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«Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 14 Biochemie, Chemie und Pharmazie der Johann Wolfgang ...»

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Regulation of EGF receptor trafficking by the

lysine deacetylase HDAC6

Dissertation

zur Erlangung des Doktorgrades

der Naturwissenschaften

vorgelegt beim Fachbereich 14

Biochemie, Chemie und Pharmazie

der Johann Wolfgang Goethe-Universität

in Frankfurt am Main

von

Yonathan Lissanu Deribe

aus Addis Ababa, Äthiopien

Frankfurt am Main 2009

D30

vom Fachbereich Biochemie, Chemie und Pharmazie

der Johann Wolfgang Goethe-Universität als Dissertation angenommen

Dekan: Prof. Dr. Dieter Steinhilber Gutachter: Prof. Dr. Volker Dötsch Prof. Dr. Ivan Dikic Datum der Disputation: 5. Juli 2010 Table of contents

Abstract

ZUSAMMENFASSUNG

1. INTRODUCTION

1.1 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)

1.1.1 Growth factors and their cognate receptors

1.1.2 ErbB family receptors

1.1.3 Mechanism of activation of EGFR

1.1.4 Signaling through EGFR

1.1.4.1 Principles of signal transduction downstream of RTKs

1.1.4.2 Signaling pathways activated by EGFR

1.1.5 Physiological functions of EGFR signaling

1.1.6 Pathological perturbations of EGFR and its signaling pathway.................17 1.1.7 Mechanisms of EGFR signal attenuation

1.2 EGFR TRAFFICKING

1.2.1 Endocytosis

1.2.1.1 Clathrin-mediated endocytosis (CME)

1.2.1.1 Clathrin independent endocytosis (CIE)

1.2.1.1.1 Caveolin mediated endocytosis

1.2.1.1.2 Dynamin-independent cdc42 regulated endocytosis

1.2.1.1.3 Internalization of large volumes of membrane – macropinocytosis and phagocytosis

1.2.1.1.4 Pathogens and the endocytic pathway

1.2.3 EGFR endocytosis

1.3 HDAC6 AND PROTEIN ACETYLATION

1.3.1 Lysine acetylation as a post-translational modification

1.3.2 Deacetylases

1.3.3 Histone deacetylase 6 (HDAC6)

1.4 MICROTUBULE DEPENDENT INTRACELLULAR TRAFFICKING

1.5 MEMBRANE BASED YEAST TWO-HYBRID SCREENING (MYTH)

1.6 RELATED ORIGINAL WORK- A CBL-CIN85 COMPLEX IN EGFR ENDOCYTOSIS...38

1.7 AIMS OF THE CURRENT STUDY

2. MATERIALS AND METHODS

2.1.2 Recombinant proteins and speciality reagents

2.1.3 Antibodies

2.1.4 Buffers, solutions and media for routine use

2.1.5 Plasmids

2.1.6 Oligonucleotides

2.1.7 Cell lines, bacterial and yeast strains

2.2 METHODS

2.2.1 Basic molecular biology techniques

2.2.1.1 Plasmid DNA transformation into E.coli

2.2.1.2 Plasmid DNA extraction from bacteria

2.2.1.3 Plasmid DNA transformation into S.cerevisae using the lithium acetate method

2.2.1.4 Plasmid DNA extraction from yeast- the lyticase approach

2.2.1.5 Site-directed mutagenesis

2.2.1.6 PCR amplification and restriction digestion of DNA

2.2.1.7 Preparation of MFα-EGFR-C-T construct

2.2.2 Membrane-based yeast two-hybrid screen

2.2.3 Cell culture methods

2.2.3.1 Cultivation of mammalian cells

2.2.3.2 Transfection of mammalian cells

2.2.3.3 Lentivirus transduction and stable shRNA expression cell line generation.... 51 2.2.3.4 Real time cell analysis (RTCA)

2.2.4 Biochemical assays

2.2.4.1 SDS-PAGE and Western blot

2.2.4.2 Immunoprecipitation

2.2.4.3 GST-GATE16 purification and GST pull down assay

2.2.4.4 In vitro kinase assay

2.2.4.5 Deacetylase assay

2.2.4.6 Ligand induced EGFR degradation

2.2.4.7 EGFR internalization assay using 125I-labelled EGF

2.2.4.8 Mass spectrometry sample preparation

2.2.5 Cell imaging studies

2.2.5.1 Immunofluorescence microscopy

2.2.5.2 Live cell imaging

2.2.6 Bioinformatic analyses

Table of contents

3. RESULTS

3.1 MYTH BASED SCREENING OF LIGAND-UNOCCUPIED EGFR.

3.1.1 Generation of bait EGFR-Cub-TF construct

3.1.2 MYTH screening using MFα-EGFR-C-T

3.1.3 Biochemical validation of putative interactions

3.2 INTERACTION BETWEEN THE CYTOPLASMIC DEACETYLASE HDAC6 AND EGFR 67

3.2.1 HDAC6 binds to EGFR

3.2.2 Mapping of EGFR and HDAC6 binding regions

3.3 HDAC6 REGULATES LIGAND-INDUCED DEGRADATION OF EGFR

3.3.1 HDAC6 overexpression slows ligand-induced degradation of EGFR.......73 3.3.2 HDAC6 knockdown accelerated the degradation of EGFR

