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Characterisation of atypical Rho GTPases of the RhoBTB family and their binding partners [Elektronische Ressource] / vorgelegt von Kristína Schenková

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142 pages
Characterisation of atypical Rho GTPases of the RhoBTB family and their binding partners Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Kristína Schenková aus Nitra, Slowakei Köln 2010 Berichterstatter/in: Prof. Dr. Angelika A. Noegel Prof. Dr. Jürgen Dohmen Tag der mündlichen Prüfung: 30. Juni 2010 Die vorliegende Arbeit wurde in der Zeit von November 2006 bis Mai 2010 unter Anleitung von Prof. Dr. Angelika A. Noegel und der Betreuung von PD Dr. Francisco Rivero am Institut für Biochemie I der Medizinischen Fakultät der Universität zu Köln und Centre for Biomedical Research, Hull York Medical School, University of Hull, Großbritannien angefertigt. 2Acknowledgement I would like to thank all people that that by any means contribute to the successful completion of this dissertation thesis. Especially I would like to thank: - PD Dr. Francisco Rivero for the opportunity to work in his group on the interesting topic, for sharing his knowledge and experience, for never ending helpfulness, for long discussions and for the opportunity to present my research at various international meetings. - Prof. Dr. Angelika A. Noegel for the opportunity to conduct my work in her renowned institute. - Prof. Dr. Jürgen Dohmen, Prof. Dr. Matthias Hammerschmidt and Prof.
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Characterisation of atypical
Rho GTPases of the RhoBTB family
and their binding partners

Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln


vorgelegt von

Kristína Schenková
aus Nitra, Slowakei


Köln 2010




















Berichterstatter/in: Prof. Dr. Angelika A. Noegel
Prof. Dr. Jürgen Dohmen





Tag der mündlichen Prüfung: 30. Juni 2010




Die vorliegende Arbeit wurde in der Zeit von November 2006 bis Mai 2010 unter
Anleitung von Prof. Dr. Angelika A. Noegel und der Betreuung von PD Dr. Francisco
Rivero am Institut für Biochemie I der Medizinischen Fakultät der Universität zu Köln und
Centre for Biomedical Research, Hull York Medical School, University of Hull,
Großbritannien angefertigt.
2Acknowledgement

I would like to thank all people that that by any means contribute to the successful
completion of this dissertation thesis. Especially I would like to thank:

- PD Dr. Francisco Rivero for the opportunity to work in his group on the interesting
topic, for sharing his knowledge and experience, for never ending helpfulness, for long
discussions and for the opportunity to present my research at various international
meetings.

- Prof. Dr. Angelika A. Noegel for the opportunity to conduct my work in her renowned
institute.

- Prof. Dr. Jürgen Dohmen, Prof. Dr. Matthias Hammerschmidt and Prof. Dr. Ludwig
Eichinger for their willingness and time to read this thesis and participate in the
committee during my exam.

- Prof. Dr. Pontus Aspenström for providing RhoBTB constructs and one cullin
construct, Prof. Dr. Reinhard Fässler for providing kindlin constructs, Dr. Manabu
Furukawa for providing cullin constructs, Prof. Dr. Martin Bähler for myosin construct
and to Dr. Michael Gmachl for providing the ubiquitin construct.

- All my colleagues and co-workers in the Institute for Biochemistry I, Medical Faculty,
University of Cologne and in the Centre for Biomedical Research, Hull York Medical
School, University of Hull for help and advices, especially to Anja and Nils. Thank you
for the great time in Hull ☺!

- My family for support.

- Vlasta for encouragement, emotional support and faith in me.

