Mitochondrial membrane binding and protein complexing of the anti-apoptotic adaptor protein GrblO. by Jennifer Hassard Department of Biology McGill University, Montreal A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master's of Science August 2001 Supervisor: Dr. David Thomas, Department of Biochemistry © Jennifer Hassard, 2001 .....--. National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington OttawaON K1A0N4 Ottawa ON K1 A ON4 canada canada Your liIe VoIJ8 rrlfénJnce Our liIe Notre rtifénlncs The author has granted a non­ L'auteur a accordé une licence non exclusive licence al10wing the exclusive permettant à la National Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies ofthis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership ofthe L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the autbor's ou autrement reproduits sans son penmsslon. autorisation. 0-612-78889-X Canada ABSTRACT GrblO is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of GrblO to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as weIl as many non-receptor molecules such as MEK1, Raf-l, and Nedd4. GrblO has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-l and the mitochondrial membrane. In this report the pattern of transient Grb10 translocation following IGF-1cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new GrblO binding partners. RESUME GrblO appartient à la famille des protéines adaptatrices Grb7 qui comprend également Grb7 et Grb14. Ces trois membres possèdent plusieurs domaines d'interaction protéine­ protéine mais n'ont pas d'activité enzymatique connue. Des études double-hybride ont montré que GrblO interagit avec des récepteurs activés par des tyrosine kinases dont le récepteur à l'insuline (IR) et le récepteur à l'IGF-I (Insulin-like Growth Factor 1) ainsi qu'avec d'autres protéines telles que MEKl, Raf-l et Nedd4. GrblO est impliquée dans la voie de signalisation anti-apoptotique régulée par IGF-I grâce à son interaction avec Raf-l et la membrane mitochondriale. Dans ce rapport nous avons étudié la translocation de GrblO après stimulation des cellules par IGF-I. Nous avons également montré l'implication d'une courte région N-terminale variable dans l'association de GrblO avec la membrane mitochondriale. Enfin, nous avons entrepris d'identifier de nouveaux partenaires de GrblO à l'aide de différentes techniques biochimiques. 11 INDEX Abstract & Résumé pag( i Index iii Acknowledgements vi 1. Introduction 1 1.1 Cellular programming for death 1 1.1.1 Morphology linked to signaling molecules 2 1.1.2 Extrinsic and intrinsic signaling pathways 3 1.1.3 Mitochondria, control centre for life or death 5 1.1.4 IGF-IR anti-apoptotic signaling 7 1.2 Orb10 and survival signaling 9 1.2.1 The Grb7family ofproteins 9 1.2.2 Grb10 binding partners 13 1.2.3 Grb10 in mitogenesis 16 1.2.4 Grb10 in anti-apoptotic signaling 18 1.2.5 Grb10 in human disease 19 1.3 Tapies of investigation in this report 21 1.3.1 Investigation ofGrb10 mitochondrial binding 21 1.3.2 The searchfor new Grb10 binding partners 22 2. Methods 24 2.1 Cloning 24 111 • 2.2 Protein preparation 25 2.3 Cell culture and transfection 26 2.4 Mitochondria isolation for immunoprecipitation & gel filtration 27 2.5 High purity mitochondria isolation for in vitro binding assays 28 2.6 In vitro binding assays 29 2.7 In vivo binding assays 30 2.8 Flag co-immunoprecipitation 30 2.9 Two-dimensional electrophoresis 31 2.9.1 1soelectric focusing 31 2.9.2 Resolution by size 33 2.9.3 Silver staining 33 2.10 Gel filtration 34 2.10.1 Mitochondrial extract separation 34 2.10.2 Column standardization 35 2.11 Western blotting 35 2.12 Fluorescence microscopy 36 3. Results 38 3.1 GrblO mitochondrial binding 38 3.1.1 Immunolocalization demonstrates a rapid Grb10 38 translocation 3.1.2 Cell counts ofimmunolocalized HeLa, a majority exhibits 39 Grb10 translocation IV 3.1.3 In vitro assays demonstrate that mitochondrial membrane 39 binding ofGrb10 occurs via the amino-terminal and is ATP-independent 3.14 In vivo assays support the results ofin vitro assays 41 3.2 Development of assays for GrblO binding partner identification 42 3.2.1 Gelfiltration confirms ideal conditionsfor Grb10 42 complexing 3.2.2 The developed binding partner assay is not efficient enough 43 to identify binding partners 4. Discussion 45 4.1 Challenges faced in this study 45 4.2 Mitochondrial membrane binding 46 4.3 Identification of Grbl0 binding partners 50 3.4 Proposed