Super-resolution analysis of PI 3-Kinase signalling network

Most cellular processes are regulated by interacting network of proteins. The tumor suppressor protein PTEN (Phosphatase and tensin homologue deleted on chromosome 10), antagonises PI 3-Kinase mediated signalling and thus regulates processes including growth and metabolism and it is this effect on PI3K signalling that seems to explain the loss of PTEN function that is observed in approximately 30% of human cancers. PTEN controls not only growth and metabolism through the AKT branch of the PI3K signalling network but it also controls cell motility and polarity independently from AKT signalling. Loss of PTEN function leads to abnormal epithelial architecture and loss of lumen formation in MDCK and NMuMG cell lines. Also, expression of PTEN in many PTEN null cells inhibits invasion in 3D matrigel culture. Previous work in our lab has shown that inhibition of Rac1 GTPase reverts the effects of PTEN loss e.g. failure of lumen formation in NMuMG. Recently, through a siRNA screen we have identified some Rac1 specific activators (Guanine nucleotide Exchange Factors or GEFs) which can revert the phenotype caused by PTEN loss. The main aim of my project is to develop FRET based reporters for these GEFs to develop new assays for their regulation and to analyse their spatio-temporal regulation by PTEN. To apply other tools to investigate the effects of PTEN on cell motility and polarity my project also uses a phospho-specific antibody screen to identify those cytoskeletal proteins whose phosphorylation may be regulated by PTEN. A deeper understanding of the signalling pathways activated in the many tumours that have lost PTEN function should help treatment selection for these patients.