ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS OF FAT1 CADHERIN

Elham Sadeqzadeh, MD MSc in Medical Biotechnology

Thesis submitted in the fulfilment of the requirements to obtain the degree of Doctor of Philosophy in Medical Biochemistry

School of Biomedical Sciences and Pharmacy University of Newcastle

August 2013 shown

Statement of Originality

This thesis contains no material which has been accepted for the award of any other degree or diploma in any University or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository** subject to the provisions of the Copyright Act 1968.

** Unless an Embargo has been approved for a determined period.

Elham Sadeqzadeh

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Acknowledgements

The fulfilment of this project would not be possible without sincere help from a number of people, whom I am utterly grateful for all they have done.

Rick (Dr. Rick F Thorne), my supervisor, who entrusted me with some great studies into understanding more about his baby, the FAT1 cadherin project. If it were not for your guidance, advice, encouragements, arguments and dares none of this would have come to exist. I am also grateful for giving me the opportunity to do a PhD project under your supervision, despite a not so good gut feeling at the start.

Gordon (Professor Gordon F Burns), my co-supervisor before retirement and a very supportive figure after that. Your presence and support during the first two years of this project and the courage you gave me to challenge things are exemplary. In the last two years I was always fortunate of having your encouraging words to lift my spirits, when things went wrong.

Charley (Dr. Charles E deBock), if it were not for your help in molecular biology experiments and your sudden and abrupt decision-making capabilities, the second and third chapters of this thesis might have taken an entirely different turn and not end up in me getting those very interesting results.

Xu (Ms. Xu Guang Yan), your help in immunocytochemistry and immunofluorescence microscopy experiments were invaluable and I do appreciate it all. I also thank all my lab- mates in the Cancer Research Unit, who made this journey more appealing, especially when experiments tend to have a mind of their own, which is not such a rare event.

Thanks to my parents who through their witty foresight prepared me for this journey in life. Thanks for teaching me how to be a better person, how to care for other people, how to be independent, how to carry my own burden in life and the philosophy of “what goes around comes around”.

Last but not least, I thank my beloved better half. You sacrificed all you had to join me here and start everything from scratch again. I was the most fortunate person for having you by my side. Your patience and grace in going through all you endured for the first few years here have been extraordinary. You have always been my best supporter and my solid ground when things went terribly wrong. I am really very grateful.

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Acknowledgement of contribution

I hereby certify that the some parts of the work embodied in this thesis has been done in collaboration with other researches, and carried out in other institutions. I have included a statement clearly outlining the extent of collaboration, with whom and under what auspices as follows.

Chapter one

The Chapter was published as a review article in Medicinal Research Reviews (Appendix 2).

 I prepared the first draft of the manuscript and some of the original artwork and all of the tables. Thereafter I undertook further editing and corrections with direction provided by my co-supervisor, Dr. Charles E. de Bock and primary supervisor, Dr. Rick F. Thorne.  Dr. de Bock prepared the artwork for figures 1.1, 1.3 and 1.4 and edited drafts versions of the manuscript.  Dr. Thorne edited the draft manuscripts and approved the final version for submission.

Chapter three

The work was published in the Journal of Biological Chemistry (Appendix 2).

 I performed the majority of the experiments for this Chapter and prepared the primary draft of the manuscript including all figures and tables.  Dr. de Bock carried out the Northern blotting experiment presented as figure 3.1.  My co-supervisor, Professor Gordon F Burns, read the drafts of the manuscript and advised relevant corrections.  Professor Andrew Boyd also provided advice on the drafts of the manuscript.  Professor Xu Dong Zhang provided melanoma cell lines used and provided advice about the experimental direction of the study.  Dr. Camila Salum Oliveira performed the knockdown experiment presented in figure 3.4A.  Dr. Thorne edited the drafts and approved the final version for submission.

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Chapter four

The work has been published in the Journal of Experimental Cell Research

 I performed the majority of the experiments for this Chapter and prepared the primary draft of the manuscript including all figures and tables.  Dr. Natalie Wojtalewicz carried out blotting experiments to detect the shed ectodomain of FAT1 in furin-overexpressing melanoma cells presented in figure 4.5A.  Dr. de Bock assisted me in the production of the stable furin overexpressing cell lines.  Dr. Matthew D. Dun and Nathan D. Smith performed mass spectrographic runs on samples I prepared. Dr. Dun also analysed data used to prepare tables in appendix 4 (tables A4.2 – A4.4).  Dr. Irmgard Schwarte-Waldhoff supervised Dr. Wojtalewicz as a post-graduate student and provided advice about experimental directions for the study.  Dr. Thorne edited the draft manuscripts and approved the final version for submission.

