Regulation of a2 Chimaerin and Associated Phosphorylation Pathways in Neuronal Signalling and Morphogenesis By Tom Jacobs Submitted to University College London for the degree of Doctor of Philosophy, Ph.D. 2004 Department of Molecular Neuroscience Institute of Neurology University College London 1 UMI Number: U602542 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602542 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ^Uo mp darling wife, ^fulie, fo r pour constant encouragement and support. ^Uo mp parents, fo r alwaps being there and for helping to get me here Acknowledgements I would like to thank Prof. Louis Lim not only for the opportunity to study for a PhD in his London lab, but also for the fantastic experience of working in Singapore and not to mention, allowing me to meet my wife, Julie. To my supervisor Dr. Chrisine Hall, thank you for all your guidance and encouragement during the course of this work. Thanks to Dr. Clinton Monfiies for the use of numerous molecular biology reagents, Dr Kate Marler and Dr Sheila Govind for initial demonstrations and training and Mathew Brown for insightful discussions. Thank you also to lab members Bhav and Sally for all your help and assistance but especially for cheering up the working environment. Thank you to all members of the Singapore lab for making me feel so welcome during my stay. Special thanks to my wife Dr Julie Edgeworth for all your emotional and professional support, encouragement and much needed motivation. Thanks to my parents, who have always supported me and put me in the position to do a PhD in the first place. 3 Abstract Abstract The generation of neuronal dendrites and axons is a highly dynamic process. These extensive structural changes are regulated by molecules such as GTPases, as well as kinases/phosphatases, which provide cyclical control mechanisms. Rho family GTPases are regulated, in part, by GAPs such as neurone-enriched chimaerin, and act through downstream kinase effectors, including cyclin dependant kinase 5 (Cdk5). a 2 chimaerin is a Rac GAP and phorbol ester receptor, which also displays Cdc42/Rac effector functions. From this study it appears a2 chimaerin exists in an auto- inhibited state in the cytosol and that DAG/phorbol ester association, or selected mutations translocates a2 chimaerin to the membrane, leading to GAP activation. a2 chimaerin GAP activity was implicated in PMA induced neurite collapse of N1E-115 neuroblastoma cells. PMA also promoted the association of a2 chimaerin with target proteins Collapsin Response Mediator Protein-2 (CRMP-2) and p35/Cdk5, possibly as a result of a conformational change. The Rac effector, Cdk5 is a proline directed, Ser/Thr kinase, whose activity contributes to neurite outgrowth, growth cone collapse and neuronal migration. Deregulation of Cdk5 in neurodegenerative diseases is implicated in pathological changes. a2-chimaerin GAP domain was shown to interact in vivo both with p35, the neuronal activator of Cdk5 and with Cdk5 itself. CRMP-2 interacted in vivo with a2 chimaerin SH2 domain, for which tyrosine phosphorylation was not essential. The functional correlates of these protein interactions of a2 chimaerin were investigated in vivo. CRMP-2, which is hyperphosphorylated in Alzheimer’s disease was identified as a novel Cdk5 substrate in vitro with CRMP-2 Ser522 identified as the target site. Phosphorylation of CRMP-2 Ser522, potentially primes for GSK-3P phosphorylation of CRMP-2 Ser518 and Thr 514 which alters CRMP-2 conformation. a2 chimaerin was not a Cdk5 substrate but may be tyrosine phosphorylated, which could potentially regulate its activity or associations in neuronal cells. 4 Table of Contents TABLE OF CONTENTS Title Page 1 Dedication 2 Acknowledgements 3 Abstract 4 Table of Contents 5-10 List of Figures 11-14 Abbreviations 15-18 Chapter 1 Introduction 19-54 1A The Cytoskeleton 20-25 1.A.1 Microtubules 21-22 1.A3 Actin Filaments 22-25 1 A.2.1 Actin binding proteins 22-23 l.A.2.2 Actin filament nucleation 23-24 IA.2.3 Actin filament depolymerisation 24 l.A.2.4 Actin filament organisation 25 1.B Rho GTPases 26-34 1.B.1 Guanine nucleotide exchange factors (GEFs) 27-28 I.B.2 GTPase activating proteins (GAPs) 28-33 IB.2.1 Rho GAP regulation ' 29-54 1 B.2.1.1 Lipid association 29-30 l.B.2.1.2 Protein interactions 30-31 1 .B.2.1.3 Phosphorylation 31 -32 l.B.2.2 Rho GAP effector functions 32-33 1.B3 Guanine nucleotide dissociation inhibitors (GDI) 33 1.B.4 Alternative mechanisms of Rho GTPase regulation 