Cholesterol inhibits assembly and 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, our understanding remains limited 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 revealed that a reduction of plasma membrane Cholesterol strongly promoted EphA2 self-assembly. Indeed, low Cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that Cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low Cholesterol increased phospho-serine levels, 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 also suggests that Cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.