The Regulation of Focal Adhesion Kinase by the scaffolding protein RACK1

294 Pages·2013·3.97 MB·English

by  Susan Dwane

The Regulation of Focal Adhesion Kinase by the scaffolding
protein RACK1

Submitted in accordance with the academic requirements to the Department of Life
Sciences, Faculty of Science and Engineering, University of Limerick in fulfilment of
the requirements for the degree of
Doctor of Philosophy
By
Susan Dwane, B.Sc.

Thesis Supervisor: Dr. Patrick Kiely, PhD
2013
To my family, with love and appreciation
TABLE OF CONTENTS
DECLARATION ............................................................................................................... I
ACKNOWLEDGEMENTS ............................................................................................. II
ABSTRACT .................................................................................................................... III
ABBREVIATIONS ......................................................................................................... V

CHAPTER 1: GENERAL INTRODUCTION ............................................................. 1
1.1 Cell Migration ........................................................................................................ 2
1.1.1 Extracellular matrix and adhesion receptors ............................................. 4
1.1.2 Receptor tyrosine kinases ......................................................................... 9
1.1.3 Non-receptor tyrosine kinases ................................................................. 12
1.1.4 Adaptor proteins ...................................................................................... 15
1.2 Focal Adhesion Kinase (FAK) ............................................................................ 18
1.2.1 FAK: Gene and expression .................................................................... 18
1.2.2 FAK: Protein structure and functional domains ..................................... 19
1.2.2.1 FAK: The N-terminal domain ........................................................... 19
1.2.2.2 FAK: The Kinase domain .................................................................. 22
1.2.2.3 FAK: The C-terminal domain ........................................................... 26
1.2.3 FAK: Role in focal adhesion complex assembly .................................... 27
1.2.4 FAK: Role in focal adhesion turnover .................................................... 33
1.2.5 FAK: Signalling in cell invasion ............................................................ 37
1.2.6 FAK: Signalling in angiogenesis ........................................................... 39
1.2.7 FAK: Signalling in cell survival ........................................................... 41
1.2.8 FAK: Role in the brain ........................................................................... 45
1.2.9 FAK: Axonal guidance and neurite outgrowth ...................................... 47
1.3 Receptor for Activated C Kinase 1 (RACK1) ..................................................... 51
1.3.1 RACK1: Gene and expression ................................................................ 51
1.3.2 RACK1: Protein structure and functional domains ................................ 52
1.3.3 RACK1: Role as scaffolding protein ...................................................... 54
1.3.4 RACK1: Role in the brain ....................................................................... 55
1.3.5 RACK1: Role in brain disorders ............................................................. 59
1.4 Aim of study ........................................................................................................ 60

CHAPTER 2: MATERIALS AND METHODS ........................................................ 62
2.1 Materials .............................................................................................................. 63
2.1.1 General Chemicals and Reagents ............................................................ 63
2.1.2 Plasmid stocks and source ...................................................................... 63
2.1.3 Antibodies ............................................................................................... 64
2.1.4 Cell Culture Reagents ............................................................................. 64
2.1.5 Cell lines ................................................................................................. 65
2.2 Methods ............................................................................................................... 66
2.2.1 Bacterial Transformation of CaCl competent cells ................................ 66
2
2.2.2 Preparation of plasmid DNA using Sigma Midi Kit ............................... 66
2.2.3 Cell Culture and Transfection ................................................................. 67
2.2.4 Differentiation of PC12 and SH-SY5Y cells .......................................... 67
2.2.5 Dissection and homogenisation of Sprague Dawley rat hippocampus ... 67
2.2.6 Preparation of cellular protein extracts ................................................... 68
2.2.7 Immunoprecipitation of proteins from cellular extracts ......................... 69
2.2.8 SDS-PAGE and Western Blotting .......................................................... 69
2.2.9 Immunofluorescence to detect protein colocalisation in cells ................ 70
2.2.10 Cell Adhesion and Cell Spreading Assay ............................................... 71
2.2.11 Neurite outgrowth assay .......................................................................... 72
2.2.12 Spot Synthesis of peptides and Overlay analysis .................................... 72
2.2.13 Alanine substitution array analysis ......................................................... 73
2.2.14 Mass Spectrometry Analysis of RACK1 interacting proteins in
hippocampus ........................................................................................... 73

