Sam Domain Inhibits Oligomerization And Auto-Activation Of Epha2 Kinase

Eph receptors are the largest family of receptor tyrosine kinases (RTKs) and bind to membrane-tethered ligands called ephrins. Eph-Ephrin interactions are involved in various biological processes, such as neural development, tissue patterning and vascular growth. Several structural studies have shown that the extracellular domain (ECD) of EphA2 binds ephrin and forms an oligomer that promotes kinase activation. However, the role of the intracellular domain (ICD) in this process remains largely unexplored. Here, we report on the unique role of the sterile α motif (SAM) domain in regulating EphA2 kinase activation. First, western blots and cell function assays indicated that deletion of the SAM domain leads to constitutive activation of the kinase. We then carried out fluorescence correlation spectroscopy (FCS) measurements to investigate the dynamics and lateral organization of EphA2 receptors in live cancer cell membranes. The FCS results showed that deletion of the SAM domain led to receptor oligomerization. This suggested that constitutive activation of the SAM-deletion construct observed in the cell functional assays were a result of receptor oligomerization. Ligand activation with ephrinA1 (EA1) induces oligomerization of the receptors, but deletion of the SAM domain led to larger oligomers than those with the full length receptor. FCS measurements were performed on cancer cells treated with two kinds of soluble EA1 ligand, dimeric EA1-Fc and monomeric EA1 (mEA1), were used. While inconsistencies exist in the literature, our FCS results demonstrated that both dimeric and monomeric EA1 activation led to oligomerization and activation of EphA2 through induction of the distinct clusters. Together, our work clarifies the unique function of SAM domain in EphA2 signaling.