Abstract
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.
Highlights
The EphA4 receptor is a member of the Eph family of receptor tyrosine kinases, which is known to control a variety of cellular functions such as cell adhesion, migration, and invasion by modifying the organization of the actin cytoskeleton [1,2,3]
We show here that the L920F gain-of-function mutation identified in a melanoma metastasis involves a leucine in the EphA4 sterile alpha motif (SAM) domain that is conserved in all Eph receptors [29]
Multiple domains in the Eph receptor extracellular and intracellular regions have been implicated in receptor– receptor association at the plasma membrane [6, 55]
Summary
The EphA4 receptor is a member of the Eph family of receptor tyrosine kinases, which is known to control a variety of cellular functions such as cell adhesion, migration, and invasion by modifying the organization of the actin cytoskeleton [1,2,3]. We observed higher FRET efficiencies for the EphA4 L920F mutant than for EphA4 WT (Fig. 4C), suggesting increased receptor association due to the L920F mutation. Comparison of FRET data for EphA4 ΔSAM and EphA4 L920F confirms the critical role of the L920F mutant SAM domain in both promoting EphA4 assembly in the absence of ligand and altering EphA4 oligomer size (Fig. S3, D and E).
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