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Table of Contents

STATEMENT OF ORIGINALITY ...... II ACKNOWLEDGEMENTS ...... III ACKNOWLEDGEMENT OF CONTRIBUTION ...... IV TABLE OF CONTENTS ...... VI LIST OF FIGURES ...... IX ABSTRACT ...... XI CHAPTER ONE - GENERAL INTRODUCTION ...... 1

1.1. INTRODUCTION ...... 2 1.2. THE FAT CADHERIN FAMILY ...... 3 1.2.1. Functions of the Drosophila Fat cadherins ...... 5 1.2.2 The vertebrate Fat cadherins...... 16 1.3. FAT CADHERINS IN DISEASE ...... 35 1.3.1. Roles for FAT cadherins in human genetic disorders ...... 35 1.3.2. FAT cadherins in cancer ...... 37 1.4. FAT CADHERINS IN THE GENOMIC ERA ...... 43 1.5. OVERVIEW AND CONCLUDING REMARKS ...... 45 1.6. HYPOTHESIS AND AIMS ...... 46 CHAPTER TWO - GENERAL MATERIALS AND METHODS ...... 48

2.1. CHEMICALS ...... 49 2.2. CELL LINES AND CULTURE ...... 49 2.1.1. Cryopreservation of cells ...... 49 2.1.2. Revival of cryopreserved cells ...... 50 2.3. MOLECULAR BIOLOGY TECHNIQUES ...... 50 2.3.1. Bacterial transformation and isolation of plasmid DNA ...... 50 2.3.2. Manipulation of DNA fragments ...... 51 2.3.3. Ligation of DNA fragments...... 51 2.4. BCA PROTEIN ASSAY ...... 52 2.5. ELECTROPHORETIC SEPARATION AND DETECTION OF PROTEINS ...... 52 2.5.1. Preparation of cell lysates ...... 52 2.5.2. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel electrophoresis) ...... 53 2.5.3. Gel transfer and Western blotting detection using enhanced chemilliumunescence 54 2.6. IMMUNOPRECIPITATION (IP) OF PROTEINS ...... 54 2.7. IMMUNOFLUORESCENCE MICROSCOPY ...... 55 2.8. CELL SURFACE BIOTINYLATION ...... 56 CHAPTER THREE - DUAL PROCESSING OF FAT1 CADHERIN PROTEIN BY HUMAN MELANOMA CELLS GENERATES DISTINCT PROTEIN PRODUCTS ...... 57

3.1. INTRODUCTION ...... 58 3.2. EXPERIMENTAL PROCEDURES ...... 61 3.3. RESULTS...... 66 vi

3.3.1. FAT1 is the major FAT cadherin expressed by human melanoma cells ...... 66 3.3.2. FAT1 is proteolytically processed before achieving cell surface expression as a non- covalent heterodimer in HaCaT cells ...... 66 3.3.3. Additional FAT1 cleavage product, p65, identified in melanoma cells ...... 73 3.3.4. p65 cleavage product is derived from full-length FAT1...... 75 3.3.5. p65 C-terminal fragment of FAT1 is membrane-associated ...... 80 3.3.6. Cellular distribution of FAT1 cadherin may reconcile with products of dual proteolytic processing ...... 82 3.4. DISCUSSION ...... 85 CHAPTER FOUR - FURIN PROCESSING DICTATES ECTODOMAIN SHEDDING OF HUMAN FAT1 CADHERIN ...... 90

4.1. INTRODUCTION ...... 91 4.2. EXPERIMENTAL PROCEDURES ...... 96 4.3. RESULTS...... 102 4.3.1. The S1 cleavage of FAT1 is catalysed by furin ...... 102 4.3.2. Over-expression of furin in LoVo cells restores the appearance of the FAT1 heterodimer on the cell surface ...... 106 4.3.3. Relative deficiency of furin is responsible for partial processing of FAT1 in melanoma cells 108 4.3.4. FAT1 is S1 cleaved between the Laminin G and EGF2 domain ...... 109 4.3.5. S1-cleavage of FAT1 regulates its ectodomain shedding ...... 111 4.4. DISCUSSION ...... 116 CHAPTER FIVE - FAT1 CADHERIN IS MULTIPLY PHOSPHORYLATED ON ITS ECTODOMAIN BUT PHOSPHORYLATION DOES NOT REQUIRE THE ATYPICAL KINASE FJX1 ...... 122

