’s thrombasthenia (Nurden 2006). The identified gene mutations reach through the entire length of the integrin subunits, resulting either in a non-functional protein (Chen et al. 1992) or in the reduction or even absence of the corresponding protein on the cell surface (Nelson et al. 2005). Platelets expressing such kind of mutated integrins are unable to bind the extracellular matrix protein fibrinogen and are thus incapable to form thrombi to close wounds. Additionally, mutations in the fibrinogen receptor β3 integrin have not only been linked to Glanzmann’s disease but also to a range of cardiac and vascular disorders, atherosclerosis, bone defects, autism and several cancers (Clemetson and Clemetson 1998; Liu et al. 2008; Schuch et al. 2014).

Interestingly, also β3 deficient mice show various defects similar to such observed in humans including bleeding disorder (Hodivala-Dilke et al. 1999), cardiovascular defects, enhanced tumor angiogenesis (Reynolds et al. 2002), age dependent osterosclerosis (McHugh et al. 2000) as well as behavioral disorders (Carter et al. 2011). Not only mutations in different integrin subunits are linked to human disorders but also the up- and down-regulation of integrin expression levels is a hallmark of diseases such as cancer (Friedrichs et al. 1995). Changes in integrin expression profiles enable cancer cells to acquire migratory and invasive features and additionally, to modify integrin- mediated downstream signaling events that in turn is accompanied by increased resistance to apoptosis and survival in a foreign extracellular milieu. Many integrins, including αvβ3, αvβ5, αvβ6, α2β1, α5β1, α6β1, and α6β4 have been associated to cancer growth and invasion (Lu et al. 2008; Bandyopadhyay and Raghavan 2009). For example, high levels of α5β1 integrin seem to correlate with low levels of transformation for certain tumors (Giancotti and Ruoslahti 1990), whereas enhanced expression of αVβ3 is likely to be positively correlated with increased malignancy in melanomas (Nip et al.

1992), ovarian (Landen et al. 2008), and cervical (Gruber et al. 2005) cancers.

It is obvious, that the loss of integrin-regulating proteins results in phenotypes resembling the loss of integrins themselves and is also connected to diseases such as the Kindler syndrome. The Kindler syndrome is an autosomal recessive inherited

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1. Introduction

dermatosis which was first described in 1954 by Theresa Kindler. It is caused by mutations in the FERMT1 gene, encoding for the integrin activating protein kindlin1 (Kindler 1954). This disorder is characterized by skin blistering as well as skin atrophy in early life, which is followed by photosensitivity and changes in pigmentation and thinning of the skin (Ashton et al. 2004). The mutation in the FERMT1 gene prevents the activation of β1- containing integrins, which leads to the detachment of epidermal cells from the basement membrane and in turn to blistering of the skin. Furthermore, keratinocytes expressing a non-functional kindlin1 show a lack of polarity, decreased proliferation and increased apoptosis, contributing to thinner and more fragile skin (Herz et al. 2006).

Also negative regulators of integrin activity have been linked to human diseases before.

Periventricular heterotopia, an X chromosome-linked brain malformation, was one of the first disease associated with mutations in the human FLNA gene encoding for the FilaminA protein (Fig. 2) (Eksioglu et al. 1996). It is assumed that periventricular heterotopia is caused by a loss of function mutation of one FLNA allele.

Figure 2. Periventricular heterotopia. Magnetic resonance imaging (MRI) images of a normal human brain A, B and of a patient with periventricular heterotopia C, D.

In contrast to the smooth ventricular surface of the normal brain, a rough zone of cortical neurons (black arrowhead) is obvious along the lateral walls of the lateral ventricles, representing neurons that have not migrated to the cortex during early brain development. A, C axial plane, B, D coronal plane; (Feng and Walsh 2004).

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FilaminA is required for the effective inactivation of integrins as well as for the dynamic regulation of filamentous actin at the leading edge of migrating cells. During cortical development the lack of FilaminA leads to the immobilization of neurons, thus preventing them from leaving the ventricular zone and in turn resulting in the disturbance of a proper formation of the six different cortical layers (Feng and Walsh 2004).

Mice lacking specific Filamins show platelet and cardiac (FilaminA), skeletal (FilaminB), and muscular defects (FilaminC), which goes along with the expression profile of these proteins. Equivalent to observation in humans, mice harboring mutations in the FLNA gene hinders neuronal migration to the cerebral cortex as well as causing cardiovascular defects. The complete loss of FilaminA in mice results in embryonic lethality accompanied by severe cardiac structural defects including ventricles, atria, and outflow tracts as well as widespread aberrant vascular patterning (Feng et al. 2006).

It is obvious that mutations and dysregulation of integrins and their associated proteins contributes to a number of serious human disorders and thus, understanding the mechanism of integrin regulation is of both physiological and pathological significance.

1.1.2. Integrin Structure and Signaling

Integrins consist of a large extracellular domain that mediates binding to a variety of ligands including extracellular matrix proteins found in basement membranes like laminin. In addition, integrins also engage fibrilar matrices like fibronectin or collagen meshworks as well as counter receptors on adjacent cells (Humphries, Byron et al.

