The L920F EphA4 Oncogenic Mutation Alters the SAM Domain Fold and Induces EphA4 Oligomerization

Abstract

The EphA4 receptor tyrosine kinase has been implicated in several cancers including melanoma, breast cancer, pancreatic cancer, and gastric cancer. Here, we have taken a multifaceted approach to uncover the molecular mechanism behind the pathogenesis due to the L920F EphA4 mutation, linked to cutaneous melanoma. This mutation resides in the core of the EphA4 sterile alpha motif (SAM) domain, a highly conserved domain located C-terminally to the intracellular kinase domain. The SAM domain is known to mediate lateral interactions among Eph receptors, so it can be expected that an oncogenic mutation in the SAM domain affects interactions. However, the L920 residue is buried in the SAM domain core, and thus the mechanism by which the L920F mutation contributes to EphA4 pathogenesis is not clear. Using a quantitative FRET assay, we found that wild-type EphA4 associates into dimers while L920F EphA4 associates into oligomers larger than dimers. Since it is known that Eph receptor oligomers are much more active than Eph receptor dimers, our findings suggest that L920F EphA4 oligomerization is the basis for the pathogenesis. MD simulations and ClusPro/PyRosetta docking simulations of EphA4 SAM domains demonstrated that L920F EphA4, unlike the wild type, engages two alternative interfaces which drives the oligomerization. Using mutagenesis, we experimentally verified the biological significance of the oligomerization interfaces in the L920F EphA4 mutant; these interfaces could be potential targets in cancer therapies. In summary, our studies show that the pathology due to the L920F EphA4 mutation is likely caused by EphA4 oligomerization and subsequent overactivation, driven by the destabilization of the SAM domain fold.