The erythropoietin hepatocellular (Eph) receptor is the largest receptor tyrosine kinase (RTK) sub-family. Unique from other RTKs, the Eph receptors exhibit their functions upon cell-cell contact, as their cognate ephrin ligands are membrane-tethered. Interestingly, when the ephrin ligands are ligated to the Eph receptors, both ephrins and Eph receptors are able to initiate downstream signalling pathways. This bidirectional signalling transduction features the complexity of Eph-mediated cellular events. As such, a clear understanding of the Eph regulatory mechanisms provides insights to diseases such as cancers, in which deregulated Eph receptor expression is frequently observed. EphA10 and EphB6, the two Eph receptors lack their catalytic activity of the kinase domains, thus categorised as “pseudokinases”. Aberrant expression of EphA10 and EphB6 has been linked to oncogenesis and cancer metastasis, respectively. Very little is known about EphA10 and EphB6, owing to their nature of being pseudokinases which are not the popular candidates in cancer therapeutics. It has been proposed that EphA10 and EphB6 can regulate other kinase-active Eph counterparts such as EphB1 and EphB4. This may resemble the mechanism of how ErbB3, a pseudokinase member of epidermal growth factor receptors, modulates the catalytic activity of ErbB1 and ErbB2.
We aim to comprehensively investigate the EphA10/EphB6-mediated signalling pathways in cancer cells using structural biology, proteomics and advanced microscopy. We have expressed and purified the EphA10 and EphB6 recombinant proteins comprising the intracellular tandem domains from insect cells. Our biochemical data suggested that both the EphA10 and EphB6 pseudokinase domains retain the ATP binding ability. The purified recombinant proteins are subjected to crystallisation trials. On the other hand, we are attempting to establish a GFP-tagged EphA10/EphB6 doxycycline-inducible expression system in cancer cell lines. Upon doxycycline induction, we will be able to perform time-course global proteomic and phosphoproteomic studies to investigate the downstream signalling pathways of EphA10/EphB6. The GFP tag of the EphA10/EphB6 will also allow us to apply the cutting-edge microscopy in order to correlate and visualize the proteomics data.