3Table of contents


1 INTRODUCTION........................................................................................ 8

1.1 The Ras superfamily of small GTPases...................................................... 8
1.2 The Rho-family............................................................................................. 9
1.3 RhoBTB proteins.......................................................................................... 10
1.3.1 Structure of RhoBTB proteins........................................................................ 11
1.3.1.1 The GTPase domain....................................................................................... 11
1.3.1.2 The proline-rich region................................................................................... 12
1.3.1.3 The BTB domain............................................................................................ 13
1.3.1.4 The C-terminal region.................................................................................... 14
1.3.2 Expression of RhoBTB proteins..................................................................... 14
1.3.3 Function of RhoBTB pr15
1.3.3.1 RhoBTB, cell growth, and apoptosis.............................................................. 15
1.3.3.2 RhoBTB and chemokine expression.............................................................. 16
1.3.3.3 RhoBTB and vesicle transport........................................................................ 16
1.3.3.4 RhoBTB and the actin filament system.......................................................... 17
1.3.4 RhoBTB in human diseases............................................................................ 18
1.4 RhoBTB proteins and proteasome-dependent degradation ................... 19
1.4.1 General overview of the proteasome-dependent degradation pathway ........ 19
1.4.2 Cullin dependent E3 ligases .......................................................................... 21
1.4.3 RhoBTB proteins as adaptors of Cul3-dependent ubiquitin ligases ............. 22
1.5 MUF1/LRRC41 ........................................................................................... 23
1.5.1 The SOCS-box .............................................................................................. 24
1.5.2 The LRR ....................................................................................................... 25
1.6 The kindlin protein family ......................................................................... 26
1.6.1 The role of kindlins in integrin activation .................................................... 27
1.6.2 Expression and localisation of kindlins ........................................................ 28
1.6.3 Kindlin in human diseases ............................................................................ 29
1.7 Uev1 .............................................................................................................. 29
1.8 Aims of the study ......................................................................................... 31

2 MATERIAL AND METHODS ................................................................. 32

2.1 Material ....................................................................................................... 32
2.1.1 Cell lines and strains ..................................................................................... 32
2.1.2 Vectors .......................................................................................................... 32
2.1.3 Oligonucleotides for siRNA ......................................................................... 32
2.1.4 Oligonucleotides for RT-PCR ...................................................................... 33
2.1.5 Oligonucleotides for PCR ............................................................................. 33
2.1.6 Constructs ..................................................................................................... 34
2.1.7 Enzymes ........................................................................................................ 35
2.1.8 Antibodies and fluorescent dyes ................................................................... 36
2.1.8.1 Primary antibodies ........................................................................................ 36
2.1.8.2 Secondary antibodies .................................................................................... 36
2.1.8.3 Fluorescent dyes ........................................................................................... 36
2.1.9 Inhibitors ....................................................................................................... 36
2.1.10. Transfection reagents .................................................................................... 37
42.1.11. Antibiotics ..................................................................................................... 37
2.1.12 Molecular weight markers ............................................................................ 37
2.1.13 Chemicals ..................................................................................................... 38
2.1.14 Kits ............................................................................................................... 38
2.1.15 Laboratory material ...................................................................................... 38
2.2 Sterilisation ................................................................................................. 39
2.3 Cell culture methods ................................................................................... 39
2.3.1 Defrosting of mammalian cell stocks ........................................................... 39
2.3.2 Passaging of mammalian cells ...................................................................... 40
2.3.3 Transfection of mammalian cells .................................................................. 40
2.3.4 Drug treatment .............................................................................................. 40
2.3.5 Determination of protein stability ................................................................. 40
2.3.6 Gene silencing .............................................................................................. 41
2.3.7 Cryostocks preparation ................................................................................. 41
2.4 Bacterial culture methods .......................................................................... 42
2.4.1 Media for bacterial cells cultivation ............................................................. 42
2.4.2 Preparation of E. coli XL-1 blue competent cells ........................................ 42
2.4.3 Transformation of E. coli XL-1 blue competent cells .................................. 43
2.4.4 Preparation of glycerol stocks ...................................................................... 43
2.5 Yeast two-hybrid system ............................................................................ 43
2.5.1 Media for yeast cells cultivation ................................................................... 43
2.5.2 Modified lithium acetate method for yeast transformation ........................... 44
2.5.3 Test of galactosidase activity ........................................................................ 44
2.6 Immunohistochemistry ............................................................................... 45
2.6.1 Fixation and permeabilisation of mammalian cells ...................................... 45
2.6.2 Immunodetection of proteins in the cells ...................................................... 45
2.6.3 Immunostaining of mitochondria ................................................................. 45
2.6.4 icrotubules .................................................................. 46
2.6.5 Immunostaining of actin filaments ............................................................... 46
2.6.6 Transferrin uptake ......................................................................................... 46
2.6.7 Microscopy and image processing ................................................................ 46
2.7 Biochemical methods .................................................................................. 47
2.7.1 Lysis of mammalian cells ............................................................................. 47
2.7.2 Immunoprecipitation of proteins with Myc-epitope or GFP-epitope tag ..... 47
2.7.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ............................... 48
2.7.4 Staining of polyacrylamide gels with Coomassie-Brilliant-Blue R 250 ...... 49
2.7.5 Transfer of proteins to membrane (Western blot) ........................................ 49
2.7.6 Staining of proteins bound to the membranes .............................................. 50
2.7.7 Immunodetection of proteins bound to the membrane ................................. 50
2.7.8 Subcellular fractionation of mammalian cells .............................................. 50
2.7.9 Ubiquitination assay ..................................................................................... 51
2.7.10 Protein expression and purification .............................................................. 52
2.7.11 Expression and purification of GST-Ubiquitin (Ub) .................................... 52
2.7.12 BCA (bicinchoninic acid) protein assay ....................................................... 53
2.7.13 GTP binding assay ........................................................................................ 53
2.8 Molecular biology methods ........................................................................ 54
2.8.1 Isolation of plasmid DNA by the alkaline method ....................................... 54
2.8.2 id DNA for transfection of mammalian cells ................... 54
2.8.3 Determination of plasmid DNA concentration ............................................. 55
2.8.4 DNA-agarose gel electrophoresis ................................................................. 56
52.8.5 Polymerase chain reaction (PCR) ................................................................. 56
2.8.6 Reverse transcription PCR (RT-PCR) .......................................................... 56
2.8.7 Elution of DNA-fragment from agarose gel ................................................. 57
2.8.8 Restriction reaction ....................................................................................... 58
2.8.9 Dephosphorylation of DNA 5´- ends ........................................................... 58
2.8.10 Ligation of vector and DNA-fragment ......................................................... 58
2.8.11 DNA-sequencing .......................................................................................... 58