5.1. INTRODUCTION ...... 123 5.2. EXPERIMENTAL PROCEDURES ...... 127 5.3. RESULTS...... 131 5.3.1. Potential ectodomain phosphorylation motifs are conserved in FAT1 cadherin ...... 131 5.3.2. FAT1 cadherin is ectodomain-phosphorylated on both serine and threonine residues 133 5.3.3. FJX1 is not the kinase that catalyses the ectodomain phosphorylation of FAT1 ...... 137 5.4. DISCUSSION ...... 142 CHAPTER SIX - GENERAL CONCLUSIONS AND FUTURE DIRECTIONS ...... 147

6.1. FRUIT FLIES AND CANCER ...... 148 6.2. THE FUNCTIONS OF FT CADHERIN DISCOVERED ...... 149 6.3. THE FUNCTIONS OF THE VERTEBRATE FAT CADHERINS ...... 151 6.4. FAT CADHERINS AND CANCER ...... 153 6.5. ABERRANT FAT1 PROTEIN PROCESSING IN CANCER ...... 154 6.6. BEYOND FAT1 HETERODIMERISATION ...... 158 6.7. THE ELUSIVE LIGAND OF FAT1 ...... 161 6.8. FUTURE DIRECTIONS OF FAT1 RESEARCH ...... 164 APPENDIX 1 - SUPPLEMENTARY DATA FOR CHAPTER ONE ...... 167 APPENDIX 2 - COPYRIGHT PERMISSIONS AND ACKNOELEDGEMENT OF CONTRIBUTION DECLARATION ...... 169 vii

APPENDIX 3 - SUPPLEMENTARY DATA FOR CHAPTER THREE ...... 181 APPENDIX 4 - SUPPLEMENTARY DATA FOR CHAPTER FOUR ...... 187 APPENDIX 5 - SUPPLEMENTARY DATA FOR CHAPTER FIVE ...... 198 REFERENCES ...... 202

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List of Figures Figure 1.1 The Fat cadherin family in Drosophila and vertebrates. 4

Figure 1.2. Mutational phenotypes of the Drosophila ft locus. 8

Figure 1.3. Functional interactions of the Fat1 cadherin cytoplasmic tail. 25

Figure1 4. Life cycle and posttranslational regulation of FAT1 cadherin 44

Figure 3.1. Expression of FAT cadherin family in human melanoma cells, melanocytes, and keratinocytes. 69

Figure 3.2. Proteolytic processing of FAT1 cadherin in keratinocytes. 70

Figure 3.3. FAT1 cadherin is cleaved and expressed as a heterodimer on cell surface of keratinocytes. 72

Figure 3.4. FAT1 cadherin occurs as both unprocessed and heterodimeric forms on cell surface of melanoma cells. 77

Figure 3.5. Proteolytic cleavage and phosphorylation of FAT1 cadherin in melanoma cells. 78

Figure 3.6. Furin is necessary for production of p430/p85 FAT1 heterodimer, whereas p65 is a cleavage product derived from unprocessed p500 molecule. 79

Figure 3.7. p65 cleavage product of FAT1 associated with alternative processing pathway is membrane-associated. 81

Figure 3.8. Schematic illustrating dual processing of mammalian FAT1 cadherin deduced from preceding experimental data. 81

Figure 3.9. Distribution of FAT1 cadherin in vitro 84

Figure 4.1. Furin is the PC responsible for S1-processing and heterodimerisation of FAT1. 105

Figure 4.2. Restoration of furin activity in furin-deficient LoVo cells results in the appearance of the FAT1 heterodimer on the cell surface. 107

Figure 4.3. Restoration of furin activity in furin-deficient LoVo cells results in the appearance of the FAT1 heterodimer on the cell surface. 110

Figure 4.4. Localisation of the S1-cleavage motif in FAT1 and the probable domains involved in heterodimerisation. 114

Figure 4.5. Heterodimerisation of FAT1 is a prerequisite for ectodomain shedding. 115

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Figure 5.1. Schematic comparing the domain organization of Drosophila Ft against human FAT4 and FAT1 cadherins with depictions of phosphorylation sites in their extracellular regions 132

Figure 5.2. FAT1 cadherin is phosphorylated on its ectodomain. 136

Figure 5.3. Assessment of the role of FJX1 in FAT1 cadherin ectodomain phosphorylation using siRNA. 138

Figure 5.4. Stable shRNA-mir-mediated knockdown of FJX1 to assess its role in FAT1 cadherin ectodomain phosphorylation. 141

