A chemical–genetic study of EphB signaling identifies kinase‐dependent developmental processes

E-mail address: msoskis@fas.harvard.edu (M. Soskis). EphB receptor tyrosine kinases regulate cell–cell contacts in a variety of processes ranging from axon guidance and topographic mapping to neuronal migration and synapse formation. EphBs bind to a group of ligands, ephrin-Bs, which span the plasma membrane, thus allowing for bidirectional signaling between cells. This complexity combined with the pleiotropic functions of EphBs throughout development has obscured the exact nature of endogenous EphB signaling. To refine our understanding of how EphBs signal, we used a chemical–genetic approach to reversibly inhibit EphB receptors in vitro and in vivo. By mutating a residue in the receptor’s ATP-binding pocket, its kinase activity is rendered sensitive to reversible inhibition by allele-specific PP1 derivatives. This approach blocks the kinase activity of EphBs while leaving their scaffolding and reverse signaling capabilities intact and can circumvent the developmental compensation that may occur after genetic lesions. Using this approach we generated knockin mice with mutations in EphB1, EphB2, and EphB3. We demonstrate that EphB kinase activity is required for several distinct processes in nervous system development. Further studies with these allelespecific kinase mice may be especially valuable in exploring the nature of EphB signaling in the adult brain.