3 RESULTS .................................................................................................... 59

3.1 Characterisation of RhoBTB proteins ...................................................... 59
3.1.1 Subcellular localisation of RhoBTB3 ........................................................... 59
3.1.1.1 RhoBTB3 partially localises at early endosomes through the C-terminal
domain............................................................................................................ 60
3.1.1.2 GFP-RhoBTB3 occasionally co-localises with transferrin .......................... 61
3.1.1.3 Overexpression of RhoBTB3 disrupts the Golgi apparatus ......................... 64
3.1.1.4 RhoBTB3 does not localise to the mitochondria .......................................... 67
3.1.1.5 Interaction of RhoBTB3 with the cytoskeleton ............................................ 69
3.1.1.5.1 Interaction of RhoBTB3 with microtubules ................................................. 69
3.1.1.5.2 Co-localisation of RhoBTB2 and RhoBTB3 does not depend on an intact
microtubule network ..................................................................................... 69
3.1.1.5.3 Interaction of RhoBTB3 with the actin cytoskeleton ................................... 72
3.1.2 The GTPase domain of RhoBTB3 does not bind GTP ................................ 72
3.1.3 Interaction of RhoBTB3 with Cul3 .............................................................. 73
3.1.3.1 RhoBTB3 interacts with endogenous Cul3 .................................................. 73
3.1.3.2 Dimerisation of RhoBTB is Cul3-independent ............................................ 74
3.1.4 Proteasomal degradation of RhoBTB3 is prevented by intramolecular
interaction ..................................................................................................... 75
3.1.5 RhoBTB3 is ubiquitinated by a Cul3-dependent ligase ............................... 76
3.2 Characterisation of MUF1/LRRC41, a binding partner of RhoBTB
GTPases ....................................................................................................... 77
3.2.1 Computational characterisation of MUF1 .................................................... 78
3.2.2 Lrrc41 mRNA is ubiquitously expressed ..................................................... 80
3.2.3 Localisation of MUF1 .................................................................................. 80
3.2.4 MUF1 interacts with all three RhoBTB proteins in vivo .............................. 84
3.2.5. RhoBTB3 has probably multiple binding sites on MUF1 ............................ 88
3.2.6. MUF1 has probably multiple binding sites on RhoBTB3 ............................ 89
3.2.7. MUF1 interacts with RhoBTB3 in a Cul3 and Cul5 independent manner ... 90
3.2.8 MUF1 is degraded in the proteasome in a Cul5 independent manner ......... 91
3.2.9 MUF1 is able to homodimerise .................................................................... 94
3.2.10 Characterisation of MUF1 interaction with potential binding partners ........ 94
3.2.10.1 MyoIXb as a potential binding partner of MUF1 ......................................... 95
3.2.10.2 RBPMS as a potential binding partner of MUF1 ......................................... 96
3.2.11 Dimerisation of cullins ................................................................................. 97
3.3 Kindlin, an interaction partner of RhoBTB3 .......................................... 100
3.3.1 RhoBTB2 and RhoBTB3 interact with kindlin-1 and kindlin-2 .................. 100
3.3.2 RhoBTB3 has probably multiple binding sites on kindlin-1 ........................ 101
3.3.3 RhoBTB3 partially co-localises with kindlin-1 and kindlin-2 ..................... 103
3.3 Uev1a, an interaction partner of RhoBTB3 ............................................. 104

