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Investigating novel PBF interactions in signal transduction
Secondary Supervisor(s): Dr Andrew Turnell
University of Registration: University of Birmingham
BBSRC Research Themes: Integrated Understanding of Health (Ageing, Pharmaceuticals)
Project Outline
Signal transduction pathways coordinate signals from both extracellular and intracellular stimuli to elicit various cellular responses via a series of molecular events, such as altered gene transcription/translation, post-translational modifications and subcellular localisation. Pituitary tumor-transforming gene-binding factor (PBF) is a ubiquitously expressed transmembrane glycoprotein with an emerging critical role in signal transduction.
PBF overexpression in vitro can induce cellular proliferation, migration, invasion, and genetic instability. Although its primary function remains unclear, our recent data reveal that PBF is a phosphoprotein with a physiological role in cellular adhesion and motility.
Tyrosine phosphorylation is a key step in signal transduction that mediates interactions between signalling molecules. The function and activity of signalling proteins that contain SH2 (Src Homology 2) domains can be modified through their binding to proteins with phosphotyrosine residues. PBF contains a highly phosphorylated tyrosine residue (Y174) and we have recently identified a number of SH2 domain-containing proteins that interact with PBF at this site. These PBF-interacting proteins are well-characterised proteins in critical signalling pathways and this project will address the hypothesis that PBF acts as an important signalling node within these pathways.
This study will investigate the interactions between PBF and these SH2 domain-containing proteins using protein-protein interaction assays such as NanoBiT, co-immunoprecipitation and GFP-TRAP assays and utilising mutant plasmids generated through cloning and mutagenesis.
The effects of manipulating PBF expression levels on related signalling proteins and downstream pathways will be investigated using siRNA knockdown, plasmid transfection, drug treatments to inhibit kinase activity/signalling pathways, Western blotting, immunofluorescence analysis and enzyme assays, and will also utilise human CRISPR-Cas9 knockout cell lines and PBF knockout mouse embryonic fibroblasts (MEFs).
Importantly, the cellular effects of PBF interactions with these signalling molecules and pathways will be investigated. These studies will involve manipulating the expression of PBF and the interacting proteins as well as downstream signalling pathways, followed by measurement of various functional endpoints including cell adhesion assays, motility assays, proliferation assays, cell cycle analysis and DNA damage evaluation.
Overall, this project will elucidate novel interactions within critical signalling pathways and increase our understanding of signal transduction in the regulation of fundamental cellular processes.
Further Reading
1) Pro-invasive Effect of Proto-oncogene PBF Is Modulated by an Interaction with Cortactin. Watkins RJ et al. J Clin Endocrinol Metab. 2016 Dec;101(12):4551-4563. doi: 10.1210/jc.2016-1932.
2) Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors. Smith VE et al. J Clin Endocrinol Metab. 2013 Jul;98(7):2876-86. doi: 10.1210/jc.2012-3640.
3) Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment. Read ML et al. Cancer Research. 2011 Oct 1;71(19):6153-64.