Overactivation of SFKs contributes to many types of cancers such as breast and colon carcinoma and chronic myelogenous leukaemia. Thus, SFKs activities in normal cells are constrained by C-terminal Src Kinase (CSK) and CSK-Homologous Kinase (CHK). Crucially, our laboratory found that in addition to phosphorylating the C-terminal regulatory tyrosine of SFKs, CHK could employ a distinct mechanism to inhibit SFKs activity involving binding of CHK to SFKs, where the binding alone is sufficient to suppress SFKs activity, referred to as noncatalytic inhibitory mechanism.
The objective of my project is to decipher the molecular basis of interaction of the SFKs member Src with CHK. Specifically I aim to define the determinants in Src mediating its binding to CHK and the allosteric site in Src that mediates the non-catalytic inhibition of Src by CHK.
Surface plasmon resonance spectroscopy and assay of the efficiency of CHK to employ the non-catalytic inhibitory mechanism to suppress Src activity were used to investigate the impacts of several point mutations mapped to the αI-helix of Src kinase domain on Src interaction with CHK and CHK inhibition of Src. The study demonstrated that all the point mutations introduced have some impacts on the binding affinity of Src to CHK. However, as the binding of CHK/Src is still very tight, my results suggest that unknown determinants located outside the Src αI helix are responsible for mediating tight binding of Src to CHK. Intriguingly, even though the mutations only lead to a slight reduction in binding of Src to CHK, two of the mutations (E517A and D518A) can significantly prevents CHK from inhibiting Src with the non-catalytic inhibitory mechanism. My results suggest that Glu-517 and Asp-518 are determinants of an allosteric site mediating the non-catalytic inhibitory activity of CHK in suppressing Src kinase activity.