University of Alberta MOAP-1: A Candidate Tumor Suppressor Protein by Jennifer Law A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science Biochemistry ©Jennifer Law Spring 2012 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. ABSTRACT Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays a key role in death receptor-dependent apoptosis and cooperates with the tumor suppressor protein Ras association domain family 1A (RASSF1A) to promote Bax activation during cell death. Although loss of RASSF1A expression is frequently observed in human cancers, it is currently unknown if MOAP-1 expression may also be affected during carcinogenesis to result in uncontrolled malignant growth. Therefore, we sought to investigate the role of MOAP-1 in cancer development. Here, we demonstrate that MOAP-1 can effectively inhibit cell proliferation both in vitro and in vivo and undergoes frequent loss of expression during carcinogenesis. The ability of MOAP-1 to suppress tumor formation requires protein kinase C (PKC)-dependent non-degradative ubiquitination at lysine residue K278 and may involve the novel MOAP-1- interacting protein receptor for activated C-kinase 1 (RACK1). Our data indicate that MOAP-1 is a candidate tumor suppressor protein regulated by PKC- dependent ubiquitination. TABLE OF CONTENTS CHAPTER 1 ........................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1. TUMOR SUPPRESSOR PROTEINS ............................................................. 2 1.1.1. Oncogenes versus tumor suppressors ....................................................... 2 1.1.2. A brief history on tumor suppressor proteins ........................................... 4 1.1.3. Classic tumor suppressor proteins pRb and p53 ....................................... 5 1.1.4. Knudson’s two-hit hypothesis ................................................................... 8 1.1.5. Loss of heterozygosity .............................................................................. 9 1.1.6. Haploinsufficiency .................................................................................... 9 1.2. RAS ASSOCIATION DOMAIN FAMILY OF PROTEINS ........................ 10 1.2.1. RASSF1 .................................................................................................. 11 1.2.2. Biological functions of RASSF1A .......................................................... 14 1.2.3. Tumor suppressor and Ras binding properties of additional RASSF family members ................................................................................................ 17 1.3. APOPTOSIS .................................................................................................. 20 1.3.1. Caspases .................................................................................................. 21 1.3.2. Pathways to apoptosis ............................................................................. 24 1.3.3. Intrinsic cell death ................................................................................... 24 1.3.3.1. Role of the mitochondria.................................................................. 24 1.3.3.2. Bcl-2 family of proteins ................................................................... 25 1.3.4. Extrinsic cell death .................................................................................. 28 1.3.4.1. Death receptors and ligands ............................................................. 28 1.3.4.2. Type I and type II cells .................................................................... 30 1.3.5. Disposal of cells following apoptosis ..................................................... 30 1.4. MODULATOR OF APOPTOSIS-1 (MOAP-1) ............................................ 31 1.4.1. Interaction of MOAP-1 with Bcl-2 family members .............................. 32 1.4.2. Cooperation of MOAP-1 with RASSF1A in death receptor-mediated apoptosis ........................................................................................................... 33 1.4.3. Regulation of MOAP-1 stability by apoptotic signals ............................ 36 1.4.4. Paraneoplastic antigens ........................................................................... 37 1.4.4.1. PNMA protein family ...................................................................... 38 1.5. RECEPTOR FOR ACTIVATED C-KINASE 1 (RACK1) ........................... 39 1.5.1. RACK proteins as PKC adaptors ............................................................ 39 1.5.2. RACK1 scaffold protein ......................................................................... 40 1.5.3. Regulation of protein translation by RACK1 ......................................... 41 1.5.4. Role of RACK1 in apoptosis .................................................................. 43 1.5.5. Involvement of PKC and RACK1 in cancer ........................................... 44 1.6. UBIQUITINATION ...................................................................................... 46 1.6.1. Ubiquitin ................................................................................................. 46 1.6.2. Ubiquitination process and enzymes ...................................................... 47 1.6.3. Ubiquitination signals ............................................................................. 48 1.6.4. Ubiquitin chain assembly ........................................................................ 50 1.6.5. The ubiquitin-proteasome system ........................................................... 52 1.6.6. Ubiquitin-mediated non-proteolytic signaling ........................................ 54 1.7. RESEARCH OBJECTIVES .......................................................................... 56 CHAPTER 2 ......................................................................................................... 57 MATERIALS AND METHODS .......................................................................... 57 2.1. Cell lines ........................................................................................................ 58 2.2. DNA transfection ........................................................................................... 60 2.2.1. DNA plasmids ......................................................................................... 60 2.2.2. DNA transfection protocol ...................................................................... 60 2.3. Protein methodology ...................................................................................... 61 2.3.1. Antibodies ............................................................................................... 61 2.3.2. Cell lysis and immunoprecipitation ........................................................ 63 2.3.3. SDS-polyacrylamide gel electrophoresis ................................................ 63 2.3.4. Gel transfer techniques............................................................................ 64 2.3.4.1. Semi-dry transfer.............................................................................. 64 2.3.4.2. Wet transfer ...................................................................................... 64 2.3.5. Western blotting ...................................................................................... 65 2.4. Fixed-cell immunofluorescence confocal microscopy .................................. 65 2.5. Calf intestinal phosphatase treatment ............................................................ 66 2.6. MTT cell proliferation assay .......................................................................... 67 2.7. Apoptosis detection assay by fluorescence-activated cell sorting ................. 67 2.8. Animal Experiments ...................................................................................... 68 2.8.1. Mouse splenocyte isolation ..................................................................... 68 2.8.2. Xenograft tumor assays ........................................................................... 69 2.9. Microarray Analyses ...................................................................................... 69 2.9.1. Oncomine meta-analysis of human cancer microarrays ......................... 69 2.9.2. MOAP-1 and RASSF1A expression analysis in neuroblastoma patients 70 2.9.3. Analysis of MOAP-1 expression in breast cancer patients ..................... 70 2.9.4. Gene expression profiling of human tumor xenografts .......................... 72 2.9.4.1. Canonical pathway and biological function analysis of xenograft gene expression changes ..............................................................................