2006). In contrast to their large extracellular domain that is followed by a single-pass transmembrane helix, integrins only exhibit a comparatively short cytoplasmic domain on average about 20-60 amino acids (Fig. 3). The cytoplasmic parts of the β chains are closely related to each other and harbor a couple of conserved motifs, whereas the cytoplasmic tail of the α subunits are more divergent in sequence and structure (Ylanne 1998; Travis et al. 2003).

The integrin α subunit is composed of a seven-bladed β-propeller followed by a thigh, a calf-1 (C1) and a calf-2 (C2) domain together shaping the integrin extracellular part (Fig.

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1. Introduction

3). Some of the propeller blade domains exhibit calcium binding EF-hand domains that allosterically can affect ligand binding (Humphries et al. 2003). Within 9 of 18 α subunits a 200 amino acid long I-domain (αI-domain) is inserted between the second and the third β-sheet that has been suggested to mediate collagen-binding functions (Larson et al. 1989; Tuckwell et al. 1995).

Furthermore the αI-domain contains a Mg2+ coordinating metal-ion dependent adhesion site (MIDAS) important for ligand binding. Besides the high homology within the extracellular αI-domains, the α integrin subunits also share one conserved GFFKR-motif in the membrane proximal region within their cytoplasmic tail. This motif is on the one hand essential for hetero-dimerization with the β subunit and on the other hand needed to lock integrins in an inactive conformation (De Melker et al. 1997). The extracellular part of the integrin β subunit is composed of a plexin-sempahorin-integrin (PSI) domain, a hybrid domain (H), an I-like domain (βI domain) and four cysteine-rich epidermal growth factor (EGF) repeats (Fig. 3). Equivalent to the αI-domain the βI domain contains a Mg2+ coordinating MIDAS site for ligand binding and an additional regulatory site adjacent to MIDAS (ADMIDAS). It is inhibited by Ca2+ or activated by Mn2+ regarding ligand binding (Humphries, Symonds et al. 2003). The β chain tails exhibit two NPxY or NPxY-like motifs essential for various integrin-mediated processes as well as conserved threonine residues (TTT or TT-like motif) associated to cell adhesion.

The apparently static receptors exhibit striking plasticity and dynamic properties since the binding of integrins to ECM proteins induces an active, high affinity (open)

Figure 3. Integrin structure.

Schematic representation of an αI-domain containing integrin heterodimer. All β subunits contain a βI domain whereas only 9 out of 18 α subunits comprise an αI- domain. Stars indicate divalent cation-binding sites (Barczyk et al. 2010).

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Figure 4. Integrin conformational changes. A Electron micrographs of negatively stained αVβ3 integrin.

First picture αVβ3 integrin in the presence of Ca2+, second representative projection average of an extended integrin with a closed headpiece in the presence of Mn2+ and third extended integrin with an open headpiece in the presence of Ca2+ and RGD-peptide. Scale bar: 100 Å. B Ribbon diagrams of the alternative conformations of the extracellular segment of αVβ3 integrin. The bent conformation is shown at the left hand side, the corresponding model of the extended conformation at the right hand side (Takagi et al. 2002).

The ligand-induced active state stimulates intracellular signaling processes that in turn initiate the cellular response to integrin-mediated cell attachment. This process is termed integrin outside-in activation and can either be triggered by ligand binding or artificially induced via manganese stimulation by binding to the MIDAS of the β subunit I-like domain (Dedhar and Hannigan 1996; Hynes 2002). Furthermore, ligand binding to integrins induces their clustering into focal complexes where a characteristic set of cytoplasmic proteins is recruited that indirectly link the short intracellular domains of these receptors to the actin cytoskeleton. The protein composition of these initial adhesion sites changes over time and these multi-protein complexes mature into larger focal adhesions (Zamir and Geiger 2001; Zaidel-Bar et al. 2004) and finally into fibrillar adhesions (Fig. 5A-C) (Geiger et al. 2001; Ciobanasu et al. 2012).

Interestingly, the change from the low affinity, inactive state (closed conformation) to the high affinity state cannot only be induced via ligand binding, but also be triggered by intracellular processes and thus is termed inside-out activation (O'Toole et al. 1994).

Integrin inside-out signaling requires dynamic and spatiotemporal regulated assembly and disassembly of focal adhesions and is achieved by an extensive interplay of proteins of the integrin adhesome, including adapter proteins, kinases, phosphatase, GTPases, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).

A B

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1. Introduction

Figure 5. Images of focal adhesions. A Fluorescence microscopic picture of immuno-fluorescently labelled fibroblasts stained for the focal adhesion-associated adapter protein paxillin (shown in green) and actin (shown in red). The green patches mark the focal adhesions located at each end of the red actin fibers; Scale bar: 10 μm. B A side view of a chicken lens cell showing the interaction of a focal adhesion with the substrate (indicated by the bracket), viewed by transmission electron microscopy; Scale bar: 500 nm; (Winograd-Katz et al. 2014). C Schematic representation of focal adhesion composition, depicting experimentally determined protein positions (Kanchanawong et al. 2010).

