Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

在正常发育期间，许多组织的祖细胞经历多能性、上皮-间充质转化、增殖、迁移和分化的进行性限制。这些事件中的大多数（如果不是全部）涉及细胞-细胞和细胞-基质粘附的修饰，并且这些粘附系统的异常修饰通常与肿瘤的形成相关。受体酪氨酸激酶的Eph家族及其配体肝配蛋白在多种癌症中经常过表达，包括乳腺癌、小细胞肺癌和胃肠癌、黑色素瘤和神经母细胞瘤。使用非洲爪蟾胚胎系统，我们已经证明，ephrinB的细胞内结构域介导的信号传导影响细胞-细胞粘附，并且这种活性可以通过与活化的FGF受体的相互作用来调节。跨膜ephrinB 1蛋白是一种双向信号分子，通过其胞质结构域发出信号以促进细胞运动进入眼区，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，ephrinB 1通过与支架蛋白Dishevelled（Dsh）的相互作用在视网膜祖细胞移动到眼区中发挥作用。我们确定FGF诱导的视网膜命运的抑制依赖于ephrinB 1细胞内结构域的磷酸化，并破坏ephrinB 1/Dsh相互作用。这调节依赖于Wnt/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和Wnt信号如何调节ephrinB 1控制眼内视网膜祖细胞运动的机制。此外，我们发现证据表明ephrinB 1信号可能通过体内细胞极性复合物调节细胞-细胞连接。这项研究的重点是评估ephrinB 1是否是一个中介或调制器的上皮细胞中的细胞连接信号使用爪蟾系统。我们确定Par极性复合蛋白Par-6是建立紧密连接所需的主要支架蛋白，与ephrinB 1相关，并受ephrinB 1调节，从而控制紧密连接。使用早期爪蟾胚胎的上皮细胞，我们发现ephrinB 1功能的丧失或获得可以破坏细胞间的接触和紧密连接。这项研究揭示了一种机制，其中ephrinB 1与活性Cdc 42竞争结合Par-6，Par-6是Par极性复合物（Par-3/Par-6/Cdc 42/aPKC）的核心支架蛋白，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ephrinB 1酪氨酸磷酸化的调节。最后，沿着我们在莫里森实验室的同事，我们已经证明ephrinB 1以EphB受体非依赖性方式与CNK 1相互作用.在培养的细胞中，ephrinB 1与CNK 1共转染增加JNK磷酸化。EphrinB 1/CNK 1介导的JNK激活通过显性负性RhoA的过表达而减少，表明JNK激活是Rho依赖性的。CNK 1单独过表达足以激活RhoA，然而，ephrinB 1和CNK 1都需要JNK磷酸化。Co-IP分析显示ephrinB 1和CNK 1作为支架蛋白连接RhoA和JNK信号组分。此外，粘附到纤连蛋白和活性Src过表达增加ephrinB 1/CNK 1结合，而药理学抑制剂抑制Src活性不仅降低ephrinB 1/CNK 1结合，而且降低JNK活化。这些数据表明Src活性增强ephrinB 1/CNK 1相互作用和下游JNK激活。EphrinB 1过表达增加了HeLa宫颈癌细胞系的细胞运动性，然而，通过siRNA消耗CNK 1消除了这种细胞迁移和伴随的JNK激活。此外，RhoA和JNK抑制剂抑制ephrinB 1介导的细胞迁移。总之，我们的研究结果表明，CNK 1介导ephrinB 1诱导的JNK激活和细胞迁移培养的癌细胞系。Eph/ephrin系统的失调与各种人类癌症中侵袭性和转移性肿瘤的促进相关。在这里，我们表明，Rho家族GTdR调节剂，Rho特异性鸟嘌呤核苷酸解离抑制剂1（RhoGDI 1），可以与ephrinB 1相互作用，这种相互作用增强后，结合同源EphB 2受体胞外结构域。缺失突变显示ephrinB 1胞内结构域的氨基酸327 - 334对于与RhoGDI 1的相互作用至关重要。用EphB 2细胞外结构域-Fc融合蛋白（EphB 2-Fc）刺激，诱导RhoA活化，并增强癌细胞在野生型肝配蛋白B1表达细胞中的迁移和侵袭。相比之下，这些Eph-Fc诱导的作用在表达突变型埃甫蛋白B1构建体（delta 327 -334）的细胞中显著减弱，所述突变型埃甫蛋白B1构建体在与RhoGDI 1相互作用时无效。此外，EphrinB 1通过siRNA的消耗抑制EphB 2-Fc诱导的RhoA活化，并减少SW 480和Hs 578 T人癌细胞系中的癌细胞迁移和侵袭。我们的研究表明，RhoGDI 1和ephrinB 1的结合促进了与肿瘤进展相关的癌细胞行为，并可能成为表达ephrinB 1的癌症的治疗靶点。

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). We determined that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1, and disrupts the ephrinB1/Dsh interaction. This modulates retinal progenitor movement that is dependent on the Wnt/planar cell polarity (PCP) pathway. These results provide mechanistic insight into how FGF and Wnt 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 determined 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. Finally, along with our colleagues in the Morrison laboratory, we have shown that ephrinB1 interacts with CNK1 in an EphB receptor- independent manner. In cultured cells, co-transfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1- mediated JNK activation is reduced by overexpression of dominant-negative RhoA, indicating that JNK activation is Rho-dependent. Overexpression of CNK1 alone is sufficient for activation of RhoA, however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-IP analysis shows that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA with JNK signaling components. Furthermore, adhesion to fibronectin and active Src overexpression increases ephrinB1/CNK1 binding, whereas inhibition of Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. These data suggest that Src activity enhances ephrinB1/CNK1 interactions and downstream JNK activation. EphrinB1 overexpression increases cell motility of the HeLa cervical cancer cell line, however, CNK1 depletion by siRNA abrogates this cell migration and the accompanying JNK activation. Moreover, RhoA and JNK inhibitors suppress ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 mediates ephrinB1-induced JNK activation and cell migration in cultured cancer cell lines. Deregulation of the Eph/ephrin system is associated with the promotion of aggressive and metastatic tumors in various human cancers. Here, we show that a Rho family GTPase regulator, Rho-specific guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the cognate EphB2 receptor extracellular domain. Deletion mutagenesis revealed that amino acids 327334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc), induces RhoA activation, and enhances cancer cell migration and invasion in wildtype-ephrinB1 expressing cells. In contrast, these Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (delta327-334) that is ineffective at interacting with RhoGDI1. Furthermore, EphrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces cancer cell migration and invasion in SW480 and Hs578T human cancer cell lines. Our study reveals that the binding of RhoGDI1 and ephrinB1 promotes cancer cell behavior associated with tumor progression and may be a therapeutic target in cancers that express ephrinB1.