64 DISCUSSION .................................................................................... 106

4.1 Subcellular localisation of RhoBTB3 ........................................................ 106
4.2 RhoBTB3 is an adaptor of Cul3-dependent ligase complexes ................ 108
4.3 Characterisation of MUF1 ......................................................................... 111
4.4 Expression and subcellular localisation of MUF1 ................................... 111
4.5 MUF1 as an adaptor for Cul5 ubiquitin ligase ........................................ 112
4.6 MUF1 is a binding partner of RhoBTB proteins ..................................... 113
4.7 MUF1 as a substrate of Cul3-RhoBTB3 ubiquitin ligase ........................ 114
4.8 Heterodimerisation of Cul3 and Cul5 ....................................................... 116
4.9 A possible model of MUF1 function and degradation ............................. 117
4.10 Kindlin is a binding partner of RhoBTB proteins ................................... 119
4.11 Uev1a is a binding partner of RhoBTB proteins ..................................... 121

5 ABSTRACT ................................................................................................ 123

6 ZUSAMMENFASSUNG ............................................................................ 125

7 REFERENCES ........................................................................................... 127

8 ABBREVIATIONS ..................................................................................... 138

Erklärung ...................................................................................................................... 140

Curriculum vitae ........................................................................................................... 141

Lebenslauf ...................................................................................................................... 142


71 Introduction

1.1 The Ras superfamily of small GTPases
The Ras superfamily, named after the most studied oncogene in human carcinogenesis,
Ras, represents a group of small guanosine triphosphatases (GTPases), which comprises
over 150 members in humans but can be found in all eukaryotes (Colicelli 2004,
Wennerberg et al. 2005). The common feature of these proteins (with few exceptions) is
their ability to bind and hydrolyse GTP due to the presence of a ~20 kDa G-domain.
The G-domain consists of a six-stranded β-sheet and five α-helices and contains four to
five conserved G-box motif elements (G1-G5), which are responsible for binding GTP.
The so-called switch domains I and II bind γ-phosphate oxygens of GTP and after GTP
hydrolysis and release of γ-phosphate, the switch domains relax into the GDP-specific
conformation (Bourne et al. 1991).
Ras proteins act as molecular switches, cycling between an active GTP-bound state
and an inactive GDP-bound state. Guanine nucleotide exchange factors (GEFs)
and GTPase activating proteins (GAPs) regulate the activation/inactivation cycle (Figure
1.1). The dissociation of GDP from the inactive GDP-bound form is promoted by
an upstream signal and conversion to the GTP-bound state is catalysed by GEFs. In the
GTP-bound state, small GTPases are active and interact with downstream effector proteins.
Hydrolysis of GTP to GDP is very slow and is accelerated by GAPs. In addition, GDP-
dissociation inhibitors (GDIs) regulate cycling of Rho and Rab GTPases between cytosol
and membranes by capturing them in both GTP- and GDP-bound states (Colicelli 2004,
Takai et al. 2001).



