Cholesterol inhibits assembly and oncogenic activation of the EphA2 receptor

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, we have a poor understanding regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments reveal that a reduction of plasma membrane cholesterol strongly promotes EphA2 self-assembly. Indeed, low cholesterol levels cause a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines reveal that low cholesterol increased phospho-serine levels in EphA2, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but it also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.