- Title
- Analysis of post-translational modifications of Fat1 cadherin
- Creator
- Sadeqzadeh, Elham
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2014
- Description
- Research Doctorate - Doctor of Philoshphy (PhD)
- Description
- 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 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 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.
- Subject
- Fat cadherin; Drosophilia; cancer; cell biology
- Identifier
- http://hdl.handle.net/1959.13/1050575
- Identifier
- uon:15178
- Rights
- Copyright 2014 Elham Sadeqzadeh
- Language
- eng
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