Background: In cancer, the over-activation of proto-oncogenic products causes normal cells to undergo malignant transformation and thus acquiring hallmarks of cancer such as over-proliferation and angiogenesis. Src is one such proto-oncogenic product which, in normal cells, is regulated by its tumour suppressor CSK-Homologous Kinase (CHK), thereby preventing Src from becoming over-activated. The over-activation of Src is known to be the underlying cause of multiple types of cancer, such as colon cancer. CHK has been shown to inhibit Src in two ways; 1) by phosphorylating the regulatory tyrosine residue in the C-terminal tail of Src, and 2) by tightly binding to and thus allosterically inhibiting Src. A number of basic residues in CHK have been found to be critical determinants of the Src-CHK interactions that mediate this tight binding. Therefore, the aim of our study is to identify the residues in Src that are important in Src-CHK interactions.
Methods: Site-directed mutagenesis will be done on the wild-type Src gene to generate several mutant constructs. The mutations are mapped to the C-terminal tail of Src which can potentially interact with the aforementioned basic residues in CHK involved in Src-CHK interactions. These constructs will be expressed and purified, following which several techniques will be used to validate their structure and function. Then, a number of assays will be performed on these mutants, including Surface Plasmon Resonance (as a binding assay) as well as assays to monitor the ability of CHK to allosterically inhibit Src and its mutants, in order to determine the impact of mutating these residues. Additionally, cell-based assays will be performed, involving the transfection of HEK293T cells with mutants cloned into pcDNA3 vector, to investigate the significance of these mutations in a cellular context. Finally, studies on CHK-deficient colon cancer cell lines, in which recombinant CHK and its mutants will be introduced via lentiviral transduction, will be done to determine the clinical significance of these mutations.