Nowadays, about 180 proteins have been identified for being part of the human integrin adhesome and close to 700 interactions between them, highlighting the intricacy and precision of this regulatory process (Zaidel-Bar et al. 2007; Zaidel-Bar and Geiger 2010).

Using three-dimensional super resolution fluorescence microscopy, Kanchanawong and colleagues could demonstrate that integrins and the actin cytoskeleton are vertically separated by an approximately 40 nm focal adhesion core complex consisting of different signaling layers and a characteristic set of signaling molecules (Kanchanawong, Shtengel et al. 2010): The integrin signaling layer composed of the cytoplasmic tail of

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transduction layer consisting of the adaptors Talin and vinculin; and the actin- regulatory layer including the adaptors zyxin, α-actinin and vasodilator-stimulated phosphoprotein (VASP) (Fig. 5C). The complex composition of focal adhesions and the resulting prospects to generate bidirectional signaling events enable cells to sense and respond to their micro environment, which in turn influences cell behavior and functions (Fig. 6).

Figure 6. Bidirectional integrin signaling. Integrin exists in different conformational states including a bent inactive, an extended or an open activated state. Integrin outside-in activation is dependent on binding of extracellular ligands to the integrin head-domain inducing a transition from a closed to an opened conformation of the β subunit I-like domain (headpiece opening). The adaptor protein Talin is recruited to focal complexes stabilizing the integrin open conformation and connecting these receptors to the actin cytoskeleton. Vinculin binding strengthens the association between Talin and F-actin generating tension which is a crucial factor for full integrin activation. Inside-out activation is dependent on the interaction of cytosolic proteins with the cytoplasmic tail of integrins. Although the exact hierarchy of recruited proteins is not clarified unanimously it is accepted that Talin and FAK are one of the first proteins to be recruited. Binding of the head domain of Talin to the β subunit results in disruption of an inhibitory salt bridge between the α and the β subunit. The co-activator kindlin enhances Talin-induced integrin activation (Bouvard et al. 2013).

1.1.3. Integrin Activators and Inactivators

Regulation of integrin activity is a fundamental process involved in a variety of physiological events including embryogenesis, maintenance of tissue integrity,

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1. Introduction

angiogenesis and immune response (Harburger and Calderwood 2009). In recent years, a lot of proteins have been identified that positively affect integrin activity. The adaptor protein Talin is one of several proteins that not only links the cytoplasmic domain of the integrin β subunits to actin filaments (Critchley 2004), but is also one of the most essential proteins indispensable for integrin inside-out activation. Besides a rod domain, Talin exhibits a head part containing a FERM (protein 4.1, ezrin, radixin, moesin) domain (subdivided into F1, F2 and F3 subdomains). By binding via the F2-F3 subdomains to the membrane proximal conserved NPxY-motif within the cytoplasmic domain of β integrin tails, Talin triggers a conformational change in the αβ integrin extracellular domain, increasing the affinity for ECM proteins (Tadokoro et al. 2003;

Calderwood 2004). It is reported that binding of the Talin head domain to the cytoplasmic domain of integrin β subunits leads to integrin activity by disrupting a salt bridge connecting the α and β integrin subunits (Campbell and Ginsberg 2004). This changes the tilt angle of the β integrin transmembrane domain (Kim et al. 2012) and in turn releases the interactions at the interface between the transmembrane domain outer membrane clasp (OMC) and the inner membrane clasp (IMC) of the α and β subunits (Shattil et al. 2010). Talin exists in an auto-inhibited conformation that can on the one hand be abolished via the interaction with phosphatidylinositol 4,5- bisphosphate (PIP2)(Martel et al. 2001; Goksoy et al. 2008) and cleavage of the inhibitory rod domain by calpain (Franco et al. 2004) or on the other hand via an agonist receptor-mediated activation of Rap1/Rap1 GTP-interacting adapter molecule (RIAM) and the subsequent recruitment of Talin to the membrane (Lee et al. 2009). Regarding Talin irreplaceable function in integrin activation, it is obvious that complete loss of Talin1 results in severe early developmental defects in mice and thus, leading to embryonic lethality at E7.5.

Other essential integrin activators are proteins of the kindlin family composed of three evolutionarily conserved proteins kindlin-1, -2 and -3. These proteins have been identified to cooperate with Talin and are essential for full integrin activation (Montanez et al. 2008; Moser et al. 2008). Similar to Talins also kindlins contain a FERM domain and via their F3 subdomain directly interact with the cytoplasmic tail of the integrin β subunit (Meves et al. 2009). Binding of the two positive regulators Talin and kindlin to integrins results in the recruitment of other cytoskeletal and signaling proteins and

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synergize to activate integrin binding to extracellular ligands. Mutations in the kindlin-1 gene have been connected to a serious skin disease the Kindler syndrome (see 1.1.1.

Integrins and Disease) (Jobard et al. 2003) and a complete loss of kindlin-2 leads to early embryonic lethality in mice at E6.5 highlighting its essential role in integrin regulation.

