The polarity regulator Scribble is involved in establishing and maintaining apical-basal polarity in epithelial cells, and was first identified as a neoplastic tumour suppressor in Drosophila. Data from cell lines and primary tissue samples suggest altered expression and/or mislocalisation of Scribble is important in tumorigenesis. Mislocalisation of SCRIB in human prostate cancer correlates with poor survival, indicating that correct localisation of Scribble is important for its function. Scribble is a membrane-associated LAP protein, and contains both leucine rich repeat (LRR) and PDZ domains. While there are many known Scribble binding partners, including other polarity regulators and many cytoskeletal, junctional and signaling proteins, the precise size and composition of the Scribble complex remains poorly understood.
We have generated a number of tagged Scribble mutant constructs, containing different domains of the protein. Using biochemical techniques and cellular assays we aim to determine the composition of the Scribble protein complex, and identify regions of the protein most important for its localisation and function. We have shown through co-immunoprecipitations and GST pulldowns that Scribble is able to interact with itself to form a dimer. Size exclusion chromatography indicates Scribble is part of a large complex of >700kDa, which is likely to contain a Scribble dimer and other Scribble binding proteins such as β-pix and β-catenin. Scribble’s LRR domain is required for dimerisation, and is also essential for correct membrane localisation.
Future studies include using FRET to determine the composition of the Scribble complex in a cellular context. We are also investigating spontaneously occurring Scribble mutations from human patients to determine whether these deleteriously affect Scribble complex formation, localisation or function. Determining the functional composition of the Scribble complex and how this is altered in different cellular contexts will enable us to further unravel the mechanism by which Scribble contributes to tumorigenesis.