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Pak5) Function AN ANALYSIS OF P21-ACTIVATED KINASE-5 (PAK5) FUNCTION BAHAREH TABANIFAR NATIONAL UNIVERSITY OF SINGAPORE 2016 AN ANALYSIS OF P21-ACTIVATED KINASE-5 (PAK5) FUNCTION BAHAREH TABANIFAR A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2016 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _________________ Bahareh Tabanifar 2016 I Acknowledgments Foremost, I would like to express my sincere gratitude to my supervisor Professor Edward Manser for the continuous support of my Ph.D study and research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance and support helped me in all the time of research and writing of this thesis. My deepest gratitude goes to my family: My beloved husband who has been there for me every step of the way… My parents for their unconditional support throughout my life and my studies. My special thanks to my mentors: Dr.Yohendran Baskaran and Dr. Ling Goh, for always being patient to answer my questions, for all of their support and help. I would like to thank the rest of my thesis committee: Professor. Shazib Pervaiz and Professor Giulia Rancati, for their encouragements and insightful comments. My sincere thanks also goes to the fellows in sGSK.IMCB lab and IMB: Dr. Jing Ming Dong, Dr.Zhoushen Zhao, Dr. Praju Vikas Anekal, Dr. Pao Chun Lin, Dr Ivan Khang Nyee TAN for their invaluable knowledge and generous guidance. Also many thanks to my friends in the lab: Ms.Elsa, Ms. Felicia and Ms.Christy for being supportive, kind and fun! I have to thank Agency for Science, Technology And Research (A*STAR) for granting me Singapore International Graduate Award (SINGA). II Table of Contents Chapter 1: Introduction ............................................................................................................. 1 1.1 Overview of Rho GTPases ............................................................................................... 1 1.2 Overview of P21 activated kinases (PAKs) ..................................................................... 2 1.2.1 Domain structure of group I and II PAKs ..................................................................... 3 1.2.2 Regulation of group I and II PAKs ............................................................................... 4 1.2.3 Expression pattern and subcellular localization of PAK5............................................. 7 1.2.4 Phosphorylation targets of PAK5 ................................................................................. 9 1.2.5 The role of PAK5 in cytoskeleton organization .......................................................... 10 1.2.6 Group II PAKs and adherens junctions ....................................................................... 11 1.2.6.1 Background on adherens junctions .......................................................................... 11 1.2.6.2 Localization of group II PAKs at cell-cell junctions ............................................... 13 1.2.7 PAK5 and the regulation of apoptosis ........................................................................ 16 1.2.7.1 Background .............................................................................................................. 16 1.2.7.2 PAK5 plays a role in cell survival ........................................................................... 19 1.2.8 The role of PAK5 on MAPK pathway ........................................................................ 22 1.2.8.1 Overview of Raf-1 kinase ........................................................................................ 22 1.2.8.2 PAK5 role on Raf-1 activation................................................................................. 23 1.2.9 The role of PAK5 on cancer progression .................................................................... 26 1.2.10 PAK5 role in neuronal vesicle trafficking ................................................................ 28 1.3 Overview of the LZTS family ........................................................................................ 28 1.3.1 The role of LZTS2 on tumour suppression ................................................................. 29 1.3.2 LZTS2 and regulation of the cytoskeleton .................................................................. 33 1.3.3 LZT2 and cell migration ............................................................................................. 34 1.3.4 The role of LZTS2 in development ............................................................................. 34 1.4 Aims of the study ........................................................................................................... 35 III Chapter 2: Materials and methods .......................................................................................... 37 2.1 Materials ........................................................................................................................ 37 2.1.1 Plasmids and siRNAs .................................................................................................. 37 2.1.2 Antibodies and reagents .............................................................................................. 38 2.2 DNA manipulation methods .......................................................................................... 38 2.2.1 Plasmid DNA purification .......................................................................................... 38 2.2.2 Polymerase Chain Reaction (PCR) and gel electrophoresis ....................................... 39 2.2.3 Preparation of competent cells for transformation via heat shock method ................. 40 2.2.4 Cloning ........................................................................................................................ 40 2.3 Protein analysis methods ................................................................................................ 42 2.3.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE) .............................................. 42 2.3.2 Western blotting .......................................................................................................... 42 2.3.3 Expression and purification of bacterial expressed recombinant proteins .................. 42 2.3.4 Antibody production ................................................................................................... 43 2.3.4.1 Coupling peptide to Cyanogen Bromide-Activated agarose for Antibody purification ........................................................................................................................... 43 2.3.4.2 Affinity purification of antibody .............................................................................. 44 2.4 Cell culture techniques and manipulations .................................................................... 44 2.4.1 Cell culture .................................................................................................................. 44 2.4.2 Transient transfection .................................................................................................. 45 2.4.3 siRNA transfection...................................................................................................... 45 2.4.4 Establishing stable lines .............................................................................................. 45 2.4.5 Immuno-precipitation ................................................................................................. 47 2.4.6 In vitro kinase assay .................................................................................................... 47 2.4.7 Immunostaining .......................................................................................................... 47 2.5 Methods for detections of protein-protein interactions .................................................. 48 IV 2.5.1 Establishing stable lines for BioID and GFP pull down analysis ............................... 48 2.5.2 Adapting cells in SILAC media .................................................................................. 48 2.5.3 Purification of biotin-labelled proteins from stable GFP-Bir*A cells ........................ 49 2.5.4 GFP Purification of proteins from stable GFP-Bir*A cells ........................................ 50 Chapter 3: Regulation of PAK5 activity ................................................................................. 52 3.1 Introduction .................................................................................................................... 52 3.2 PAK5 contains a functional auto inhibitory domain ...................................................... 54 3.3 PAK5 is likely a Cdc42-specific effector ...................................................................... 54 3.4 PAK5 auto-regulatory domain can inhibit PAK4 .......................................................... 59 3.5 Different cellular distributions of PAK4 and PAK5 ...................................................... 61 3.6 The non-catlytic domain of PAK5 is sufficient for puncta formation ........................... 62 3.7 Establishing the regions of PAK5 involved in self-association ..................................... 67 3.8 PAK5 self-association contributes to its high basal
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