EphA regulation of Rho GTPases through the exchange factor ephexin during axon guidance

During the development of the nervous system, neurons project their axons over long distances to find and connect with their eventual synaptic targets. In the progress of an axon toward its targets, the tip of the axon, the growth cone, is guided by a variety of extracellular signals. Receptors on the surface of the growth cone receive these signals and translate them into changes in the organization of actin and myosin components of the cytoskeleton, and hence into changes in growth cone shape, speed, and direction. The Rho family of small GTPases has been implicated in the regulation of various aspects of actin and myosin organization. In neurons, the Rho family members Cdc42 and Rac1 induce axon extension while RhoA induces axon retraction. However, if and how Rho GTPases are regulated during axon guidance events was previously unknown. This thesis explores the signalling pathways through which one class of axon guidance signals, the ephrin-As, exert their effects on the actin cytoskeleton through ephexin, a novel activator of Rho GTPases.; Ephrin-As are cell-surface bound molecules which bind to and activate the EphA family of receptor tyrosine kinases and exert an inhibitory or repulsive effect on growth cones. A screen for proteins that interact with the intracellular region of one EphA family member, EphA4, was performed to identify molecules that might function in EphA signalling pathways. This screen revealed ephexin, a member of the Dbl family of guanine nucleotide exchange factors (GEFs) that activate Rho family GTPases by catalyzing their conversion from the inactive GDP-bound form to the active GTP-bound form. Ephexin expression is high in regions of the nervous system undergoing neuronal differentiation and process outgrowth and is similar to that of EphA receptors, consistent with a role in EphA-regulated axon guidance. Overexpression of ephexin can lead to the activation of RhoA, Cdc42, and Rac1 in fibroblasts and neurons, and can directly activate RhoA and Cdc42 in vitro. In fibroblasts and neurons, EphA activation shifts ephexin activity toward RhoA. A mutant form of ephexin blocks ephrin-A-induced growth cone retraction in neurons, implying that ephexin is a required component of EphA signalling pathways.; Next, the molecular mechanisms by which EphA receptors regulate the activity of ephexin were investigated. Cloning and sequencing of the other mammalian ephexin-related GEFs revealed a conserved site for phosphorylation by the cytoplasmic tyrosine kinase Src in the N-terminal region of ephexin. Src directly phosphorylates ephexin at this site in vitro. A novel assay for GEF activity that takes advantage of the ability of GEFs to bind stably to nucleotide-free forms of their substrate GTPases was developed and used to show that overexpression of Src, as well as inhibition of a tyrosine phosphatase, activates ephexin's activity toward RhoA. Furthermore, as expected, Src activation of ephexin requires the Src phosphorylation site on ephexin. These results demonstrate that ephexin is regulated by tyrosine phosphorylation. In addition, in a transfectable cell culture model for EphA signalling, EphA4 activation leads to rapid induction of ephexin's interaction with RhoA. These findings raise the possibility that ephrin-A activation of EphA receptors lead to the phosphorylation and activation of ephexin via Src.