Molecular analysis of insulin action using high throughput genetic screens Poh Sim Khoo Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Garvan Institute of Medical Research University of New South Wales 31st March 2011 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Khoo First name: Poh Sim Other name/s: Abbreviation for degree as given in the University calendar: PhD School: St Vincent's Cllinical School Faculty:Medicine Title: Molecular analysis of insulin action using high throughput genetic screens Abstract 350 words maximum: (PLEASE TYPE) The insulin-stimulated uptake of glucose by muscle and adipose is vital for the maintenance of glucose homeostasis in the body. This uptake primarily occurs through the action of the insulin-regulatable glucose transporter GLUT4, which is rapidly translocated to the plasma membrane in response to the insulin signal. However, in the insulin resistant state, insulin is unable to effect a normal biological response in its target tissues, characterized by decreased glucose uptake as a result of defects in insulin signaling and attenuated GLUT4 translocation. Elucidating the molecular causes underlying insulin resistance is important for the development of therapeutics for this disease. Identifying the components involved in the propagation or regulation of insulin signaling is an important step in understanding insulin resistance. To date, the upstream components of the signaling pathway are well established, demonstrating the importance of the insulin receptor, IRS proteins and PI3K/Akt signaling axis in this process. As a result much work has focused on defects at the point of IRS in the development of insulin resistance. However, it has recently been suggested that defects associated with insulin resistance occur independently of IRS. Therefore, identifying the sites that this dysfunction occurs at is of great interest in understanding this disease. The purpose of this study is to discover novel proteins involved in the regulation of the insulin signaling and GLUT4 translocation. A GLUT4-overexpressing HeLa cell line was developed and optimized for use in high throughput screening for regulators of insulin stimulated GLUT4 translocation. Insulin stimulation caused GLUT4 translocation to the plasma membrane, and the activation of the PI3K/Akt signaling pathway in these cells. Using this cell line, I performed an siRNA screen of kinase and DUB libraries which identified a number of novel targets that may play a role in the regulation of insulin stimulated GLUT4 translocation. In conclusion, this assay can be used to identify novel regulators of insulin stimulated GLUT4 translocation, which may potentially represent targets for drug development in the treatment of insulin resistance Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). .... .... Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ................................................................. Date ................................................................. Acknowledgements I would like to thank my supervisor at the Garvan, David James. I feel privileged to have been able to work on a challenging project, and it would not have been possible without his vision and guidance. I would also like to thank my co-supervisor Jackie Stoeckli for her great practical advice and for teaching me techniques in the lab that I have used during my PhD. At Genentech, I would like to thank Richard Scheller for the opportunity to work in his lab. Thanks to my supervisor Jagath Junutula, I am extremely grateful for his help, advice and enthusiasm. I would also like to thank my colleagues in the lab; Karen Ervin, Sunil Bhakta, Siler Panowski and Coen Kuijl for the friendship and laughter, and for making it a great environment to be in. Thanks to James Lee for his work with the optimization and execution of the siRNA transfections, Jean-Philippe Stephan for all of his help with running the screens and the image analysis and Eric Torres for his help with the data analysis. Their help with the siRNA screens was invaluable. Finally thanks to my friends and family for believing in me and supporting me through this time, it is very much appreciated. Table of Contents Summary 1 Chapter 1 – General Introduction 3 Insulin and the maintenance of glucose homeostasis 4 The GLUT4 glucose transporter 4 Insulin-stimulated GLUT4 translocation 6 PI3K-dependent insulin signaling 6 PI3K 7 Akt 7 AS160 8 aPKC (λ, ζ) 9 PI3K-independent signaling 10 Contraction stimulated GLUT4 translocation 12 Increased intracellular calcium 12 AMPK 13 Convergence of insulin and contraction signaling pathways leading to GLUT4 translocation 14 Insulin resistance 15 Inflammation and insulin resistance 16 JNK 16 IKKβ 16 ERK 17 P62 signaling adaptor 17 SOCs proteins 18 GSK3 18 PKCθ 19 Tyrosine and serine phosphatases 19 PIKfyve 20 Hypothesis and aims 21 Chapter 2 – Materials and Methods 22 Reagents, Antibodies and Constructs 23 Cell Culture and Transfections 23 Generation of HeLa cells stably expressing HA-GLUT4 24 siRNA Transfection (screening) 24 Quantitative GLUT4 translocation assay 24 IN Cell image acquisition 25 Image analysis 25 Transferrin uptake assay 25 Immunofluorescence microscopy 26 Taqman analysis for gene knockdown 26 Preparation of cell lysates 27 Western blotting 27 Chapter 3 – Development of a GLUT4 translocation assay in HeLa cells 29 Introduction 30 Results 34 HeLa cells stably expressing HA-GLUT4 exhibit insulin stimulated GLUT4 translocation 34 HA-GLUT4-HeLa cells express the majority of genes expressed in human adipose and muscle tissue 34 Localization of GLUT4 in the HA-GLUT4-HeLa cell line 37 Insulin, IGF1 and EGF treatment of HA-GLUT4-HeLa cells induces GLUT4 translocation 39 Comparison of insulin signaling in HA-GLUT4-HeLa cells and 3T3-L1 adipocytes 40 PI3K and Akt inhibitors block insulin-stimulated GLUT4 translocation in both 3T3-L1 adipocytes and HA-GLUT4-HeLa cells 41 siRNA-mediated knockdown of Akt and PI3K inhibits GLUT4 translocation in HA-GLUT4-HeLa cells 41 Development of GLUT4 translocation assay for high throughput screening using the HA-GLUT4-HeLa cell line 43 Discussion 47 Chapter 4 – Identifying kinase regulators of insulin signaling and GLUT4 translocation by siRNA screening 51 Introduction 52 The insulin receptor – kinase activity and regulation by phosphorylation 52 Phosphorylation of IRS proteins and modulation of the insulin signal 53 Kinase regulation of insulin signaling and GLUT4 translocation independent of IRS phosphorylation 54 Results 55 Primary kinase screen 55 Secondary screen 60 Tertiary screen 66 Discussion 75 Kinases known to function in insulin signaling and GLUT4 translocation 75 Kinases that interact with regulators of insulin signaling 77 Kinases related to ERK and JNK signaling 79 Kinases involved in regulation of trafficking 79 Metabolic proteins 80 Miscellaneous 81 Conclusion 81 Chapter 5 – Identifying DUB or ULP regulators of insulin signaling and GLUT4 translocation by siRNA screening 83 Introduction 84 Ubiquitin and Ubiquitin-like proteins 84 Ubiquitin system functions 85 Ubiquitin-like proteins and insulin signaling 87 Results 90 Primary screen 90 Secondary screen 96 Tertiary screen 100 Discussion 106 Proteins regulating protein stability and degradation 106 Proteins involved in mRNA processing and protein translation 107 Proteins involved in vesicle trafficking 109 Miscellaneous functions 109 Conclusion 110 Chapter 6 – General Discussion 112 Development and optimization of the HA-GLUT4-HeLa cell line 113 Involvement of the ERK/MAPK pathway in insulin signaling