Function of the cell adhesion molecule JAM-A in tumour cells

Junctional adhesion molecule A (JAM-A) is a member of the immunoglobulin super family involved in regulation of cell migration and cell adhesion. It was recently shown that JAM-A, tetraspanin CD9 and vitronectin receptor integrin αVβ3 form a trimeric complex in endothelial cells. Complex-bound JAM-A regulates migration and basic fibroblast growth factor (bFGF)-mediated angiogenesis of endothelial cells via mitogen-activated kinase (MAPK) signalling (Peddibhotla et al., 2013). In this thesis, it was analysed if this complex is expressed in the breast ductal carcinoma cell line MCF-7 and if the complex is involved in tumour cell migration.It is shown in this thesis that JAM-A forms a tetrameric complex in MCF-7 cells comprising tetraspanins CD9, CD81 and vitronectin receptor integrin αVβ5 (JAM-A – Tspan – ITGB5 complex) in which the two tetraspanins link JAM-A to integrin αVβ5 independently from each other. CD81 and integrin αVβ5 are described as novel interaction partners of JAM-A. Depletion of the CD9/CD81 which disconnects JAM-A from integrin αVβ5 increases vascular endothelial growth factor (VEGF)-induced ERK1/2-MAPK (extracellular signal-regulated kinase1/2-MAPK) activation. This effect is mediated by JAM-A, suggesting a negative regulatory role of JAM-A in VEGF-mediated MAPK signalling. Investigations to identify signalling proteins upstream of ERK showed that tyrosine kinase c-Src is associated with the tetrameric complex in MCF-7 cells. Depletion of JAM-A increased c-Src activity, indicating that JAM-A regulates the activity of c-Src associated with the complex. Analyses of the subcellular localisation revealed that the tetrameric complex localises to lamellipodia of MCF-7 cells, showing for the first time a role of JAM-A outside of cell-cell contacts in lamellipodia. Importantly, it can be shown that loss of JAM-A increases Abelson interactor-1 (Abi1) activity, a subunit of the WAVE-regulatory complex (WRC), linked to lamellipodia formation, suggesting that complex-associated JAM-A regulates lamellipodia. Disruption of the complex resulted in increased collective and single cell migration, supporting a regulatory function of the tetrameric complex during cell migration. This indicates that complex-mediated signalling regulates WRC activity to balance lamellipodia formation and cell migration.In order to analyse the negative regulatory function of JAM-A, c-Src was identified to mediate JAM-A tyrosine 280 (Y280) phosphorylation in vitro. In MCF-7 cells, JAM-A is shown to be Y280-phosphorylated at lamellipodia after VEGF treatment. c-Src negative regulator CSK2(C-terminal Src kinase) specifically interacts with Y280-phosphorylated JAM-A and is found to being associated with the tetrameric complex in MCF-7 cells, suggesting that JAM-A negatively regulates c-Src activity by recruitment of CSK to the tetrameric complex.Together the results show that JAM-A, CD9, CD81 and integrin αVβ5 form a tetrameric complex in MCF-7 cells to balance c-Src activity and thus, MAPK signalling and WRC activity in lamellipodia. Regulation might occur via recruitment of CSK to the tetrameric complex through JAM-A which regulates integrin αVβ5-associated c-Src activity. The data show a new function of JAM-A limiting WRC activity in lamellipodia and thus, migration of MCF-7 cells which could be highly relevant for processes such as tumour cell migration and invasion.