CHAPTER 3: DEVELOPMENT OF NEURONAL MODEL SYSTEM TO STUDY
THE RELATIONSHIP BETWEEN RACK1 AND FAK ......................................... 75
3.1 Abstract ................................................................................................................ 76
3.2 Introduction .......................................................................................................... 77
3.3 Results .................................................................................................................. 82
3.3.1 Fractionation of hippocampus from Sprague Dawley rats. .................... 82
3.3.2 Localisation of FAK and RACK1 in fractionated hippocampus from
Sprague Dawley rats. .............................................................................. 82
3.3.3 Optimisation of matrices for differentiation of SH-SY5Y cells. ............ 85
3.3.4 Optimisation of growth factor media for differentiation of SH-SY5Y
cells. ........................................................................................................ 87
3.3.5 Confirmation of differentiation parameters for SH-SY5Y cells. ............ 89
3.3.6 Optimisation of matrices for differentiation of PC12 cells. .................... 91
3.3.7 Confirmation of morphological differentiation of SH-SY5Y and PC12
cells. ........................................................................................................ 95
3.3.8 Confirmation of biochemical differentiation of SH-SY5Y and PC12
cells. ........................................................................................................ 97
3.3.9 Immunofluorescent staining of growth cones in SH-SY5Y and PC12
cells. ........................................................................................................ 99
3.4 Discussion .......................................................................................................... 101

CHAPTER 4: TO IDENTIFY AND CHARACTERISE NOVEL RACK1
INTERACTING PROTEINS: FOCUS ON COFILIN-1 ........................................ 106
4.1 Abstract .............................................................................................................. 107
4.2 Introduction ........................................................................................................ 108
4.3 Results ................................................................................................................ 111
4.3.1 Identification of RACK1 interacting proteins using Mass Spectrometry
analysis .................................................................................................. 111
4.3.2 Grouping of proteins found ................................................................... 111
4.3.3 Mass spectrometry data for Cofilin-1 ................................................... 113
4.3.4 RACK1 binds to Cofilin-1 in hippocampus and SH-SY5Y cells ......... 116
4.3.5 RACK1 colocalises with Cofilin-1 in undifferentiated and differentiated
SH-SY5Y cells ...................................................................................... 116
4.3.6 FAK binds to Cofilin-1 in hippocampus and differentiated SH-SY5Y
cells ....................................................................................................... 119
4.3.7 FAK colocalises with Cofilin-1 in differentiated SH-SY5Y cells ........ 121
4.3.8 The interaction between RACK1 and FAK is regulated by
differentiation ........................................................................................ 121
4.3.9 Cofilin-1 overexpression does not affect FAK activity ........................ 123
4.3.10 IGF-1 stimulation affects PP2A mediated dephosphorylation of Cofilin-1
in SH-SY5Y cells. ................................................................................. 126
4.3.11 Identification of Cofilin-1 interaction with RACK1 domain
truncations ............................................................................................. 131
4.3.12 Confirmation of Cofilin-1 interaction with WD4 domain of RACK1 .. 131
4.3.13 Identification of Cofilin-1 interaction site on RACK1 peptide arrays .. 134
4.3.14 Modelling of RACK1 WD4 domain ..................................................... 134
4.4 Discussion .......................................................................................................... 138

