Schuck Ryan J, Ward Alyssa E, Sahoo Amita R, Rybak Jennifer A, Pyron Robert J, Trybala Thomas N, Simmons Timothy B, Baccile Joshua A, Sgouralis Ioannis, Buck Matthias, Lamichhane Rajan, Barrera Francisco N
Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, USA.
Department of Physiology and Biophysics, Case Western Reserve University, School of Medicine, Cleveland, TN, USA.
Commun Biol. 2025 Mar 11;8(1):411. doi: 10.1038/s42003-025-07786-6.
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.
受体酪氨酸激酶EphA2通过促进转移来驱动癌症恶性发展。EphA2可以处于不同的自组装状态：单体、二聚体和寡聚体。然而，我们对EphA2的哪种状态负责驱动促转移信号传导了解甚少。为了解决这一局限性，我们开发了SiMPull-POP，这是一种用于准确量化膜蛋白自组装的单分子方法。我们的实验表明，质膜胆固醇的减少强烈促进EphA2的自组装。事实上，低胆固醇水平对EphA2配体ephrinA1-Fc产生类似的影响。这些结果表明胆固醇抑制EphA2组装。在不同细胞系中的磷酸化研究表明，低胆固醇会增加EphA2中磷酸丝氨酸的水平，这是致癌信号的特征。对胆固醇用于抑制EphA2组装和活性的机制的研究表明存在一种反式效应，即EphA2在β-肾上腺素能受体活性下游被蛋白激酶A磷酸化，而胆固醇也会抑制这种活性。我们的研究不仅为EphA2致癌功能提供了新的机制见解，还表明胆固醇作为一种分子保护机制，可防止EphA2不受控制的自组装和激活。
