EphB regulates L1 phosphorylation during retinocollicular mapping

Interaction of the cell adhesion molecule L1 with the cytoskeletal adaptor ankyrin is essential for topographic mapping of retinal ganglion cell (RGC) axons to synaptic targets in the superior colliculus (SC). Mice mutated in the L1 ankyrin-binding motif (FIGQY1229H) display abnormal mapping of RGC axons along the mediolateral axis of the SC, resembling mouse mutant phenotypes in EphB receptor tyrosine kinases. To investigate whether L1 functionally interacts with EphBs, we investigated the role of EphB kinases in phosphorylating L1 using a phospho-specific antibody to the tyrosine phosphorylated FIGQY1229 motif. EphB2, but not an EphB2 kinase dead mutant, induced tyrosine phosphorylation of L1 at FIGQY1229 and perturbed ankyrin recruitment to the membrane in L1-transfected HEK293 cells. Src family kinases mediated L1 phosphorylation at FIGQY1229 by EphB2. Other EphB receptors that regulate medial-lateral retinocollicular mapping, EphB1 and EphB3, also mediated phosphorylation of L1 at FIGQY1229. Tyrosine1176 in the cytoplasmic domain of L1, which regulates AP2/clathrin-mediated endocytosis and axonal trafficking, was not phosphorylated by EphB2. Accordingly mutation of Tyr1176 to Ala in L1-Y1176A knock-in mice resulted in normal retinocollicular mapping of ventral RGC axons. Immunostaining of the mouse SC during retinotopic mapping showed that L1 colocalized with phospho-FIGQY in RGC axons in retinorecipient layers. Immunoblotting of SC lysates confirmed that L1 was phosphorylated at FIGQY1229 in wild type but not L1-FIGQY1229H (L1Y1229H) mutant SC, and that L1 phosphorylation was decreased in the EphB2/B3 mutant SC. Inhibition of ankyrin binding in L1Y1229H mutant RGCs resulted in increased neurite outgrowth compared to WT RGCs in retinal explant cultures, suggesting that L1–ankyrin binding serves to constrain RGC axon growth. These findings are consistent with a model in which EphB kinases phosphorylate L1 at FIGQY1229 in retinal axons to modulate L1–ankyrin binding important for mediolateral retinocollicular topography.