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Subcellular localization and kinetics of the activation of Armadillo/beta-catenin in developing epithelia

Reference	BB/D004802/1
Principal Investigator / Supervisor	Professor Alfonso Martinez Arias
Co-Investigators / Co-Supervisors	Professor Kathryn Lilley
Institution	University of Cambridge
Department	Genetics
Funding type	Research
Value (£)	314,764
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	30/11/2008
Duration	36 months

Abstract

Armadillo/beta-catenin (Arm/ beta-cat) is a modular protein with a double activity as a mediator of cell adhesion and cytoskeletal activity and also as a nuclear effector of Wnt signalling. These functions are mediated by different pools: the interactions between cell adhesion and the cytoskeleton are mediated by a stable form associated with cadherin at the cell surface while Wnt signalling relies on an unstable soluble pool. In the absence of Wnt signalling the scaffolding protein Axin assembles a complex in which GSK3beta phosphorylates Arm/ beta -cat and primes it for proteasome mediated degradation. In the presence of Wnt, Axin is destroyed and dephosphorylated Arm/beta-cat accumulates and translocates into the nucleus. Mutations in the GSK3 beta phosphorylation sites of Arm/ beta-cat generate hyperactive signalling forms of this protein. However, surprisingly, these forms can still be subject to regulation by Axin and other proteins e.g Notch (unpub. obs.). While there is abundant information about the molecular activity of the Axin based destruction complex, there is very little known about its subcellular localisation, how this is regulated by Wnt signalling and the significance of this localisation for the signalling process. Here we propose to carry out a detailed quantitative and cell biological analysis of Drosophila Armadillo in relation to Wnt signalling. These experiments have two goals. First to make a description of the biochemical parameters of the activation of Arm/ beta-cat in an epithelium which should identify rate liming steps and the sensitivity and robustness of the process. Second to place the process of activation of Arm/ beta-cat in a subcellular context and determine whether it takes place in the cytosol or, as suggested by some preliminary evidence, in some membrane bound structures. And third, to use a combination of cell biology and proteomics to identify novel proteins involved in the regulation of Arm/ beta-cat stability and activity. Our studies will also focus on Axin because of its close functional association to Arm/ beta-cat. In mammals, mutations that lead to the activation of beta-catenin are associated with several pathological situations, in particular multiple forms of cancer. Therefore understanding the process of activation of beta-catenin, identifying rate limiting steps and proteins involved in this process are important first steps towards the identification of targets of clinical significance. The importance of the activation of beta-catenin for biomedical research is further underscored by its involvement in the establishment and maintenance of various kinds of stem cells. Our interest in using Drosophila stems from its excellent track record as a reference for the analysis of Wnt signalling in vertebrates; what is true of Drosophila might be enhanced and complicated in vertebrates, but is true of vertebrates.

Summary

Embryonic development involves the generation of thousands of different kinds of cells and their spatial organization into specific tissues and organs. These events rely on the expression of cell type specific molecules that confer identity to individual cells and help their assembly into an organism. During this process, the acquisition of molecular identities and their tranformation into shapes and sizes has to be coordinated. This requires interactions between cells mediated by molecular signals and sensors and the processing of these signals by an intracellular protein network. Wnt proteins are an important kind of cellular signals which have specific cellular receptors and, with regard to the generation of cell fates, a dedicated intracellular signalling network whose output is the activity of a protein called Armadillo in fruit flies and beta-catenin in vertebrates.When a cell receives a Wnt signal it activates Armadillo/beta-catenin and this protein conveys information to the nucleus that results in the expression of particular genes. Armadillo/beta-catenin has other functions in addition to its role in Wnt signalling and it is likely that the intracellular location of different pools of this protein determines its activity. Remarkably the functioning and regulation of Armadillo and beta-catenin is the same which allows us to use the enormous experimental potential of Drosophila in the confidence that what we are looking at is of general significance. The Wnt signal and its effector Armadillo/beta-catenin participate in a very large number of the processes that transform one cell into an organism and also have recently been shown to regulate important properties of stem cells. Furthermore, specific mutations that elevate the signalling ability of beta-catenin are associated with and diagnostic of several types of Cancer. These observations make it important to understand the details of the mechanism that leads to the activation of beta-catenin. While much has been learnt from genetic approaches we know very little about the process itself. In particular the concentrations of the different elements of the signalling network, their location inside the cell and how it relates to other processes which might modulate it. The aim of this research is to fill these gaps and in providing a quantitative and cellular account of the activity of Armadillo/beta-catenin, to characterize the biochemical ccordinates of the process and to identify new regulatory elements.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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