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.
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