Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

在正常发育过程中，许多组织的祖细胞都经历了多能性、上皮细胞向间质细胞转变、增殖、迁移和分化的进行性限制。大多数，如果不是全部，这些事件涉及细胞-细胞和细胞-基质粘附的修饰，这些粘附系统的异常修饰通常与肿瘤的形成有关。Eph家族的受体酪氨酸激酶及其配体ephrin在多种癌症中经常过度表达，包括乳腺癌、小细胞肺癌和胃肠道癌、黑色素瘤和神经母细胞瘤。利用非洲爪蟾胚胎系统，我们已经证明了由ephrinB细胞内结构域介导的信号传导影响细胞间的粘附，并且这种活性可以通过与激活的FGF受体相互作用来调节。跨膜ephrinB1蛋白是一种双向信号分子，通过其细胞质域发出信号，促进细胞运动进入视野，而成纤维细胞生长因子受体（FGFR）的激活抑制这些运动和视网膜命运。在非洲爪蟾胚胎中，ephrinB1通过与支架蛋白disheveled （Dsh）的相互作用，在视网膜祖细胞进入视野的过程中发挥作用。然而，FGFR调节这种细胞运动的机制尚不清楚。在这里，我们提出的证据表明，fgfr诱导的视网膜命运的抑制依赖于细胞内ephrinB1区域的磷酸化。我们证明了ephrinB1中酪氨酸324和325的磷酸化破坏了ephrinB1/Dsh的相互作用，从而调节了依赖于平面细胞极性（PCP）途径的视网膜祖细胞运动。这些结果提供了FGF信号如何调节ephrinB1控制视网膜祖细胞在视野内运动的机制见解。此外，我们发现证据表明ephrinB1信号可能通过细胞极性复合物在体内调节细胞-细胞连接。本研究的重点是利用Xenopus系统评估ephrinB1在上皮细胞中是细胞-细胞连接信号的介质还是调节剂。我们提出的证据表明，Par极性复合物蛋白Par-6是建立紧密连接所需的主要支架蛋白，它与ephrinB1结合并受ephrinB1调节，从而控制紧密连接。利用早期非洲爪蟾胚胎上皮细胞，我们发现ephrinB1功能的丧失或获得可以破坏细胞间的接触和紧密连接。这项研究揭示了ephrinB1与活性Cdc42竞争与Par-6结合的机制，Par-6是Par极性复合物（Par-3/Par-6/Cdc42/aPKC）的中心支架蛋白，并破坏紧密连接相关蛋白（ZO-1, Cingulin）的定位。这种竞争影响紧密连接的形成，并受ephrinB1酪氨酸磷酸化的调节。

During normal development progenitor cells of many tissues undergo progressive restriction of pluripotency, epithelial-to-mesenchymal transition, proliferation, migration, and differentiation. Most, if not all, of these events involve modifications of cell-cell and cell-matrix adhesion, and abnormal modifications of these adhesion systems are often associated with the formation of tumors. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are frequently over-expressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Using the Xenopus embryonic system, we have demonstrated that signaling mediated by the intracellular domain of ephrinB affects cell-cell adhesion, and that this activity can be modulated by interaction with an activated FGF receptor. The transmembrane ephrinB1 protein is a bi-directional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 within ephrinB1 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity (PCP) pathway. These results provide mechanistic insight into how FGF signaling modulates ephrinB1 control of retinal progenitor movement within the eye field. Moreover, we found evidence that ephrinB1 signaling may regulate cell-cell junctions through a cell polarity complex in vivo. This study focused on assessing whether ephrinB1 is a mediator or modulator of cell-cell junction signaling in epithelial cells using the Xenopus system. We presented evidence that the Par polarity complex protein, Par-6, which is a major scaffold protein required for establishing tight junctions, associates with ephrinB1 and is regulated by ephrinB1, resulting in the control of tight junctions. Using the epithelial cells of early stage Xenopus embryos, we showed that loss- or gain-of function of ephrinB1 can disrupt cell-cell contacts and tight junctions. This study reveals a mechanism where ephrinB1 competes with active Cdc42 for binding to Par-6, a scaffold protein central to the Par polarity complex (Par-3/Par-6/Cdc42/aPKC) and disrupts the localization of tight junction-associated proteins (ZO-1, Cingulin). This competition affects formation of tight junctions, and is regulated by tyrosine phosphorylation of ephrinB1.