EphB regulates L1 phosphorylation during retinocollicular mapping.
EphB regulates L1 phosphorylation during retinocollicular mapping.
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DOI：
10.1016/j.mcn.2012.05.001
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发表时间：
2012-06
期刊：
影响因子：
--
通讯作者：
Maness PF
中科院分区：
文献类型：
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作者：
Dai J;Dalal JS;Thakar S;Henkemeyer M;Lemmon VP;Harunaga JS;Schlatter MC;Buhusi M;Maness PF
细胞粘附分子 L1 与细胞骨架接头锚蛋白的相互作用对于视网膜神经节细胞 (RGC) 轴突到上丘 (SC) 突触目标的地形图绘制至关重要。 L1 锚蛋白结合基序 (FIGQY1229H) 突变的小鼠显示 RGC 轴突沿 SC 中外侧轴的异常映射，类似于 EphB 受体酪氨酸激酶的小鼠突变表型。为了研究 L1 是否与 EphB 功能性相互作用，我们使用酪氨酸磷酸化FigQY1229基序的磷酸特异性抗体研究了 EphB 激酶在磷酸化 L1 中的作用。 EphB2（但不是 EphB2 激酶死亡突变体）在FigQY1229 处诱导 L1 酪氨酸磷酸化，并扰乱 L1 转染的 HEK293 细胞中锚蛋白向膜的募集。 Src 家族激酶通过 EphB2 介导FIGQY1229 处的 L1 磷酸化。调节内侧-外侧视网膜小球映射的其他 EphB 受体 EphB1 和 EphB3 也介导 FigQY1229 处 L1 的磷酸化。 L1 细胞质结构域中的酪氨酸 1176 调节 AP2/网格蛋白介导的内吞作用和轴突运输，但不被 EphB2 磷酸化。因此，L1-Y1176A 敲入小鼠中 Tyr1176 突变为 Ala 导致腹侧 RGC 轴突的视网膜小球映射正常。视网膜专题图谱中小鼠 SC 的免疫染色表明，L1 与视网膜受体层 RGC 轴突中的磷酸-FIGQY 共定位。 SC裂解物的免疫印迹证实野生型中L1在FIGQY1229处被磷酸化，但L1-FIGQY1229H (L1Y1229H)突变体SC中没有磷酸化，并且EphB2/B3突变体SC中L1磷酸化降低。与视网膜外植体培养物中的WT RGC相比，L1Y1229H突变型RGC中锚蛋白结合的抑制导致神经突生长增加，这表明L1-锚蛋白结合有助于限制RGC轴突生长。这些发现与EphB激酶磷酸化视网膜轴突中FIGQY1229位点的L1以调节对中外侧视网膜小球地形重要的L1-锚蛋白结合的模型一致。
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.