Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

EphrinB 与替代信号通路之间的串扰机制

• 批准号: 8937682
• 负责人: Ira Daar
• 金额: $ 64.01万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937682
• 关键词: Adhesions; Affect; Axon; Benign; Binding; Biological Models; Breast; CNKSR1 gene; Cancer cell line; Cell Cycle Progression; Cell Polarity; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Colon; Complex; Congenital Abnormality; Cultured Cells; Cytoplasmic Tail; Data; Development; Dominant-Negative Mutation; Embryo; Embryonic Development; Eph Family Receptors; Ephrin B Receptor; Ephrins; Epithelial Cells; Event; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Fibronectins; Frequencies; Hela Cells; Human; Intercellular Junctions; Laboratories; Ligand Binding; Ligands; Link; MAPK8 gene; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of gastrointestinal tract; Mediating; Mediator of activation protein; Membrane; Modification; Movement; Neoplasm Metastasis; Neural Crest Cell; Neuroblastoma; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Positioning Attribute; Process; Prostate; Protein Tyrosine Kinase; Proteins; Repression; Research; Retinal; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Staging; Stem cells; System; Tight Junctions; Tissues; Transfection; Tumor Angiogenesis; Tumor Cell Invasion; Tumor Stem Cells; Tyrosine Phosphorylation; Vertebrates; Visual Fields; Xenopus; angiogenesis; cell motility; embryonic stem cell; epithelial to mesenchymal transition; gain of function; hindbrain; human disease; in vivo; inhibitor/antagonist; insight; lung small cell carcinoma; melanoma; member; migration; neoplastic cell; overexpression; pluripotency; protein complex; retinal progenitor cell; rho; skeletal; stem cell fate specification; tumor; tumorigenesis

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 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 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.

在正常发育过程中，许多组织的祖细胞经历了多能性、上皮向间充质转化、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-基质黏附的改变，这些黏附系统的异常改变通常与肿瘤的形成有关。Eph家族受体酪氨酸激酶及其配体--肾上腺素经常在多种癌症中过度表达，包括乳腺癌、小细胞肺癌和胃肠道肿瘤、黑色素瘤和神经母细胞瘤。利用非洲爪哇胚胎系统，我们已经证明了由eaffinB胞内结构域介导的信号影响细胞与细胞之间的黏附，并且这种活性可以通过与激活的成纤维细胞生长因子受体的相互作用来调节。跨膜的ewitinB1蛋白是一种双向信号分子，通过其细胞质结构域发出信号，促进细胞运动进入眼场，而成纤维细胞生长因子受体(FGFR)的激活抑制了这些运动和视网膜的命运。在非洲爪哇胚胎中，eparinB1通过与支架蛋白disheveled(Dsh)的相互作用，在视网膜前体细胞进入眼场中发挥作用。我们确定FGFR诱导的视网膜命运的抑制依赖于ewitinB1细胞内结构域的磷酸化，并破坏ewitinB1/Dsh的相互作用。这调节依赖于平面细胞极性(PCP)途径的视网膜前体细胞的运动。这些结果提供了对成纤维细胞生长因子信号如何调控视网膜前体细胞在眼野内运动的控制的机械性洞察。此外，我们发现有证据表明，在活体中，ewitinB1信号可能通过细胞极性复合体来调节细胞间的连接。这项研究的重点是利用非洲爪哇系统评估ewitinB1是否是上皮细胞中细胞-细胞连接信号的中介者或调节者。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，它与ewitinB1结合，并受ewitinB1的调节，导致紧密连接的控制。利用非洲爪哇早期胚胎的上皮细胞，我们发现ewitinB1功能的丧失或获得可以破坏细胞与细胞之间的接触和紧密连接。本研究揭示了ePhrinB1与活性CDC42竞争结合PAR-6的机制，PAR-6是PAR极性复合体(PAR-3/PAR-6/CDC42/aPKC)的中心支架蛋白，并扰乱紧密连接相关蛋白(ZO-1、Cinglin)的定位。这种竞争影响紧密连接的形成，并受ewitinB1酪氨酸磷酸化的调节。最后，与我们在莫里森实验室的同事一起，我们已经证明ePhinB1以不依赖于EphB受体的方式与CNK1相互作用。在培养细胞中，与CNK1共转染可增加JNK的磷酸化。显性负性RhoA的过度表达降低了EPhinB1/CNK1介导的JNK激活，表明JNK的激活是Rho依赖的。仅CNK1的过表达就足以激活RhoA，而JNK的磷酸化则需要ewitinB1和CNK1。共IP分析表明，ewitinB1和CNK1是连接RhoA和JNK信号元件的支架蛋白。此外，与纤维连接蛋白的黏附和活性的Src过表达增加了ewitinB1/CNK1的结合，而药物抑制剂抑制Src的活性不仅减少了ewitinB1/CNK1的结合，而且还减少了JNK的激活。这些数据表明，Src活性增强了ewitinB1/CNK1的相互作用和下游JNK的激活。在HeLa宫颈癌细胞系中，过表达EPhinB1增加了细胞的运动能力，然而，通过siRNA耗尽CNK1可以抑制这种细胞迁移和伴随的JNK激活。此外，RhoA和JNK抑制剂可抑制ewitinB1介导的细胞迁移。综上所述，我们的研究结果表明，CNK1介导了ewitinB1诱导的JNK激活和培养的癌细胞系中的细胞迁移。