Signaling Mechanisms of EphrinB1 in Cell Adhesion, Migration and Invasion

我们目前的研究兴趣是旨在研究Eph受体酪氨酸激酶及其ephrin配体信号事件影响细胞-细胞粘附和形态发生运动的机制。从这些信号转导途径的阐明，我们可以提高我们对肿瘤发生的认识。细胞-细胞粘附系统在细胞的正常发育和形态发生中起着重要作用。这种粘附系统的失活被认为在癌症侵袭和转移中起关键作用。非洲爪蟾胚胎非常适合研究这些过程，因为青蛙具有很好的特征和不变的细胞命运图，并且在实验中可以很容易地追踪细胞谱系。突变受体、配体和其他蛋白质可以在胚胎中异位表达。因此，它们对信号转导、运动和分化的影响可以在发育中的脊椎动物中进行形态学、组织学和生化评估。我们的实验室目前正在利用非洲爪蟾卵母细胞和胚胎系统以及人类培养的细胞系研究非洲爪蟾Eph受体酪氨酸激酶和ephrinB跨膜配体在细胞信号传导和功能中的作用。目前，我们的重点放在这些Eph家族成员发送影响形态发生运动的信号的机制上。Eph家族成员与许多发育过程的调控有关，并且在转移性癌症中被发现不受调控，例如前列腺癌、卵巢癌、乳腺癌、结肠癌、神经母细胞瘤、肺癌和黑色素瘤。我们的实验室继续进行这些研究，检查控制细胞粘附和细胞运动的ephrinB1的近端和远端信号。我们发现有证据表明ephrinB1通过其胞内结构域与Dishevelled （dsh）相互作用，并共同选择Wnt/平面细胞极性（PCP）途径，控制视网膜祖细胞进入视野的运动。通过生化分析和功能增益或损失实验，我们的数据表明，在眼视野形成过程中，dsh与ephrinB1相关，并通过PCP通路下游成员介导ephrinB1信号传导。因此，我们使用视野作为模型系统来理解ephrinB1如何控制细胞运动。最近，我们研究了ephrinB1影响细胞-细胞连接的机制。越来越多的证据表明，在细胞的适当迁移以及细胞和组织边界的形成中需要ephrin配体。这些过程都依赖于细胞粘附系统，在细胞发育过程中正常的形态发生过程以及侵袭和转移过程中起着至关重要的作用。尽管ephrinB配体是双向信号分子，但在上皮细胞中，ephrinB1通过其胞内结构域信号调控细胞-细胞粘附的确切机制尚不清楚。我们还发现，在非洲爪蟾胚胎发育过程中，高度相关的Eph配体ephrinB2蛋白的减少会导致神经管闭合缺陷。这种ephrinB2蛋白水平的下降是在flotilin -1支架蛋白的缺失上观察到的，flotilin -1支架蛋白是一种新发现的ephrinB2结合伙伴。在flotilin -1表达缺失的情况下，ephrinB2蛋白水平的急剧下降是特异性的，部分原因是金属蛋白酶ADAM10对切割的敏感性增加。上述结果表明，flotilin -1通过ADAM10调控ephrinB2蛋白水平，是爪蟾胚胎正常神经管形态发生所必需的。尽管ephrin信号传导与颅神经嵴（CNC）细胞的迁移有关，但目前尚不清楚ephrinB信号传导如何影响这一事件。我们提供的证据表明，TBC1d24是一种推测的Rab35- gtpase激活蛋白（Rab35 GAP），通过支架dishevelzed （Dsh）与ephrinB2复合物，并介导影响CNC细胞接触抑制运动（CIL）的信号。此外，我们发现在迁移CNC时，ephrinB2与TBC1d24相互作用，进而通过Rab35负性调节E-Cadherin在这些细胞中的循环。当与同源的Eph受体结合时，ephrinB2被酪氨酸磷酸化，从而破坏ephrinB2/Dsh/TBC1d24复合物。该复合物的溶解导致质膜上e -钙粘蛋白水平增加，导致CIL丢失，并抑制CNC迁移。我们的研究结果表明，TBC1d24是ephrinB2通过CIL控制CNC细胞迁移的关键参与者。我们还完成了一项研究，该研究与我们之前关注的调节ephrinB蛋白水平的机制及其对发育的影响有关。Rab11Fip5是非规范Wnt/PCP信号通路的靶点，并通过与Rab11的相互作用调节蛋白质向膜的再循环。Rab11及其在神经管闭合中的作用受PCP信号的控制，这是Rab11相关核内体的顶端积累所必需的。我们能够确定Rab11Fip5在将ephrinB1循环到发育中的前脑膜中起关键作用。此外，ephrinB1和Rab11Fip5之间的相互作用是由Rab11蛋白介导的，是维持膜上ephrinB1和维持这些端脑细胞增殖能力的关键。这些结果提供了自闭症谱系障碍候选基因产物Rab11fip5和ephrinB1之间的一种新的机制联系，并表明通过Rab11/Rab11fip5复合体对ephrinB1的适当再循环控制着端脑的形成。

Our current research interests are aimed toward examining the mechanism by which Eph receptor tyrosine kinases and their ephrin ligands signal events affecting cell-cell adhesion and morphogenetic movements. From the elucidation of these signal transduction pathways we may improve our understanding of oncogenesis. The cell-cell adhesion system plays a major role in normal development and morphogenesis. Inactivation of this adhesion system is thought to play a critical role in cancer invasion and metastasis. The Xenopus embryo is well suited for investigations of these processes because the frog has a well characterized and invariant cell fate map and cell lineage can be easily traced during experiments. Mutant receptors, ligands, and other proteins can be ectopically expressed in embryos. Thus, their effects on signal transduction, motility, and differentiation can be assessed morphologically and histologically as well as biochemically in a developing vertebrate. Our laboratory is currently investigating the role of the Xenopus Eph receptor tyrosine kinases and ephrinB transmembrane ligands in cell signaling and function using the Xenopus oocyte and embryo systems, as well as human cultured cell lines. At present, our emphasis is placed upon the mechanism by which these Eph family members send signals affecting morphogenetic movements. Members of the Eph family have been implicated in regulating numerous developmental processes and have been found to be deregulated in metastatic cancers, for example, prostate, ovarian, breast, colon, neuroblastoma, lung, and