An essential hallmark of integrins is their ability to fine-tune their affinity for their ligands. Integrins are not passively returning to their inactive conformation, hence it is obvious that not only integrin activation but also their inactivation has to be precisely controlled. A direct mechanism of integrin inhibition involves the binding of a protein to the integrin cytoplasmic tail that in turn disturbs association of integrin activators. So far a few factors are known that stabilize the integrin “off” state. Filamin is large rod- shaped actin cross-linking protein known to interact with several proteins via its 24 immunoglobulin-like domains (Stossel et al. 2001). Abolishing of the auto-inhibition of Filamin via tension-induced binding to actin exposes several binding sites for different focal adhesion proteins as well as for integrins (Pentikainen and Ylanne 2009). Since Filamin and Talin share overlapping binding sites, it becomes obvious that these proteins compete for binding to integrin tails (Fig. 7A, E). FilaminA-mediated displacement of Talin from the β integrin tail stabilizes the inactive conformation (Kiema et al. 2006). Interestingly, depletion of both proteins in cells completely restores integrin activity, indicating that the switch between Talin and Filamin binding to integrins is crucial for integrin activity (Nieves et al. 2010). Hence, it is plausible that complete loss of the negative integrin regulator Filamin (FilaminA) leads to embryonic lethality in mice at E14.5 (Feng, Chen et al. 2006).

Other integrin inhibitors like the PTB-containing (phosphotyrosine binding domain) proteins including docking protein 1 (DOK1) and integrin cytoplasmic domain- associated protein (ICAP1) have also been reported to compete with Talin and kindlin for β integrin tail binding (Bouvard et al. 2003; Wegener et al. 2007; Millon-Fremillon et al. 2008). DOK1 binds to the same region on the β integrin tail as Talin and ICAP1 shares a similar binding region with kindlin (Fig. 7B, C, E). Thus, binding of the respective inactivator leads to the expulsion of the corresponding activator. Another integrin regulator called SHARPIN binds directly to a conserved IMC region within the integrin α subunits and therefore, represents the first integrin regulator affecting the α tail (Rantala et al. 2011).

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1. Introduction

Figure 7. Integrin inhibitors and their mechanism of action. Binding of the integrin inactivators Filamin, ICAP1, Dok1, SHARPIN and MDGI (mammary-derived growth inhibitor) to the α or β subunit results in displacement of the positive regulators Talin and kindlin. A Epidermal growth factor-activated ribosomal protein S6 kinase2 (RSK2)-mediated phosphorylation of Filamin on Ser2152 promotes integrin binding and Talin displacement. B Binding of Krev interaction trapped 1 (KRIT1) to ICAP1 prevents Integrin-ICAP1 interaction and Talin displacement. C Src-mediated integrin tyrosine phosphorylation (within proximal NPxY motif) enhances DOK1 binding and Talin displacement. D MDGI and SHARPIN bind to the integrin α subunit. SHARPIN prevents recruitment of Talin and kindlin to integrins. E The α and β binding sites of integrin regulators are depicted. The residue of the inner membrane clasp between the α and β subunit are colored in green, the conserved NPxY motifs in blue (Bouvard, Pouwels et al. 2013).

SHARPIN binds to the highly conserved WKXGFFKR sequence within the integrin α subunit and thereby prevents the recruitment of Talin and kindlin. It was shown that the last arginine residue of the WKXGFFKR motif, forming the inhibitory salt bridge with the corresponding aspartic acid residue within the β subunit, is not required for SHARPIN binding. SHARPIN binds to integrins with an intact salt bridge and thus might stabilize

A B

D C

E

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1.1.4. Site-Specific Phosphorylation of Integrins and Their Regulators

To date a multiplicity of proteins has been identified that either directly (more than 40) or indirectly (more than 230) interact with the cytoplasmic tail of integrins (Legate and Fassler 2009; Zaidel-Bar and Geiger 2010). Thus, it is obvious, that there is the necessity of a spatiotemporal regulation of these interactions by different mechanisms.

In recent years more and more focus has been placed on phosphorylation events affecting integrin affinity and integrin-mediated down-stream signaling. Indeed, the cytoplasmic tail of integrins is comparatively short but nevertheless it accommodates plenty of phosphorylation sites potentially serving as posttranslational switches that regulate binding of different molecules (Fig. 8).

The β integrin subunit exhibits conserved phosphorylation sites which turned out to serve as a dynamic mechanism to conduct adaptor binding to integrins. The conserved tyrosine residues of both NPxY motifs were decoded to modulate integrin activation.

These tyrosine residues can be phosphorylated in a Src-dependent manner (Sakai et al.

