A cancer mutation promotes EphA4 oligomerization and signaling by altering the conformation of the SAM domain

The Eph receptor tyrosine kinases and their ephrin ligands regulate many physiological and pathological processes. EphA4 plays important roles in nervous system development and adult homeostasis, while aberrant EphA4 signaling has been implicated in neurodegeneration. EphA4 may also affect cancer malignancy, but the regulation and effects of EphA4 signaling in cancer are poorly understood. A correlation between decreased patient survival and high EphA4 mRNA expression in melanoma tumors that also highly express ephrinA ligands suggests that enhanced EphA4 signaling may contribute to melanoma progression. A search for EphA4 gain-of-function mutations in melanoma uncovered a mutation of the highly conserved leucine 920 in the EphA4 sterile alpha motif (SAM) domain. We found that mutation of L920 to phenylalanine (L920F) potentiates EphA4 autophosphorylation and signaling, making it the first documented EphA4 cancer mutation that increases kinase activity. Quantitative Föster resonance energy transfer and fluorescence intensity fluctuation (FIF) analyses revealed that the L920F mutation induces a switch in EphA4 oligomer size, from a dimer to a trimer. We propose this switch in oligomer size as a novel mechanism underlying EphA4-linked tumorigenesis. Molecular dynamics simulations suggest that the L920F mutation alters EphA4 SAM domain conformation, leading to the formation of EphA4 trimers that assemble through two aberrant SAM domain interfaces. Accordingly, EphA4 wild-type and the L920F mutant are affected differently by the SAM domain and are differentially regulated by ephrin ligand stimulation. The increased EphA4 activation induced by the L920F mutation, through the novel mechanism we uncovered, supports a functional role for EphA4 in promoting pathogenesis.