33-34 l.C The Rho GTPases and the cytoskeleton 34-37 l.C .l Actin dynamics 34-36 1.C2 Microtubule dynamics 36-37 5 Table of Contents 1.D Central Nervous System (CNS) Development 37-54 1.D.1 Neuronal Migration 38-40 l.D.1.1 Cyclin dependent kinase 5 (Cdk5) 38-40 1JX2 Neurite outgrowth 40-47 l.D.2.1 Rho GAPs 42-43 1.D 22 Collapsin response mediator protein 2 (CRMP-2) 43-44 l.D.2.3 Cyclin dependent kinase 5 (Cdk5) 45-47 1J>3 Axonal Guidance 47- 52 l.D.3.1 Semaphorins 49-51 l.D.3.2 Regulation of guidance cues 51-52 1.D.4 Neurodegenerative Diseases 52-54 1.E Aims 54 Chanter 2 Materials & Methods 55-78 2.A Materials 56-58 2A.1 Laboratory Reagents 56 2.A.2 DNA Manipulation Reagents 56 2.A3 Tissue Culture Reagents 56 2A.4 Protein Work Reagents 57 2 .A 3 Antibodies 57 2.A.6 cDNA Constructs 58 2.A.7 Oligomer Synthesis ans cDNA sequenecing 58 2.B Methods 58-78 2.B.1 DNA Manipulation 58-66 2.B.1.1 Competent bacterial transformation 58 2.B.1.2 Plasmid DNA mini preparations 59-60 2.B.1.3 Plasmid DNA midi preparations 60-61 2.B.1.4 Phenol/Chloroform extraction 61 2.B.1.5 Ethanol precipitation 62 2.B.1.6 Quantitation of DNA 62 2.B.1.7 Restriction digestion of DNA 62 2.B.1.8 Agarose gel electrophoresis 63 6 Table of Contents 2.B. 1.9 Visualisation of DNA with ethidium bromide 63 2.B.1.10 Site directed mutagenesis 63-65 2.B. 1.11 Identification of positive mutant clones 66 23.2 Cell Culture 67 23.2.1 Cell stocks 67 233 Morphological Analysis 68-70 23.3.1 Coverslip preparation 68 2.B.3.2 Transfection on coverslips 68 2.B.3.3 Immunostaining 68-69 2.B.3.4 Fixed cell imaging 69 2.B.3.5 Live cell imaging 70 2.B.4 Protein W ork 70-75 23.4.1 Recombinant protein production 70-71 2.B.4.1.1 GST Fused 71 23.4.13 Non Tagged Protein 71 23.4.2 Mammalian cell90mm plate transfection 71 2.B.4.3 Cell treatment 72 2.B.4.3.1 Pervanadate 72 2.B.4.3.2 Phorbol 12-myristate 13-acetate (PMA) 72 2.B.4.4 Cell lysis 72-73 23.4.5 Immunoprecipitation/GST-pulldown 73 2.B.4.6 Rac-GTP assay 74 2.B.4.7 Cell fiactionation 74-75 2.B.4.8 In vitro kinase assays 75 2.B5 Protein Analysis 75-78 2.B.5.1 Protein quantification 75 23.5.2 SDS-polyacrylamide gel electrophoresis 76-77 2.B.5.3 Commassie blue staining 77 2.B.5.4 Western transfer (semi dry blotting) 77 2.B.5.5 Immunoblotting 78 7 Table of Contents Chapter 3 Results I 79-101 (x2 Chimaerin: GAP Regulation and Neuronal Morphology 80 3.A a2 Chimaerin Location 81-85 3.A.1 Mutations in the SH2 domain and GAP domain of a2 Chimaerin alter subcellular distribution 81-83 3A.2 PMA translocates a2 chimaerin 83-85 3.B a2 Chimaerin GAP Activity and Neuroblastoma Cell Morphology 86-96 3.B.1 PMA causes retraction of a2.10 neuroblastoma cells accompanied by a decrease in Rac-GTP 86-88 3 B 2 PMA causes retraction of N1E-115 neuroblastoma cells which can be inhibited with the expression of GAP inactive a 2 chimaerin 88-92 3.B.3 a2 Chimaerin GAP activity inhibits neuritogenesis of N1E-115 neuroblastoma cells when membrane targeted 92-94 3.B.4 GAP inactive a2 Chimaerin expression induces neurite outgrowth inNlE-115 neuroblastoma cells 94-96 3.C PMA Activates a2 Chimaerin GAP activity 96-100 3.C.1 PMA alters a2 chimaerin distribution in Cos-7 Cells 96-98 3.C.2 a2 Chimaerin inhibits PMA induced lamellipodia in Cos-7 Cells 98-100 3.D Summary of Results I 100-101 Chapter 4 Results II 102-126 a2 Chimaerin and P35/Cdk5 103 4A.1 p35 interacts with a2 Chimaerin C-terminus 104 4.A.2 a2 Chimaerin is not an in vitro p25/Cdk5 substrate 105-106 4.A.3 a2 Chimaerin interacts separately with p35 and Cdk5, but not with the active p35/Cdk5 complex 106-109 4A.4 a2 Chimaerin does not regulate p35/Cdk5 in vitro 109-110 4A.5 PMA promotes o2 Chimaerin binding with p35 111-112 8 Table of Contents 4A.6 <x2 Chimaerin C-terminal gap domain is sufficient for Cdk5 association 112-114 4A.7 PMA increases binding of a2 Chimaerin with p35/Cdk5 114-118 4.A.8 p35 or CRMP-2 co-expression does not regulate a2 chimaerin induced morphology 118-121 4A.9 p35 expression in neuroblastoma cells dissociates a 2 chimaerin from the membrane fraction 122-123 4A.10 Cdk5 Kinase activity is not required for neuritogenesis of N1E-115 neuroblastoma cells 123-125 4.B Summary of Results II 125-126 Chapter 5 Results III 127-160 A2 Chimaerin SH2 Domain and Associated Phosphorylation Pathways 128 5.A Phosphotyrosine binding is not required for GAP inactive a2 Chimaerin induction of neurites 129-130 5.B a2 Chimaerin and CRMP-2 130-171 5.B.1 PMA increases CRMP-2 association with a2 chimaerin in vivo 130-132 5.B.2 a2