2001; Bledzka et al. 2010). Phosphorylation of the membrane distal NITY motif within β3 integrin disrupts kindlin-2 binding (Bledzka, Bialkowska et al. 2010), whereas phosphorylation of the membrane proximal NPLY motif leads to impaired Talin association (Oxley et al. 2008). In contrast, phosphorylation of the proximal NPLY motif within β3 integrin enhances binding of the negative regulator DOK1 to the integrin tail (Oxley, Anthis et al. 2008). Not only the tyrosine residues of both NPxY motifs were

Figure 8. Amino acid sequence of some α and β integrin cytoplasmic tails. The motif needed for stabilizing the integrin inactive state (salt bridge formation) is shaded in red. The α chain motif, containing a serine residue important for outside-in activation, is colored in yellow. The conserved NPxY motifs within the β chain are shaded in blue and the conserved threonine residues in green. Potential phosphorylation sites are shown in red (Fagerholm et al. 2004).

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1. Introduction

identified to affect integrin activity but also the threonine residues within the cytoplasmic tail of the β subunit. In humans, the Thr789 in β1 integrin is conserved across all integrin tails except for the β1D isoform, whereas Thr788 is replaced by serine residues within β3, β5 and β6. Mutational studies revealed the first indication for the importance of these residues (T788A/T789A) within the cytoplasmic tail of β1 integrin, negatively affecting cell spreading and integrin activity (Wennerberg et al. 1998; Nilsson et al. 2006). In this regard it was also shown that T cell receptor complex activation can trigger phosphorylation of β7 integrin threonine residue leading to enhanced β7- mediated cell adhesion (Hilden et al. 2003). Furthermore, the phosphorylation status of Thr758 of β2 as well as Thr783 and Thr785 of β7 integrin seems to be important for integrin function, since modification of this residues have been identified to strongly decrease FilaminA association (Kiema, Lad et al. 2006; Takala et al. 2008). So far, kinases that have been identified to play a role in phosphorylation of these threonine residues within the β integrin subunit are the protein kinase C (PKC), the calcium/calmodulin- dependent protein kinase II (CaMKII), Akt and 3-phosphoinositide dependent protein kinase-1 (PDK1). Angiotensin-dependent activation of PCKε in rat cardiac fibroblasts results in the association of this kinase with β1 integrin, accompanied by increased β1 Thr788/789 phosphorylation as well as enhanced cell adhesion (Stawowy et al. 2005).

Also CaMKII has been found to interact with β1 integrin in normal human epithelial cells and is thought to phosphorylate Thr788/789 during mitosis, but contradictory, resulting in reduced cell adhesion (Takahashi 2001; Suzuki and Takahashi 2003). T779 within the platelet β3 integrin (which is homologous to β1 integrin T789) was found to be phosphorylated by Akt and PDK1 in vitro and inhibits outside-in signaling of this receptor (Kirk et al. 2000), indicating that phosphorylation of these residues might differ among integrins.

However, not only phosphorylation of integrins themselves has been shown to influence integrin affinity but also phosphorylation of their regulators modulates integrin activity.

Phosphorylation of FilaminA via RSK2 and phosphorylation of ICAP1 via the CaMKII at a specific serine and threonine residue triggers binding of these proteins to β2 and β1 integrin, respectively, and hereby stabilizing the integrin “off” state (Gawecka et al.

2012; Millon-Frémillon et al. 2013).

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1.2. Phosphatases

Protein phosphorylation is a fast and efficient tool to control cell response to internal and external cues. It is the most common form of reversible posttranslational modifications (PTM) and plays a key role in controlling a multiplicity of cellular processes such as differentiation, proliferation, apoptosis, migration or metabolism (Manning et al. 2002). Impressively, from the estimated 20,000-25,000 human protein- coding genes approximately 17,000 proteins have at least one annotated residue in the Phosphosite database (Hornbeck et al. 2012), highlighting the importance of this modification. Thus, it is obvious that abnormal phosphorylation profiles are the cause or the consequence of different human diseases like cancer, diabetes and neurodegenerative or inflammatory disorders (Table 1) (Cohen 2001; Gee and Mansuy 2005; Easty et al. 2006; Tonks 2006).

Table 1. Disease caused by mutations in specific protein kinases and phosphatases (Cohen 2001).

The majority of protein phosphorylation in eukaryotic cells occurs predominantly on the hydroxyl-containing amino acids serine (Ser), threonine (Thr) and tyrosine (Tyr). Serine is the predominant target with about 86 %, followed by phosphorylation events at threonine residues up to 11.8 % and finally about 1.8 % of protein phosphorylation

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1. Introduction

occurs at tyrosine residues (Olsen et al. 2006). Phosphorylation of proteins is a common mechanism for controlling the behavior of a protein, such as affecting its subcellular distribution or leading to its activation or inactivation. The addition or the removal of a phosphate group to or from a molecule, respectively, is accomplished by antagonizing activities of protein kinases and phosphatases. The human genome encodes about 518 putative protein kinases that can be classified into 428 serine/threonine kinases and 90 tyrosine kinases (Lander et al. 2001; Johnson and Hunter 2005).

Figure 9. Classification of protein phosphatase superfamily. (1) Phosphatases were first classified into six families according to the catalytic domain InterPro annotation. (2) Each family was further subdivided into classes according to their preferred substrates or literature annotation (3) (Sacco et al. 2012).