melanoma. Our laboratory has continued these studies examining proximal and distal signaling from ephrinB1 that controls cell adhesion and cell movement. We found evidence that ephrinB1 signals via its intracellular domain to control retinal progenitor movement into the eye field by interacting with Dishevelled (dsh), and co-opting the Wnt/planar cell polarity (PCP) pathway. Using biochemical analysis and gain or loss of function experiments, our data suggest that dsh associates with ephrinB1 and mediates ephrinB1 signaling via downstream members of the PCP pathway during eye field formation. Thus, we have used the eye field as a model system for understanding how ephrinB1 controls cell movement. Recently, we have examined the mechanisms by which ephrinB1 affects cell-cell junctions. A body of evidence is emerging that shows a requirement for ephrin ligands in the proper migration of cells, and the formation of cell and tissue boundaries. These processes are dependent on the cell cell adhesion system, which plays a crucial role in normal morphogenetic processes during development, as well as in invasion and metastasis. Although ephrinB ligands are bi- directional signaling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. We also have shown, that a decrease in a highly related Eph ligand, ephrinB2 protein, causes neural tube closure defects during Xenopus embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo. Although Eph-ephrin signaling has been implicated in the migration of cranial neural crest (CNC) cells, it is still unclear how ephrinB transduces signals affecting this event. We provide evidence that TBC1d24, a putative Rab35-GTPase activating protein (Rab35 GAP), complexes with ephrinB2 via the scaffold Dishevelled (Dsh), and mediates a signal affecting contact inhibition of locomotion (CIL) in CNC cells. Moreover, we found that in migrating CNC, ephrinB2 interacts with TBC1d24, which in turn negatively regulates E-Cadherin recycling in these cells via Rab35. Upon engagement of the cognate Eph receptor, ephrinB2 is tyrosine phosphorylated, which disrupts the ephrinB2/Dsh/TBC1d24 complex. The dissolution of this complex leads to increasing E-Cadherin levels at the plasma membrane, resulting in loss of CIL, and inhibition of CNC migration. Our results indicate that TBC1d24 is a critical player in ephrinB2 control of CNC cell migration via CIL. We have also completed a study that pertains to our previous focus on mechanisms that regulate ephrinB protein levels and the resulting effects on development. Rab11Fip5 is a target of the non-canonical Wnt/PCP signaling pathway and regulates recycling of proteins to the membrane through its interaction with Rab11. Rab11 and its role in neural tube closure has been shown to be under the control of PCP signaling, which is required for the apical accumulation of the recycling Rab11-associated endosomes. We were able to determine that Rab11Fip5 plays a critical role in cycling ephrinB1 to the membrane in the developing forebrain. Moreover, the interaction between ephrinB1 and Rab11Fip5 is mediated by Rab11 protein and is key to maintaining ephrinB1 at the membrane and maintaining the proliferative capacity of these telencephalic cells. These results provide a novel mechanistic connection between the candidate autism spectrum disorder gene product, Rab11fip5, and ephrinB1, and indicate that proper recycling of ephrinB1 through the Rab11/Rab11fip5 complex controls proper telencephalon formation.