Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

29-POS Board 8 CHOLESTEROL BLOCKS ONCOGENIC ACTIVITY AND SELF-ASSEMBLY OF THE EPHA2 RECEPTOR Ryan Schuck 1 ; Alyssa E Ward 1 ; Amita R Sahoo 5 ; Jennifer A Rybak 3 ; Robert J Pyron 3 ; Thomas N Trybala 2 ; Timothy B Simmons 1 ; Joshua A Baccile 2 ; Ioannis Sgouralis 4 ; Matthias Buck 5 ; Rajan Lamichhane 1 ; Francisco N Barrera 1 ; 1 University of Tennessee , BCMB, Knoxville , TN, USA 2 University of Tennessee , Chemistry , Knoxville , TN, USA 3 University of Tennessee , GST, Knoxville , TN, USA 4 University of Tennessee , Mathematics, Knoxville , TN, USA 5 Case Western Reserve University, Physiology and Biophysics, Cleveland, OH, USA The EphA2 receptor exerts a profound influence on cancer malignancy. EphA2 has two modes of activation that have opposite effects on cancer outcomes. EphA2 acts as a tumor suppressor when it is phosphorylated at cytoplasmic tyrosines after binding of Ephrin ligands, which triggers receptor monomers to self-assemble. On the other hand, EphA2 can be activated in the absence of ligand when kinases phosphorylate key serine residues. Under these conditions, EphA2 triggers oncogenic signaling that drives an invasive phenotype that leads to metastasis. We hypothesized that specific cellular mechanisms must exist to keep EphA2 in check by maintaining its optimal self-assembly levels and preventing its oncogenic activation by kinases. However, improved methods to quantify protein oligomers are needed to rigorously evaluate this idea. Here, we developed a single-molecule method, SiMPull-POP, which achieves high sensitivity to quantify oligomeric populations of membrane proteins. Our approach captures the native environment of the eukaryotic cell membrane in DIBMA-derived nanodiscs (DIBMALPs). SiMPull-POP delivers single-molecule sensitivity to quantify protein interactions via photobleaching analysis. We used SiMPull-POP to discover that cholesterol (Chol) is a potent inhibitor of EphA2 assembly. Additionally, we observed that Chol regulates EphA2 activity by preventing oncogenic phosphorylation of key residue Ser897. We performed cell biology experiments that suggest that Chol controls EphA2 serine phosphorylation by regulating protein kinase A activity via changes in the levels of the second messenger cAMP. Based on the data presented, we propose that the presence of cholesterol in the plasma membrane acts as a safety mechanism that prevents uncontrolled self-assembly and oncogenic activation of EphA2.


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