Figure 1.1: Regulation of activity of small GTPases. In the active state, the GTPase binds GTP and
interacts with effectors of signalisation. GAPs accelerate the hydrolysis of bound GTP. In GDP-bound state,
small GTPases are inactive. GEFs catalyse the release of GDP. Due to the higher cytosolic concentration of
GTP than GDP, the GTPase can again bind GTP. Taken from Colicelli (2004).
8The Ras superfamily can be subdivided into five families according to the sequence
and known functions of their members: Ras (Rat sarcoma oncoproteins), Rho (Ras
homologous proteins), Rab (Ras-like proteins in brain), Ran (Ras-like nuclear protein), Arf
(ADP-ribosylation factor) and Miro (mitochondrial Rho) (Colicelli 2004, Wennerberg
et al. 2005). Members of the Ras superfamily are involved in a variety of cellular processes
like gene expression (Ras, Rho), regulation of cell proliferation, differentiation
and survival (Ras), actin organisation and cell cycle progression (Rho), vesicular transport
and trafficking of proteins (Rab, Arf), transport between nucleus and cytoplasm
and microtubule organisation (Ran) (Colicelli 2004, Takai et al. 2001, Wennerberg
et al. 2005).

1.2 The Rho-family
The Rho family is characterised by an insertion (so-called Rho insert) of usually 13
residues with high sequence variability between the fifth ß-strand and the fourth α-helix in
the GTPase domain (Valencia et al. 1991). To date, 21 proteins of the Rho family have
been described in vertebrates (Figure 1.2): Cdc42-like (Cdc42, TC10, TCL, Chp/Wrch2,
Wrch1), Rac-like (Rac1-3, RhoG), Rho-like (RhoA-C), Rnd (Rnd1-2, Rnd3/RhoE), RhoD
(RhoD und Rif), RhoH/TTF and RhoBTB (RhoBTB1-3) (Wennerberg and Der 2004).
RhoBTB3 is very often not considered as a member of the Rho family because of its
divergent GTPase domain. Members of the Rho family are present from lower eukaryotes
up to mammals and have not been identified in eubacteria and archaea.
The most studied Rho GTPases are RhoA, Rac1 and Cdc42. The members of the Rho, Rac
and Cdc42 subfamilies are involved in regulation of cytoskeleton reorganisation in
response to extracellular signals. Rho proteins are responsible for the formation of stress
fibres and focal adhesions, Rac proteins for the formation of lamellipodia and Cdc42
proteins are involved in filopodia formation. They also have been implicated in many other
cytoskeleton-dependent processes like cell growth (G1 cell cycle progression), cytokinesis,
morphogenesis, cell-cell interaction, cell polarity and cell migration. In addition, Rho
proteins are involved in cellular processes such a membrane trafficking, endocytosis
and gene expression (Jaffe and Hall 2005, Takai et al. 2001). Other Rho GTPases have
been also identified in cytoskeleton-dependent processes like loss or formation of stress
fibres (Rnd1, Rnd3, RhoD and Rif), focal adhesions (Rnd1, Rnd3, and RhoD), cell
migration and cell-cell adhesion (Rnd3), formation of Cdc42-independent filopodia (Rif)
9and cell migration and cytokinesis (RhoD) (Vega and Ridley 2007). For others, like Rnd2
and RhoBTB1, 2 and 3 an effect on the actin cytoskeleton has not been observed.



Figure 1.2: Phylogenetic tree of the Rho family of small GTPases. The family can be divided into six
subfamilies: RhoA-related, Rac-related, Cdc42-related, Rnd proteins, RhoH/TTF and RhoBTB proteins. Note
that RhoBTB3 is not shown in this tree because of its divergent GTPase domain. Taken from
Burridge and Wennerberg (2004).

Some of the members of the Rho family are referred to as atypical Rho GTPases because
their structure and functional characteristics differ from those of the classical ones.
The atypical GTPases are Rnd proteins, RhoH, Wrch1, Chp/Wrch2 and RhoBTB
(Aspenström et al. 2007). One of the most striking features that make these Rho GTPases
atypical is the difference in the cycling between the GTP- and the GDP-bound state. For
example, RhoH has been shown to be constitutively in the GTP-bound state (Li et al.
2002), as well as Rnd1 and Rnd3 (Chardin 2006). Wrch1 is predominantly in the GTP-
bound state (Saras et al. 2004, Shutes et al. 2004). RhoBTB proteins seem to be even more
different – RhoBTB2 does not bind GTP (Chang et al. 2006) and it was shown recently
that RhoBTB3 can bind and hydrolyse ATP (Espinosa et al. 2009).

1.3 RhoBTB proteins
The RhoBTB subfamily constitutes the more recent addition to the Rho family. It was
identified during the study of Rho-related protein-encoding genes in Dictyostelium
discoideum (Rivero et al. 2001). In humans, the RhoBTB subfamily is composed of three
10

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