In contrast to the abundance of kinases the fully sequenced human genome is thought to contain only about 200 phosphatases, subdivided into 108 putative tyrosine phosphatases (PTPs), roughly 30 serine/threonine phosphatases (phosphoprotein phosphatases (PPPs) as well as metal-dependent protein phosphatases (PPM)), 21 phosphatases containing a haloacid dehalogenase-like hydrolase domain (HADs), 37

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enzymes harboring a phosphatidic acid phosphatase or inositol polyphosphate-related domain (LPs) as well as 5 phosphatases with a NUDIX hydrolase domain (NUDT) (Fig. 9) (Alonso et al. 2004; Sacco, Perfetto et al. 2012).

Regarding the ratio of protein kinases to phosphatases it becomes comprehensible, why over the past decades the scientific community has focused on kinases. While the number of protein tyrosine kinases compared to tyrosine phosphatases is almost fairly balanced, there is a striking difference in the quantity of serine/threonine kinases to phosphatases. Therefore, phosphatases have been considered for a long time for being promiscuous, unregulated enzymes that play a non-specific role in controlling phosphoprotein homeostasis in vivo. However, besides the approximately 200 identified proteins containing a phosphatase catalytic domain there are also about 56 regulatory subunits identified to date.

Figure 10. The complexity of PP2A regulation. PP2A predominately exist as heterotrimer composed of conserved A and C subunits and variable B subunits. This complex is regulated in multiple ways, including regulation of heterotrimer assembly, microbial toxins (e.g. okadaic acid or microcystin), protein inhibitors such as SET or CIP2A, and phosphorylation of the B and C subunits to regulate activity, assembly, and targeting. At the right hand side the regulating B subunits are depicted, corresponding to at least 15 different genes, each with multiple splice variants (Virshup and Shenolikar 2009).

All 13 members of the PPP family of serine/threonine phosphatases receive regulation and substrate specificity within the cell through combinatorial interactions between

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1. Introduction

their conserved catalytic subunits and various numbers of regulatory subunits, together forming an active holoenzyme. PP2A for example, belonging to the PPP family of serine/threonine phosphatases typically exist as heterotrimer composed of a catalytic C- , a structural A- and a regulatory B-type subunit (Fig. 10).

The core complex, comprising the catalytic and the structural subunit, can exist independently or can be associated with the regulatory components via the structural A- type subunit (Janssens et al. 2008; Virshup and Shenolikar 2009). Hence, regulated binding and the dynamic exchange of the B-type subunits represents a beneficial mechanism for controlling PP2A activity, including substrate selectivity as well as targeting the enzyme within the cell. In contrast to the PPP, members of the PPM family do not associate with regulatory B-type subunits, but instead harboring additional interaction domains and conserved sequence motifs. One member of the Mn2+/Mg2+- dependent PPM family is the Protein Phosphatase 2C (PP2C). PP2C represents a large family of highly conserved protein phosphatases with 15 distinct PP2C genes encoded in the human genome (Lammers and Lavi 2007), all characterized by the conserved PP2C- like domain. Interestingly, homologs of human PP2Cs can be found in almost all phyla, spanning from plants, bacteria, and yeast, to nematodes, insects, and mammals (Schweighofer et al. 2004), highlighting their particular need in regulating key cellular signaling events. Unlike the PPP family, PP2C has a large number of isoforms encoded by different genes. These different isoforms are marked by specific sequences and domain organizations giving the PP2C isoforms distinct functions, subcellular distribution and expression pattern. It is assumed that these additional interacting sites probably contribute to substrate specificity and selectivity. Nevertheless, further research is needed to unravel the molecular determinants affecting the mode of action of this class of protein phosphatases.

1.2.1. The Serine/Threonine Phosphatase PPM1F

PPM1F belongs to the PP2C family of metal-dependent serine/threonine protein phosphatases. So far, two genes, PPM1F and PPM1E, have been identified (Koh et al.

2002). PPM1F, also known as POPX2 (Partner of PIX2), CaMKPase or hFem-2, was first

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specifically affecting multifunctional CaMKs (Ishida et al. 2003). Afterwards, PPM1E (alias POPX1 or CaMKP-N) was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor (PIX), sharing 52 % sequence identity in the catalytic domain to PPM1F (Manser et al. 1998; Takeuchi et al. 2001; Koh, Tan et al.

2002). The cytosolic PPM1F with a relative molecular mass of around 50 kDa is ubiquitously expressed, while the larger 84 kDa, nuclear PPM1E is predominately found in brain and testis (Takeuchi, Ishida et al. 2001). Recently, Zhang and colleagues could solve the crystal structure of the full-length Caenorhabditis elegans Fem-2 (cFem-2), the homolog of the human PPM1F (Zhang et al. 2013). cFem-2 is composed of an N-terminal domain and a C-terminal PP2C-like domain. Equivalent to other members of the PP2C family (Shi 2009), the C-terminal domain harbors a central β sandwich formed by two sets of antiparallel β sheets. One set of β sheets is flanked by two α helices in an antiparallel fashion, whereas the other set is flanked by three α helices (Fig. 11A).