further work 51 5. References 53 Figures included at end 1 - 12 v AC K N 0 W LED G MEN TS l graciously thank Dr. David Thomas for supervision, guidance, and funding during the progress of this research. Thank you to Dr. André Nantel for invaluable encouragement, supervision, and expertise (and an endless supply of movie trailers) throughout this research. Dr. Nantel provided the pAN series of vectors, sorne of which were used for cellular transfections and from which many new vectors were sublconed. Dr. Anne-Pascale Bouin is thanked for her many hours of instruction and help with co-immunoprecipitation and 2-dimensional electrophoresis (2-D) techniques. l also thank Fizah Buch for her dedication to the completion of experiments using gel filtration and her enthusiasm and energy directed towards tbis project. Fizah performed preliminary standard runs and aIl cell runs using the gel filtration system with my supervision as part of her independent research project for the McGill Department of Anatomy and Cell Biology. Anne Marcil is graciously thanked for her continued support and for my introduction to mammalian cell culture. Thank you to Daniel Dignard for sequence data and help with interpretation, along with providing good lunchtime humour. André Nantel, Anne-Pascale Bouin, Micbiei Sho, Cunle Wu, and Marc Pelletier are aIl appreciated for lively discussions concerning research direction and for help reviewing tbis manuscript. Finally, l also thank Dr. Claude Jakob for a memorable introduction to the Eukaryotic Genetics group at the Biotechnology Research Institute. VI 1. INTRODUCTION 1.1 Cellular programming for death The programmed path to cellular suicide, called apoptosis, is crucial for the development, daily maintenance, and survival of the mammalian organism. Apoptosis plays an essential role in the shaping of organs, limbs, and digits during fetal development. Using apoptotic signaling, the human body continually removes old cells to be replaced with new ones and restricts the proliferation of unwanted autoimmune lymphocytes. Apoptosis can also be used by the body to remove cells infected by a pathogen. The study of pathways that lead to apoptosis has become increasingly important in the light of the many human conditions that can arise when such signaling goes off track. Cancer, of significant importance in the field of apoptosis research, can be the result of insufficient apoptotic control. It is now demonstrated that in many circumstances the loss of proper apoptotic signaling is responsible for resistance to chemotherapeutic drugs (O'Gorman & Cotter, 2001). On the other hand, excessive apoptosis is linked with Alzheimer's Disease (AD), Huntington's Disease (HD), and Amyotrophic Lateral Sc1erosis (ALS) which manifest as the selective and detrimentalloss of certain neuronal cell types within specific regions of the brain and peripheral nervous system (Nijhawan, et al., 2000). As our understanding of apoptotic signaling increases, so does our understanding of these related conditions, and perhaps so will our ability to better treat them. 1 1.1.1 Morphology linked to signaling molecules Apoptosis is associated with a number of morphological characteristics. As the cell begins to shrink and lose its shape, its outer membrane eventually undergoes blebbing resulting in popcom-like shaped cells. Shrinking and budding of the nucleus is seen in conjunction with chromatin condensation and eventual DNA fragmentation. Finally, the cell fragments into apoptotic bodies that are engulfed by neighbouring cells. As opposed to necrosis, apoptosis is a tidy and specifically controlled means for a cell to die. The cellular signaling involved in apoptosis is complex and relies on a balance between pro- and anti-apoptotic signaIs. Two groups of proteins are of great importance to apoptosis, the caspases and the Bcl-2 family members. Caspases, fittingly dubbed the "central executioners" (Hengartner, 20(0), are cysteine proteases (having a cysteine residue within the enzyme active site) which cleave after aspartic acid residues (Thomberry, et aL, 1997). Cellular protein cleavage by caspases results in many of the morphological changes mentioned above. For instance, fragmentation of nuclear DNA is known to be a result of the caspase-activated DNase (CAD) (Nagata, 20(0). Blebbing of the cellular membrane has been attributed to caspase cleavage activation of the p21-activated kinase (PAK2) (Rudel & Bokoch, 1997).