As protein tyrosine kinases play important roles in the cellular signalling pathways, protein tyrosine kinases can act as either tumour suppressors or proto-oncogenes. C-Src, the first proto-oncogenic protein tyrosine kinase, has been found to be upregulated in colorectal cancer. Only one activating SRC mutation has been reported in cancers (Irby et al, 1999). The C-terminal Src Kinase (CSK) family members, CSK and CSK-Homologous Kinase (CHK), act as upstream negative regulators of Src Family Kinases (SFKs) by phosphorylating a conserved tyrosine at its C-terminal tail. Following phosphorylation of its C-terminal tail tyrosine, a SFK, adopts a closed inactive conformation via intramolecular interactions between the phosphotyrosine at the tail with its SH2 domain as well as the SH2-kinase linker towards the SH3 domain. However, despite being phosphorylated at the C-terminal tail, a SFK may still possess kinase activity when the tyrosine at its activation loop is phosphorylated. Quantitative measurements of the messenger RNA and protein expression levels of CSK, CHK and SRC genes in various colon cancer cell lines have detected expression of CSK and SRC proteins but found CHK to be missing in a few colorectal cancer cell lines (Zhu et al, 2008). In addition, we have previously reported that CHK has a unique ability to inhibit SFK activity via allosteric inhibition, a characteristic absent in other SFK regulator (Chong et al, 2004; Chong et al, 2006). This  highlights the uniqueness of CHK. As the crystal structure complex of CSK:c-Src has been solved (Levinson et al, 2008), we used this as template to identify the key residues in CHK important for binding and inhibiting SFK. A key residue in the α-FG loop of CHK has been pointed out to be a key determinant in inhibiting SFK. Kinase inhibition studies and Surface Plasmon Resonance were employed. In order to understand how mutations affect the structure of CHK, we apply biophysical methods to further shed light on the structure of CHK kinase domain.