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ABSTRACT

First identified in Drosophila as a tumour-suppressor gene, Fat cadherin (Ft) and the closely related Fat2 (Ft2) have been identified as giant members of the cadherin superfamily. Ft engages the Hippo signalling pathway during development and both receptors have been shown to function in different aspects of cell polarity and migration. There are four vertebrate homologues, Fat1-Fat4, all closely-related in structure to Drosophila ft and ft2. Over the past decade knock-out mouse studies, genetic manipulation and large sequencing projects have aided our understanding of the function of vertebrate Fat cadherins in tissue development and disease. The majority of studies of this family have focused on Fat1, with evidence now showing it can bind to ENA/VASP, β-catenin and Atrophin proteins to influence cell polarity and motility; Homer1 and 3 proteins to regulate actin accumulation in neuronal synapses; and Scribble to influence the Hippo signalling pathway. Fat2 and Fat3 can regulate cell migration in a tissue specific manner and Fat4 appears to influence both planar cell polarity and Hippo signalling recapitulating the activity of Drosophila Ft. Knowledge about the exact downstream signalling pathways activated by each family member remains in its infancy, but it is becoming clearer that each may have tissue specific and redundant roles in development. Importantly there is also evidence building to suggest that Fat cadherins may be lost or gained in certain cancers.

This thesis represents the first in-depth biochemical investigation of human FAT1 cadherin, particularly its comparative expression in normal versus cancer cells. The first chapter studied the expression profile of all FAT cadherins in a panel of 20 cultured melanoma cells where all melanoma cell lines variably, but universally express FAT1 at the mRNA level and less commonly Fat2, Fat3 and Fat4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA levels relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that human FAT1 was site-1 (S1) cleaved into a non-covalent heterodimer before achieving cell surface expression. A similar processing event had been reported in Drosophila Ft indicating that this was an evolutionary conserved mechanism. The use of inhibitors also established such cleavage is catalysed by a member of the proprotein convertase family, likely furin. However, in melanoma cells the non-cleaved pro-form of FAT1 was also expressed on the cell surface together with the S1-cleaved heterodimer. The appearance of both processed and non-processed forms of FAT1 on the cell surface demarked two possible biosynthetic pathways. Moreover FAT1 processing in melanoma cells generated a potentially functional proteolytic product in melanoma cells: a persistent 65kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. Localisation studies of xi

FAT1 both in vitro and in vivo showed melanoma cells display high levels of cytosolic FAT1 protein whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. The mechanisms deriving the unprocessed FAT1 and the p65 product were then further investigated to uncover the potential biological activities of these cancer specific products.

The second chapter investigated the mechanisms behind dual processing of FAT1 in cancer cells including the mechanism of FAT1 heterodimerisation. Generally the S1 processing step and accompanying receptor heterodimerisation is thought to occur constitutively but the functional significance of this process in transmembrane receptors has been unclear and controversial. Using siRNA against a number of different proprotein convertases it was established that the S1-cleavage of FAT1 is catalysed only by furin. Mass spectrographic analysis identified the precise location of the cleavage site occurring between the laminin G and the second EGF domain on the extracellular domain of FAT1, consistent with an evolutionarily conserved region found in Drosophila DE-cadherin known to be involved in heterodimerisation. Utilising furin overexpressing studies in melanoma together with the furin deficient LoVo cells, indicated the likely reason behind partial heterodimerisation of FAT1 was deficiency in furin activity. Moreover, it was also determined from these experiments that only the heterodimer form of FAT1 was subject to a second cleavage step (S2) and subsequent release of the extracellular domain. This indicated that S1-processing was a prerequisite for FAT1 ectodomain shedding and established a general biological precedent with implications for the shedding of other transmembrane receptors that undergo heterodimerisation. Part of this work also established an ELISA assay against the extracellular domain of FAT1 that may find utility to investigate shed FAT1 as a potential new cancer biomarker in blood.

Previous studies in Drosophila had shown that the interaction between Ft and its ligand, the large cadherin Dachsous (Ds) is regulated through ectodomain phosphorylation mediated by the atypical kinase, Four-jointed (Fj). The third chapter investigated the process of ectodomain phosphorylation of FAT1 on the basis that this important regulatory mechanism may be conserved. Using the known Fj-phosphorylation motif, in silico analyses were undertaken to determine if phosphorylation sites were conserved in human FAT cadherins. This search identified nine potential sites in FAT1 as potential substrates for the sole homologue of Fj in humans, FJX1. Using general antibodies against phospho-serine and phospho-threonine it was revealed that the extracellular domain of FAT1 was multiply phosphorylated on these residues. However, silencing FJX1 using either siRNA or stable shRNA transduction did not indicate any role for FJX1 in FAT1 ectodomain phosphorylation. Nevertheless, given that many regulatory processes are conserved between Drosophila and xii

vertebrate Fat cadherins, the establishment that ectodomain phosphorylation occurs in FAT1 provides the strong likelihood that this process will be important in regulating the interaction of FAT1 with its presently unknown ligand. This knowledge may therefore provide an essential starting point for identifying the ligand of FAT1 and in helping to understand how their interaction is regulated between cells.

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