3.4 HDAC6 MODULATES THE KINETICS OF EGFR INTRACELLULAR TRAFFICKING...75

3.4.1 HDAC6 has no effect on EGFR internalization

3.4.2 Knockdown of HDAC6 accelerates the delivery of EGF late endosomes.77

3.5 ACETYLATION AND RECEPTOR TRAFFICKING

3.5.2 Acetylation of α-tubulin is increased upon EGF stimulation

3.5.3 Acetylation of α-tubulin Lys40 is important for efficient motility of endosomes

3.6 PHOSPHORYLATION OF HDAC6 MODULATES ENZYMATIC ACTIVITY..................84 3.6.1 HDAC6 is phosphorylated on serine and tyrosine residues

3.6.2 Phosphorylation of HDAC6 by EGFR inhibits deacetylase activity..........86

3.7 EFFECTS OF HDAC6 ON EGFR MEDIATED CELLULAR RESPONSES

3.7.1 Downregulation of HDAC6 alters Akt and ERK signalling pathways.......87 3.7.2 HDAC6 knockdown cells have stunted response to EGF stimulation.......88





4. DISCUSSION

4.1 MYTH SCREEN

4.2 HDAC6 IS A NOVEL EGFR INTERACTOR

4.3 HDAC6 STABILIZES LIGAND-INDUCED DEGRADATION OF EGFR

4.4 HDAC6 PLAYS A PIVOTAL ROLE IN THE POST-ENDOCYTIC TRAFFICKING OF EGFR

4.5 EGF INDUCES ACETYLATION OF -TUBULIN

4.6 ACETYLATION OF -TUBULIN MODULATES MOTILITY OF ENDOSOMES................98 Table of contents

4.7 PHOSPHORYLATION OF HDAC6 BY EGFR MODULATES ITS DEACETYLASE

ACTIVITY

4.8 HDAC6 DEPLETION RESULTS IN PERTURBATIONS IN CELLULAR PHYSIOLOGY..101

5. SUMMARY AND FUTURE OUTLOOKS

6. REFERENCES

ABBREVIATIONS

LIST OF ORIGINAL PUBLICATIONS

ACKNOWLEDGMENTS

CURRICULUM VITAE

ERKLÄRUNG

Abstract Abstract Epidermal growth factor (EGF) receptor belongs to the broad family of enzymatic receptors called receptor tyrosine kinases (RTKs). Generally, the binding of a ligand to these receptors leads to activation of their intracellular kinase activity that sets in motion a cascade of signaling events. In order to ensure appropriate responses to physiological stimuli, the cell is endowed with the ability to regulate signal transduction via numerous mechanisms such as dephosphorylation of the RTK and its substrates as well as downregulation of the RTK.

Activation of EGFR is a potent mitogenic (proliferative) and motogenic (cell motility) signal that plays crucial roles during embryonic development and maintenance of adult tissue. EGFR signaling is primarily regulated by ligand-induced receptor internalization with subsequent degradation in lysosomes. While the complex of proteins that are recruited to EGFR after its activation is well understood, proteins that interact with the receptor in the absence of ligand binding are still not systematically studied.

With the goal of identifying novel binding partners of non-activated EGFR, a membrane based yeast-two hybrid screen (MYTH) was conducted. MYTH is based on the principle of in vivo reconstitution of the N-terminus (Nub) and C-terminus (Cub) halves of ubiquitin once brought into close proximity. A chimeric protein consisting of EGFR fused to Cub and a transcription factor was used as a bait to screen Nub-tagged cDNA library. Analysis of resultant yeast transformants revealed a total of 87 proteins to interact with EGFR. Of these only 11 were previously shown to bind to EGFR. A majority of the other proteins were shown to interact with the receptor by yeast retransformation. Fifteen were confirmed to bind to EGFR by coimmunoprecipitation assays in mammalian cells.

One of the novel EGFR interactors identified in the screen was histone deacetylase 6 (HDAC6). This deacetylase is localized in the cytoplasm and known to deacetylate tubulin, HSP90 and cortactin. The juxtamembrane region of EGFR binds to the Cterminus of HDAC6. Functionally, overexpression of wild type HDAC6 stabilized ligand-induced degradation of the receptor. On the other hand, deacetylase deficient or EGFR binding compromised mutants of HDAC6 were able to stabilize EGFR only partially. Downmodulation of HDAC6 expression by RNAi markedly accelerated

Abstract

degradation of the receptor. Taken together, HDAC6 is a negative regulator of EGFR downregulation that is dependent on its deacetylase activity and ability to bind to the receptor.