Figure 11. Overall structure of cFem-2. A The N-terminal domain of cFem-2 is shown in cyan and the C- terminal domain in purple. The Mg2+ ions are depicted as spheres and helices in the N-terminal region are marked. B The catalytic core of cFem-2 and the residues involved in Mg2+ coordination, shown as sticks, are illustrated. Mg2+ ions and water molecules are marked as blue and red spheres, respectively. Metal- oxygen coordination bonds are depicted as red dashed lines (Zhang, Zhao et al. 2013).

PPM1F and its homologs require manganese or magnesium ions (Mn2+/Mg2+) for their enzymatic activity. Resolving the crystal structure of cFem-2 discovered two Mg2+ ions in the catalytic center. Both are hexa-coordinated by oxygen atoms from amino acids and water molecules (Fig. 11B), conformable with the structure of the human PP2Cα (Shi 2009). Notably, comparing the residues involved in phosphate interaction and in

A B

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1. Introduction

the coordination of metal ions are highly conserved among different Fem-2 homologs, reaching from different nematode species to the human PPM1F (Fig. 12). Mutations of these residues involved in metal ion coordination all abolish or dramatically impair phosphatase activity (Harvey et al. 2004; Oliva-Trastoy et al. 2007; Zhang, Zhao et al.

2013). It has also been found that PPM1F phosphatase activity is regulated by oxidation and reduction at the cysteine residue 359, that upon reduction, e.g. by 2- mercaptoethanol, leads to an increase in enzymatic activity (Baba et al. 2012). Unlike the C-terminal domain of PPM1E that seems to control its catalytic activity (Sueyoshi et al.

2012; Ishida et al. 2013), the N-terminal domain of PPM1F does not directly regulate its phosphatase activity but rather acts as a protein interaction or scaffold domain (Zhang, Zhao et al. 2013; Phang et al. 2014).

Figure 12. Sequence alignment of different Fem-2 homologs. The conserved residues contributing to phosphate and Mg2+ binding are highlighted with red stars. Residues with 100 % homology, over 75 % homology, and over 50 % homology are colored in dark blue, purple, and light blue, respectively (Zhang, Zhao et al. 2013).

One of the main substrates identified so far are the multifunctional CaMKs, including CaMKI, II, and IV (Ishida et al. 1998; Fujisawa 2001; Ishida, Shigeri et al. 2003). PPM1F has been shown to deactivate the auto-phosphorylated CaMKII by dephosphorylating Thr286 within the kinase activation loop in vitro (Ishida, Kameshita et al. 1998; Harvey,

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Banga et al. 2004). In return, PPM1F can be phosphorylated by CaMKII in the presence of poly-L-lysine resulting in a 2-fold increase in its enzymatic activity. Accordingly, the activity of PPM1F seems to be regulated through phosphorylation by its target enzyme (Kameshita et al. 1999). PPM1F is also known to be an inhibitor of members of the p21- activated kinases (PAK) family of serine/threonine kinases. In vitro studies have demonstrated that PPM1F dephosphorylates key residues within the activation loop of PAK such as Thr422 (Koh, Tan et al. 2002). Equivalent to PPM1E also PPM1F can interact with the Rho-GEF PIX and thus block efficiently PAK-mediated biological effects such as actin reorganization and cell migration downstream of the Rho-GTPases Cdc42 and Rac. In this regard, it was also shown that PPM1F can inhibit actin stress fiber breakdown mediated by active Cdc42 (Koh, Tan et al. 2002). Another identified PPM1F interaction partner is the formin protein mDia1. PPM1F can negatively affect mDia1- and RhoA-dependent transcription (Hill et al. 1995) mediated by serum response factor (SRF) (Xie et al. 2008). Recently, Phang and colleagues discovered that PPM1F also can dephosphorylate the KIF3 kinesin motor complex (KIF3A) at the serine residue 690. It was hypothesized that the PPM1F-mediated dephosphorylation of KIF3A keeps this protein in an auto-inhibited conformation, resulting in an impaired transport of N- cadherin and β-catenin to the membrane and hereby losing cell-cell contacts sides (Phang, Hoon et al. 2014). PPM1F might also be involved in the regulation of cytoskeleton dynamics through the regulation of MAP kinases (MAPK1/3) and glycogen synthase kinase 3 (GSK3) activities, since silencing of PPM1F alters the phosphorylation level of these kinases and decreases their activity (Zhang et al. 2013). Furthermore, PPM1F has been detected to be highly expressed in invasive cancer and its depletion dramatically reduces cancer cell motility and invasiveness, accompanied by the loss of stress fibers, reduced focal adhesions and β1 integrin expression (Susila et al. 2010).

Finally, PPM1F has also been linked to cell death, since its overexpression induces caspase-dependent apoptosis in mammalian cells (Tan et al. 2001).

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1. Introduction

1.3. Aims of the Work

The Ph.D. project entitled “Identification of Negative Regulators of Integrin-Mediated Cell Adhesion” aimed at a detailed understanding of the regulatory network of focal adhesion proteins that affect integrin activation in a negative manner.

