The regulation of cell-cell adhesion by GTPase activating proteins

Adherens junctions (AJs) are responsible for the adhesion of cells to their neighbours and influence a range of cellular processes. The formation and maintenance of AJs is governed by members of the Rho subfamily of small GTPases. Activation of the GTPases Rac1 and RhoA is necessary for the formation of cell-cell contacts in keratinocytes. However, how the GTPases themselves are regulated is not well understood. Rho GTPase activating proteins (GAPs) inactivate GTPases by promoting the hydrolysis of GTP to GDP. Whilst a large number of proteins containing Rho GAP domains have been identified, knowledge of this large family of proteins remains limited. It is predicted that multiple Rho GAPs are required in order to coordinate precise processes that are necessary for the formation and maintenance of cell-cell contacts to occur. However, relatively few Rho GAPs have been identified as regulators of cell-cell contacts. The aims of this project are to 1) identify GAPs involved in the regulation of cell-cell contact formation in keratinocytes and 2) to specifically investigate the role of CdGAP in junction formation and maintenance. To this end, 20 GAPs have been identified from a siRNA screen as potential regulators of junction formation, the vast majority of which have not previously been linked to this process. Subsequently, two Rho-specific GAPs have been investigated to probe their roles in the regulation of Rho in both junction formation and maintenance. The second part of this thesis focuses on the Rac and Cdc42-specific GAP CdGAP, which I demonstrate is an important regulator of AJs in keratinocytes. I show that CdGAP is able to interact with the scaffold protein Ajuba. Ajuba is known to localise to cell-cell adhesions, where it is necessary for maintaining Rac activation at junctions. However, Ajuba itself has no catalytic activity. My data suggests cooperation between CdGAP and Ajuba allows Rac levels to be fine-tuned at AJs. Overall, this data identifies numerous Rho GAPs as potential regualtors of junction formation, and confirms for the first time the importance of three Rho GAPs, ARAP1, ARHGAP6 and CdGAP, in the regulation of cell-cell contact formation and maintenance. Ultimately, this work provides further insight into the regulation of AJs by GAPs and enhances our understanding of how scaffold proteins, such as Ajuba, are able to influence this tightly controlled process.Open Acces