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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Aberrations in BMP signaling have recently been implicated as a cause of human cancer. Here we demonstrate and define the tumor suppressive properties of BMP4. Consistent with its potential role in a tumor suppressor pathway, BMP4 treatment eliminated the tumorigenic potential of an undifferentiated human cancer cell line. This loss of tumorigenicity was accompanied by an increase in apoptosis, alterations in cell cycle profile, and an increase in cell size. Interestingly, human colon cancer cells were resistant to the growth-suppressive properties of BMP4. To identify putative downstream mediators of BMP4-mediated tumor suppression, Affymetrix Genechips were employed to identify BMP4-regulated genes. The human BMP4 transcriptome was characterized by the modulation of many genes well known to play important roles in differentiation and development, including the induction of numerous genes involved in Wnt signaling. Modulation of Wnt gene expression by BMP4 had several functional consequences--BMP4 treatment led to activation of TCF reporters; complete activation of at least one BMP4-responsive gene required TCF sites; and treatment with a Wnt ligand was sufficient to mimic several of the phenotypic effects of BMP4 treatment. These data demonstrate the tumor suppressive properties of BMP4 signaling, show that colon cancer cells are resistant to BMP4-induced differentiation and growth suppression, further define the BMP4 transcriptome, and raise the intriguing possibility that interactions between the Wnt and BMP signaling pathways may play an important role in differentiation and tumor suppression.

Human somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells. This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice. Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well-defined mutation in an endogenous gene. These modified cells can be studied both in vitro and in vivo. This unit presents protocols for human somatic cell gene targeting.

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insightinto the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.