In recent years, more and more focus has been placed on phosphorylation events affecting integrin activity as well as integrin-mediated down-stream signaling. The cytoplasmic tail of integrins comprises conserved phosphorylation sites, which turned out to serve as a molecular switch to regulate adaptor binding to integrins. For sure, not only integrins themselves have been identified to be regulated via phosphorylation, but also integrin effector proteins are controlled via their phosphorylation state. Since a lot of kinases have been identified affecting integrin activity modulation, we defined their antagonists -protein phosphatases- for being a reasonable target controlling integrin affinity in a negative way. Therefore, we ask the following questions:

1. Do further proteins exist that regulate integrin activity in a negative manner and in particular are protein phosphatases of the human integrin adhesome involved in the integrin activity control?

2. What is/are the target/s of the candidate phosphatase and how is the underlying molecular mechanism regulated?

3. Does the candidate phosphatase affect integrin-dependent cellular processes?

4. Does the candidate phosphatase also impact on integrin activity in vivo and consequently affects physiological events?

First, an shRNA-based (small hairpin RNA) functional screening approach was used to identify a candidate protein negatively affecting integrin-mediated cell adhesion as well as integrin activity. Furthermore, this project included a candidate approach in human cells to elucidate the role of the novel regulator in integrin-mediated cell adhesion and its mode of action. The role of the identified candidate protein was also evaluated in an in vivo mouse model.

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A detailed understanding of the regulatory network of focal adhesion proteins that affect integrin activation is highly worthwhile and will possibly open up new starting points for novel anti-inflammatory drugs with a promising therapeutic target.

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2. Results

2. Results

2.1. An ShRNA-Based Screening Approach Identified PPM1F as a Negative Regulator of Integrin-Mediated Cell Adhesion

Binding of the cytoplasmic adapter protein Talin to the conserved NPxY motif in the cytoplasmic tail of the β integrin subunit is critical for integrin inside-out activation.

Talin is composed of an N-terminal globular head domain that is allosterically repressed via a flexible rod domain and thereby kept in an auto-inhibited conformation (Goksoy, Ma et al. 2008). In 1999, Calderwood and colleagues could show that expression of a Talin fragment containing the head domain led to a constitutively active variant of this protein able to activate integrin αIIbβ3 (Calderwood et al. 1999). In a preliminary proof- of-concept experiment, we observed that expression of the Talin head domain (Talin1 aa1-433) in HEK 293T cells (human embryonic kidney cells) resulted in increased cell adhesion. We speculated that depletion of a negative regulator of integrin activity should also lead to enhanced cell adhesion to extracellular matrix proteins to a similar extent as the overexpression of the Talin head domain. HEK cells expressing the constitutively active Talin variant exhibited increased integrin activity which was reflected via enhanced cell adhesion to the integrin ligand collagenI but not to the integrin- independent substrate Poly-L-lysine (Fig. 13A, B). To identify novel integrin regulators affecting integrin activity in a negative manner, a lentivirus-based system was used to silence phosphatases of the human integrin adhesome in HEK cells. Adhesion potential of these stable knockdown cells was evaluated on the integrin ligands collagenI and fibronectin and the control substrate Poly-L-lysine. The screening results yield a first indication of three interesting candidates with regard to the serine/threonine phosphatase PPM1F, the tyrosine phosphatase PTP-PEST, as well as the receptor-like tyrosine phosphatase alpha (RPTPα). Knockdown of these phosphatases led to increased cell adhesion compared to control cells (Fig. 13C). RPTPα has been directly linked to αVβ3 integrin-cytoskeleton connection before and is required for the force-dependent formation of focal complexes (von Wichert et al. 2003). Also, PTP-PEST has been connected to integrin regulation and cell adhesion previously (Souza et al. 2012).

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In contrast, PPM1F has not been directly linked to integrin regulation before. Moreover, PPM1F-depleted cells exhibited the most prominent increased adhesiveness compared

Figure 13. Identification of PPM1F as a negative regulator of integrin activity. A HEK cells were transfected as indicated and seeded onto collagenI (1 µg/ml) or onto Poly-L-lysine (10 µg/ml) for 40 min. Non-adherent cells were removed via washing and remaining cells were quantified after crystal violet staining. Values were normalized to total number of seeded cells. Bar represent mean ± SD of 3 wells. B HEK cells were transfected as indicated and whole cell lysates were analyzed via western blotting. C HEK cells were transduced with lentiviral particles encoding shRNAs targeting the indicated phosphatases. Stable cell lines were seeded onto integrin ligands (1 µg/ml collagenI, 0.8 µg/ml fibronectinIII9-12) or onto Poly-L-lysine. Adherent cells were quantified and values were normalized to total number of seeded cells. Bar represent mean

± SD of 3 wells. Representative pictures are shown in D. E Expression of focal adhesion proteins in control and PPM1F knockdown cells was analyzed by western blotting.

Coll=CollagenI; Poly-L=Poly-L-lysine; FN=Fibronectin

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