(R2967) Structural and Functional Studies of the Effects of Phosphorylation on Ephrin Receptor Tyrosine Kinase, Epha2, and the Relationship With Its SAM Domain as an Autoinhibitor

Pravesh Shrestha (Case Western Reserve University)| Zhen-Lu Li (Case Western Reserve University)| Amita Sahoo (Case Western Reserve University)| Xiaojun Shi (Department of Medicine, MetroHealth)| Fatima Javier (Case Western Reserve University)| Deanna Bowman (University of Akron)| Jeannine Mueller-Greven (Case Western Reserve University)| Belinda Willard (Proteomics Center, Cleveland Clinic Foundation)| Bing-Cheng Wang (Department of Medicine, MetroHealth)| Adam Smith (University of Akron)| Matthias Buck (Case Western Reserve University)

Eph receptors are the largest subfamily of membrane-bound RTK family. Eph receptors have significant roles during embryonic development, cell maturation, and adulthood. The role of EphA2 in axon guidance and synaptogenesis is well established. While normally a repulsive signal, a non-canonical cell migration promoting activity has been observed by non-canonical unliganded EphA2 signaling mechanism. Here we probed the effects of phosphorylation on intracellular domain (ICD) interactions of EphA2 in solution and bound to membranes. Results from this study indicate that deletion of sterile α motif (SAM) domain leads to an increased binding between kinase domains. Interestingly, upon oligomerization, reduced kinase activity is observed, compared to that of monomeric state of EphA2 ICD. Thus, intriguingly, while deletion of SAM domain increases oligomerization in case of phosphorylated ICD, it appears that such persistent protein-protein interactions are not required for kinase activity in vitro. Mutation study of the linker region between kinase domain and SAM domain give insight into its regulatory role for EphA2 activity. Also docking and all atom simulations only support a weakly bound kinase dimer in solution. The activation likely involves an allosteric mechanism by disrupting SAM domain-kinase domain and/or SAM domain-membrane interactions.