Imaging studies revealed that HDAC6 does not affect internalization of EGFR from the plasma membrane but rather influences the post-endocytic trafficking of the receptor-ligand complex to lysosomes. Pulse-chase experiments using fluorophoretagged EGF showed that EGFR is transported faster towards the peri-nuclear region and delivered to late endosomes rapidly in HDAC6 depleted cells.

HDAC6 is demonstrated to act, at least partly, by regulating the acetylation of tubulin. Upon EGFR activation, acetylation of -tubulin on lysine 40 is progressively increased as shown by mass spectrometry and immunoblotting. Forced expression of a dominant negative mutant of -tubulin, but not wild type -tubulin, led to reduced speed and processive movement of early endosomes in GFP-Rab5 expressing cells. In a surprising twist, EGFR is able to phosphorylate HDAC6 on Tyr570.

Phosphorylation of Tyr570 and Ser568 leads to inactivation of the deacetylase function of HDAC6 as shown by in vivo and in vitro assays. In summary, HDAC6 diminishes EGFR downregulation by slowing the transport of intracellular vesicles.

The inhibitory effect is removed once HDAC6 is phosphorylated on key residues.

In line with these findings, two recent reports have shown that hyper-acetylation of tubulin induced by inhibition of HDAC6 increases the transport of brain derived neurotrophic factor and JNK interacting protein-1 in different cell systems. Acetylated microtubules are more efficient in recruiting motor proteins like kinesin-1 and dynein.

These findings indicate that HDAC6 plays an important regulatory role in intracellular trafficking pathways. However, several outstanding issues still remain unresolved. How does acetylation of microtubules influence vesicular trafficking? In this regard, the temporal and spatial dynamics of -tubulin acetylation following EGFR activation should be studied. Furthermore, whether HDAC6 affects the trafficking of other endocytic cargos and additional organelles is an interesting question to address.

Zusammenfassung Zusammenfassung Einleitung Der epidermale Wachstumsfaktor-Rezeptor (EGFR) gehört zu der umfassenden Familie enzymatisch aktiver Rezeptoren, die als Rezeptortyrosinkinasen (RTKs) bezeichnet werden. RTKs stellen eine große Gruppe der Zelloberflächenrezeptoren dar, die eine intrinsische Tyrosinkinaseaktivität aufweisen. Sie katalysieren infolge extrazellulärer Stimuli den Transfer der gamma-Phosphatgruppe eines ATP-Moleküls auf Hydroxylgruppen von Tyrosinresten3. Die allgemeine Struktur von RTKs lässt sich in eine extrazelluläre Ligandenbindedomäne, eine einzige Transmembrandomäne und eine zytoplasmatische, katalytisch aktive Region gliedern, auf die der für die Regulation der Signalweiterleitung verantwortliche C-Terminus folgt.

Die Bindung eines Liganden an diese Rezeptoren führt im allgemeinen zur Aktivierung ihrer intrazellulären Kinaseaktivität, die eine Kaskade von Signalvorgängen in Gang setzt. Im Fall des EGFR bindet der Ligand (EGF) als Monomer an den Rezeptor in einem Verhältnis von 2:2 und induziert Konformationsänderungen, die zur Dimerisierung des Rezeptors führen. Daraufhin werden Signalwege innerhalb der Zelle eingeleitet, indem Proteine, die eine Phosphotyrosinbindedomäne aufweisen, an phosporylierte Tyrosinreste im Cterminalen Bereich des Rezeptors binden. Die Aktivität des EGFR sowie anderer RTKs ist streng reguliert, um eine angemessene und physiologische Antwort auf einen Stimulus zu generieren.

Mehrere Mechanismen wie die Dephosphorylierung des Rezeptors und weiterer Komponenten des Signalweges stellen sicher, dass die Signalweiterleitung entsprechend beendet wird. Der wichtigste Mechanismus, um die Weiterleitung des Signals herunterzuregulieren, besteht jedoch in der schnellen Endozytose und dem anschließendem Abbau sowohl des Rezeptors als auch des Liganden. Die Aktivierung infolge der Bindung eines Liganden führt zur Anhäufung des Rezeptors in von Clathrin überzogenen Einstülpungen (CCPs) auf der Plasmamembran, die sich anschließend durch Bildung von „Clathrin bedeckten Vesikeln“ (CCVs) abschnüren.

Der EGFR wird über CCVs, frühe Endosomen und multivesikuläre Körper schließlich zum Abbau ins Lysosom gebracht. Das Schicksal der Fracht entscheidet sich auf der Ebene des frühen Endosoms: entweder kommt es zur Rückführung an die Plasmamembran oder zum Abbau im Lysosom. Welcher Weg eingeschlagen wird,

Zusammenfassung

hängt von der Ubiquitinierung der Fracht ab, die von einem Proteinkomplex (endosomal-sorting complex required for transport (ESCRT)) erkannt wird.

Mikrotubuli und die an diese assoziierten Motorproteine wie Dynein sind für den Transport früher Endosomen zu späten Endosomen essentiell und somit auch für den Abbau der Fracht.



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