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 upon the cell-cell adhesion system that plays a critical role in normal morphogenetic processes during development, as well as in invasion and metastasis1–9. 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 has remained elusive. Here, we present evidence that ephrinB1 associates with the Par polarity complex protein, Par-6, a scaffold protein required for establishing tight junctions, and can compete with the small GTPase Cdc42 for an association with Par-6. This competition results in inactivation of the Par complex, resulting in the loss of tight junctions. Moreover, the interaction between ephrinB1 and Par-6 is disrupted upon tyrosine phosphorylation of the intracellular domain of ephrinB1. Thus, we have identified a mechanism by which ephrinB1 signaling regulates cell-cell junctions in epithelial cells, and this may impact how we devise therapeutic interventions regarding these molecules in metastatic disease.

The Eph (erythropoietin-producing hepatoma) family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB (Eph receptor interactor B) protein is a bidirectional signaling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase, residing on another cell. A reverse signal can be transduced into the ephrinBexpressing cell via tyrosine phosphorylation of its conserved Cterminal cytoplasmic domain. Although some insight has been gained regarding how ephrinB may send signals affecting cytoskeletal components, little is known about how ephrinB1 reverse signaling affects transcriptional processes. Here we report that signal transducer and activator of transcription 3 (STAT3) can interact with ephrinB1 in a phosphorylation-dependent manner that leads to enhanced activation of STAT3 transcriptional activity. This activity depends on the tyrosine kinase Jak2, and two tyrosines within the intracellular domain of ephrinB1 are critical for the association with STAT3 and its activation. The recruitment of STAT3 to ephrinB1, and its resulting Jak2-dependent activation and transcription of reporter targets, reveals a signaling pathway from ephrinB1 to the nucleus.FGF receptor ͉ Eph receptor ͉ neuroepithelial cells ͉ tyrosine phosphorylation ͉ Jak2 M embrane-anchored ephrinB (erythropoietin-producing hepatoma interactor B) proteins are ligands for EphB receptors and have been involved in many biological processes such as cell adhesion, neural crest migration, brain segmentation, and vasculogenesis (1-3). Upon cell-to-cell contact, ephrinB1 not only sends a forward signal through the activation of its cognate receptor, but it also rapidly undergoes tyrosine phosphorylation at the C terminus and recruits the adaptor protein growth-factor-receptor-bound protein 4 (Grb4)/Nck adaptor protein 2 (Nck2), resulting in an increase of focal adhesion kinase (FAK) catalytic activity as well as cell rounding (4). In addition, the C-terminal PDZ-binding motif of ephrinB1 is able to mediate reverse signaling by recruitment of PDZ-RGS3, a GTP exchange factor that regulates the migration of cerebellar granule cells (5). Moreover, it has been reported that ephrinB1 signals through its intracellular domain to control retinal progenitor cell movement into the eye field by interacting with the scaffold protein Dishevelled (6). Thus, several signaling molecules have been identified to transmit reverse signaling of ephrinB1 by either phosphorylation-dependent or -independent means. ephrinB1 can be phosphorylated in response to binding a cognate Eph receptor (7,8) or the tight junction protein Claudin (9) or in response to FGF receptor activation (10). Among the six tyrosine residues present in the intracellular domain of ephrinB1, one resides in a well conserved YXXQ motif at the C terminus and is rapidly phosphorylated upon engagement of t...

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