Signaling Mechanisms of EphrinB1 in Cell Adhesion, Migration and Invasion

我们目前的研究兴趣是研究Eph受体酪氨酸激酶及其ePhin配体信号影响细胞-细胞黏附和形态发生运动的机制。通过对这些信号转导途径的阐明，可以提高我们对肿瘤发生的认识。细胞-细胞黏附系统在正常发育和形态发生中起着重要作用。这种黏附系统的失活被认为在癌症的侵袭和转移中起着关键作用。非洲爪蛙胚胎非常适合研究这些过程，因为这种青蛙有一个很好的特征和不变的细胞命运图，而且在实验期间可以很容易地追踪到细胞谱系。突变的受体、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从形态和组织学以及生化方面进行评估。我们的实验室目前正在利用非洲爪哇的卵母细胞和胚胎系统以及人类培养的细胞系来研究非洲爪哇Eph受体酪氨酸激酶和eparinB跨膜配体在细胞信号和功能中的作用。目前，我们的重点放在这些Eph家族成员发出影响形态发生运动的信号的机制上。Eph家族的成员参与了许多发育过程的调控，并被发现在转移性癌症中被解除调控，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，检测控制细胞黏附和细胞运动的ewitinB1的近端和远端信号。我们发现有证据表明，ewitinB1通过其细胞内结构域与Disheveled(Dsh)相互作用，并共同选择Wnt/平面细胞极性(PCP)途径，控制视网膜前体细胞进入眼场。利用生化分析和功能得失实验，我们的数据表明，在眼场形成过程中，dsh与ewitinB1联系在一起，并通过PCP通路的下游成员介导ewitinB1信号传递。因此，我们使用眼场作为一个模型系统来理解ewitinB1是如何控制细胞运动的。最近，我们研究了ewitinB1影响细胞-细胞连接的机制。大量证据表明，在细胞的正常迁移以及细胞和组织边界的形成过程中，需要有肾上腺素配体。这些过程依赖于细胞黏附系统，细胞黏附系统在发育过程中的正常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。虽然ewitinB配体是双向信号分子，但ewitinB1通过其胞内结构域调节上皮细胞细胞间黏附的确切机制尚不清楚。我们证明蓝精灵通过与Eph1受体配体的拮抗相互作用来调节中胚层/外胚层边界的组织分离，Eph受体配体在调节胚胎生殖层的分离中起着关键作用。E蛋白B1是S-2降解的靶标；然而，S-1与E蛋白B1的相互作用阻止了与S-B的结合，并阻止了E-B 1的泛素化和降解，因为它是S-1的一个相当弱的底物。抑制胚胎中胚层中SMurf1的表达会导致eparinB1介导的该组织与外胚层分离的丧失，这可以通过同时抑制SMurf2的表达来挽救。我们相信，这些发现极大地拓宽了我们对蓝精灵泛素连接酶和eferinB调控的概念和认识，它们在胚胎发育过程中影响组织排斥和边界维持。此外，由于eparinB和蓝精灵都与癌症进展密切相关，这项工作可能会在考虑对转移性疾病中的这些分子进行干预时产生影响。我们还发现，在非洲爪哇胚胎发育过程中，一种高度相关的Eph配体ewitinB2蛋白的减少会导致神经管闭合缺陷。EwitinB2蛋白水平的这种下降在Flotillin-1支架蛋白的丢失上观察到，Flotillin-1支架蛋白是一种新发现的ewitinB2结合伙伴。在缺乏Flotillin-1表达的情况下，ewitinB2蛋白水平的急剧下降是特殊的，部分原因是金属蛋白酶ADAM10对切割的敏感性增加。这些发现表明，Flotillin-1通过ADAM10调节ewitinB2蛋白水平，并且是非洲爪哇胚胎适当的神经管形态发生所必需的。虽然Eph-eaffin信号与脑神经脊细胞的迁移有关，但ewitin B如何转导影响这一事件的信号仍不清楚。我们提供的证据表明，一种可能的Rab35-GTP酶激活蛋白(Rab35 GAP)TBC1D24通过支架杂乱(Dsh)与ewitinB2形成络合物，并在CNC细胞中介导影响接触性运动抑制(CIL)的信号。此外，我们还发现，在迁移的Cnc中，ewitinB2与TBC1D24相互作用，进而通过Rab35负向调节这些细胞中E-钙粘附素的循环。当与同源Eph受体结合时，ewitinB2被酪氨酸磷酸化，从而破坏ewitinB2/Dsh/TBC1D24复合体。这种复合体的溶解导致质膜上E-钙粘附素水平的增加，导致CIL的丢失，并抑制了NC的迁移。我们的结果表明，TBC1D24在通过CIL调控cnc细胞迁移过程中起着关键作用。

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 demonstrate that the Smurfs regulate tissue separation at mesoderm/ectoderm boundaries through antagonistic interactions with ephrinB1, an Eph receptor ligand that has a key role in regulating the separation of embryonic germ layers. EphrinB1 is targeted by Smurf2 for degradation; however, a Smurf1 interaction with ephrinB1 prevents the association with Smurf2 and precludes ephrinB1 from ubiquitination and degradation, since it is a substantially weaker substrate for Smurf1. Inhibition of Smurf1 expression in embryonic mesoderm results in loss of ephrinB1-mediated separation of this tissue from the ectoderm, which can be rescued by the coincident inhibition of Smurf2 expression. This system of differential interactions between Smurfs and ephrinB1 regulates the maintenance of tissue boundaries through the control of ephrinB protein levels.We believe that these findings significantly broaden our concept and knowledge of the Smurf ubiquitin ligases and ephrinB regulation that affects tissue repulsion and border maintenance during embryogenesis. Furthermore, since both ephrinBs and Smurfs have been strongly implicated in cancer progression, this work may have implications when considering interventions regarding these molecules in metastatic disease. 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.