Double Inhibition And Activation Mechanisms Of Ephexin Family RhoGEFs

Eph receptors are the largest family of tyrosine kinase receptor proteins,playing a bidirectional role in signal transduction by interacting with their ligand Ephrin expressed at the surface of neighboring cells.Ephrin-Eph-mediated signaling pathway regulates cytoskeleton dynamics by modulating downstream Rho GTPase activity,and plays important roles in diverse cellular processes including cell migration,cell adhesion,cell polarity,cell morphology and so on.Ephexin family proteins,the Dbl guanosine exchange factors(GEFs),act as downstream RhoGEFs of the Ephrin-Eph signaling pathway,which activate Rho GTPase and regulate cytoskeleton remodeling.They play key roles in cell proliferation and migration,cell differentiation,neural development,signal transduction,and metabolic homeostasis.Numerous studies have shown that mutations in the gene encoding the Ephexin family are closely related to cancers and brain disorders.Ephexin family consists of five members,which can switch between autoinhibition and activation states to spatiotemporally control the activity of Rho GTPase.Ephexin family proteins all contain a catalytical DH-PH domain followed by a SH3 domain,except Ephexin5.It has been reported that the removal of the SH3 domain greatly enhanced the GEF activity of Ephexin4,suggesting that the SH3 domain could inhibit Ephexin4 GEF activity.Moreover,SH3 mediated autoinhibition is also present in Ephexin1 and Ephexin3.More interestingly,a conserved ?-helix inhibition peptide(IH)preceding the DH domain of Ephexin 1-3 also inhibit their respective RhoGEF activity.These findings suggest that the regulation of Ephexin family activity may involve multiple autoinhibition and activation modes,although the underlying molecular mechanisms remain unclear.In this thesis,we study in detail the autoinhibited conformation of Ephexin4.We solve two crystal structures:1)SH3 domain-mediated autoinhibited conformation,mainly containing DH domain,PH domain,SH3 domain and an unexpected ?-helix at the carboxy-terminal(HC),abbreviation for DPSH;2)the double autoinhibited Ephexin4,mainly including the DPSH and N-terminal IH-mediated autoinhibition mode,abbreviation for IDPSH.Via structural analysis and in vitro GEF assay,we find that mutation of the key residues at the inhibition interface(e.g.,R706D,Y220D)can effectively activate the activity of Ephexin4.Importantly,a mutation of Ephexin4-R706L,identified in patients with seminoma,can significantly enhance the activity of Ephexin4,providing a possible structural basis for the pathogenesis of the associated disease.The structural and amino acid sequence analysis also suggest that the double autoinhibited conformation of Ephexin4 might also existed in other Ephexin family members.Ephexinl also possesses a similar double autoinhibited conformation according to our in vitro GEF assay.In addition,we predict and confirm that another RhoGEF-SGEF(ARHGEF26)with a similar domain organization to Ephexin4 is also in a double autoinhibition state.How is the double autoinhibitory state of Ephexin4 activated?We find that the PDZ domain of Dlgl(PDZ1 or PDZ2)can bind to the PDZ binding motif(PBM)of Ephexin4,and this interaction can activate the activity of Ephexin4 for RhoG Structural analysis shows that PBM is very close to SH3-HC.We speculate that the PDZ-PBM interaction may create steric hindrance clash,which would relieve the inhibited interaction between DH and SH3-HC,thus allowing RhoG to bind to the DH domain and complete the catalytic reaction.On the other hand,we find that substitution of the conserved tyrosine Y220 in the IH to aspartic acid(i.e.,Y220D)effectively activates the GEF activity of Ephexin4 towards RhoG,suggesting that phosphorylation at this site might be the possible mechanism for activation of IH-mediated autoinhibition.Further,we confirm the double autoinhibition and activation mechanisms of Ephexin4 at the cellular level through transwell-based cell migration assay.Interestingly,Ephexin4 adopts a double inhibitory conformation in which N-and C-terminal inhibition may operate independently,provide a multilayered regulatory system for spatiotemporal control of enzymatic activities of Ephexins in response to distinct external stimuli.In summary,we elucidate the double autoinhibition and activation of the Ephexin family RhoGEFs,via combination of biochemistry,biochemistry,structural biology,and cell biology approaches.Moreover,since the two inhibitory elements operate independently,multiple upstream signals might also independently relieve each inhibitory mode to fine-tune the GEF activity of Ephexins during cell signaling.We also offer possible clues for the pathological dysfunction of the Ephexin family RhoGEFs.