Signaling Mechanisms of EphrinB1 in Cell Adhesion, Migration and Invasion

Division Of Basic Sciences - Nci

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Abstract

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.