The Role of c-Src in E-Cadherin Activity

Robert Mclachlan (2009). The Role of c-Src in E-Cadherin Activity PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Robert Mclachlan
Thesis Title The Role of c-Src in E-Cadherin Activity
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2009-10
Thesis type PhD Thesis
Supervisor Professor Alpha Yap
Total pages 138
Total colour pages 23
Total black and white pages 115
Subjects 06 Biological Sciences
Abstract/Summary Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signalling, being enriched in tyrosine-phosphorylated proteins, tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signalling, however, is complex and incompletely understood. In my thesis I have tested the hypothesis that c-Src contributes positively to cadherin biology by functioning as part of an adhesion activated cell-signalling pathway. I found that c-Src is active at both established and reforming cell-cell contacts, and c-Src can be activated by homophilic ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signalling pathway serves positively to support cadherin function, while quantitative changes in signal strength may result in qualitative differences in functional outcome. Finally, my data implicated PI3-kinase signalling and cortactin as potential targets for cadherin-activated c-Src signalling. By inhibiting protein tyrosine phosphatases with pervanadate, I found that tyrosine phosphatase activity and not just protein binding was required to stimulate Src activity in response to cadherin ligation. I identified the tyrosine phosphatase RPTPα as a possible regulator of cadherin-activated Src signalling. RPTPα localises to cell-cell adhesions and it is found in a complex with E-cadherin and c-Src. Furthermore, knockdown of RPTPα disrupted the integrity of cadherin-based contacts and the activity of Src at these cell-cell contacts. This suggests that in response to cadherin-homophilic ligation PTP activity is required to stimulate Src signalling. Finally, I identified a novel pathway by which aberrant growth factor signalling could be downregulating cadherin function and promoting the invasion of epithelial cells. Stimulating cells with high levels of EGF revealed that aberrant epidermal growth factor signalling could disrupt cadherin-activated cell signalling. The integrity of cadherin-based contacts and the activity of Src at the cell-cell contacts were both disrupted in the presence of high levels of EGF. Analysis of E-cadherin and RPTPα immunoprecipitates suggested that activation of cadherin-bound EGFR might disrupt Src activation by displacing E-cadherin-RPTPα binding. Finally, analysing the subcellular distribution of these proteins revealed that, in response to high levels of EGF, E-cadherin, β-catenin, EGFR and pEGFR are internalised together in phospho-cortactin-rich endosomal-like structures. Therefore I propose that E-cadherin adhesion activates a cell-signalling pathway involving c-Src that functions to dynamically regulate the actin cytoskeleton and to maintain the adhesive strength of cell-cell adhesions. Perturbation of cadherin-activated Src signalling downregulates cadherin function and promotes the disassembly of cell-cell adhesive contacts. The concept of a cadherin-activated Src signalling pathway provides a new way to think about cadherin biology. Instead of merely functioning as passive glue holding two cells together, E-cadherin functions as an adhesion-activated signalling receptor. Dysregulation of E-cadherin-activated Src signalling and downregulation of cell-cell adhesions could be a mechanism promoting the invasion and metastasis of epithelial tumours.
Keyword E-cadherin
Cell-Cell Adhesion
Protein Tyrosine Phosphatase
Receptor Crosstalk
Additional Notes Pages to be printed in colour are 21, 23, 24, 26, 58, 62-63, 70-72, 74, 77, 88-91, 100-105, 116.

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Created: Tue, 02 Feb 2010, 15:33:17 EST by Mr Robert Mclachlan on behalf of Library - Information Access Service