CHAPTER 5: REGULATION OF FAK ACTIVITY BY AGAP2 AND ARF1 .... 145
5.1 Abstract .............................................................................................................. 146
5.2 Introduction ........................................................................................................ 148
5.3 Results ................................................................................................................ 151
5.3.1 RACK1 binds to FAK in hippocampus and PC12 cells ....................... 151
5.3.2 RACK1 colocalises with FAK in differentiated PC12 cells ................. 153
5.3.3 Y52F mutation of RACK1 affects neurite outgrowth in PC12 cells .... 153
5.3.4 Mass spectrometry data for Arf1 and AGAP2 ...................................... 158
5.3.5 RACK1 binds to AGAP2 in hippocampus and neuronal cell lines ...... 161
5.3.6 RACK1 colocalises with AGAP2 in differentiated SH-SY5Y cells .... 161
5.3.7 RACK1 binds to Arf1 in hippocampus and colocalises with Arf1 in SH-
SY5Y cells ............................................................................................ 163
5.3.8 RACK1 preferentially interacts with inactive Arf1 .............................. 167
5.3.9 Complex formation for regulating FAK activity .................................. 169
5.3.10 Activation of FAK is promoted by inactive Arf1 ................................. 171
5.3.11 Cell adhesion is promoted by inactive Arf1 .......................................... 171
5.3.12 Effect of knockdown of RACK1 on complex formation ...................... 173
5.3.13 Investigation of Arf1 interaction site on RACK1 using RACK1 domain
truncations ............................................................................................. 176
5.3.14 Identification of Arf1 interaction sites on RACK1 peptide arrays ....... 178
5.3.15 Identification of Arf1 interaction sites on RACK1 alanine substitution
arrays ..................................................................................................... 178
5.3.16 Modelling of RACK1 WD3 and WD7 domains ................................... 180
5.4 Discussion .......................................................................................................... 183

CHAPTER 6: GENERAL DISCUSSION ................................................................. 188
6.1 The importance of FAK activity in cell migration ............................................ 189
6.2 Development of neuronal model system ........................................................... 190
6.3 Identification of Cofilin-1 as RACK1 interacting protein ................................. 191
6.4 Importance of Arf1 and AGAP2 in regulating FAK activity ............................ 193
6.5 Implications for Regulation of FAK activity ..................................................... 196

BIBLIOGRAPHY ....................................................................................................... 200
APPENDIX A .............................................................................................................. 268
DECLARATION

I hereby declare that I am the sole author of this thesis and that it has not been submitted
for any other University or higher institution, or for any other academic award in this
University. References and acknowledgements have been made, where necessary, to the
work of others.

Signature: Date:
-------------------------------------------------------------------- ---------------------------
Susan Dwane
Department of Life Sciences
Faculty of Science and Engineering
University of Limerick

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ACKNOWLEDGEMENTS
Firstly, I would like to thank Dr. Pat Kiely for being a wonderful supervisor and friend.
The encouragement and guidance you have given to me over the past 3 and a half years
is greatly appreciated. I have learned so much from you. Thanks to Dr. Paddy McHale
and Dr. Virginie Gautier for believing in me and encouraging me to pursue further
study. Health Research Board Ireland is acknowledged for financial support during this
study.
Thanks to all the members of the Kiely lab, both past and present, for all the
support and laughter along the way. A big thank you to Edel for always being there for
me. I couldn’t have done it without you! Thanks to Maeve and Miranda (my original lab
buddy!) for their proofreading skills; Michael for his help with Pymol and Craig for the
laughs and support when things wouldn’t work. We’ll always have Potter Saturdays! I
would also like to thank all the members of the ROC lab for the first year of my PhD in
UCC. In particular, thanks to Mary and Cedric for your continued support and
friendship.
Thank you to my family for all their love and support and even learning the
name of my thesis! Mom, thanks for the hugs and for always being at the end of the
phone ready to listen. Caelen, for always being there when I need you and for all the
warnings not to procrastinate during my write up! Melanie, thanks for always making
me smile and for all the laughs at random things that no one else understands!
Finally, thank you to Stefan, for being there through it all. Every step of the way,
you supported me and I will always be grateful for that.
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