The Smurf ubiquitin ligases regulate tissue separation via antagonistic interactions with ephrinB1.

The formation of tissue boundaries is dependent on the cell-cell adhesion/repulsion system that is required for normal morphogenetic processes during development. The Smad ubiquitin regulatory factors (Smurfs) are E3 ubiquitin ligases with established roles in cell growth and differentiation, but whose roles in regulating cell adhesion and migration are just beginning to emerge. Here, 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.

EphrinB1 interacts with CNK1 and promotes cell migration through c-Jun N-terminal kinase (JNK) activation.

The Eph receptors and their membrane-bound ligands, ephrins, play important roles in various biological processes such as cell adhesion and movement. The transmembrane ephrinBs transduce reverse signaling in a tyrosine phosphorylation-dependent or -independent, as well as PDZ-dependent manner. Here, we show that ephrinB1 interacts with Connector Enhancer of KSR1 (CNK1) in an EphB receptor-independent manner. In cultured cells, cotransfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1-mediated JNK activation is reduced by overexpression of dominant-negative RhoA. Overexpression of CNK1 alone is sufficient for activation of RhoA; however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-immunoprecipitation data showed that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA and JNK signaling components, such as p115RhoGEF and MKK4. Furthermore, adhesion to fibronectin or active Src overexpression increases ephrinB1/CNK1 binding, whereas blocking Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. EphrinB1 overexpression increases cell motility, however, CNK1 depletion by siRNA abrogates ephrinB1-mediated cell migration and JNK activation. Moreover, Rho kinase inhibitor or JNK inhibitor treatment suppresses ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 is required for ephrinB1-induced JNK activation and cell migration.

Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways.

The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. We demonstrate that both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. ephrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field.

Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor.

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cgamma binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met-mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor.

Low-molecular-weight protein tyrosine phosphatase is a positive component of the fibroblast growth factor receptor signaling pathway.

Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) has been implicated in the regulation of cell growth and actin rearrangement mediated by several receptor tyrosine kinases, including platelet-derived growth factor and epidermal growth factor. Here we identify the Xenopus laevis homolog of LMW-PTP1 (XLPTP1) as an additional positive regulator in the fibroblast growth factor (FGF) signaling pathway during Xenopus development. XLPTP1 has an expression pattern that displays substantial overlap with FGF receptor 1 (FGFR1) during Xenopus development. Using morpholino antisense technology, we show that inhibition of endogenous XLPTP1 expression dramatically restricts anterior and posterior structure development and inhibits mesoderm formation. In ectodermal explants, loss of XLPTP1 expression dramatically blocks the induction of the early mesoderm gene, Xbrachyury (Xbra), by FGF and partially blocks Xbra induction by Activin. Moreover, FGF-induced activation of mitogen-activated protein (MAP) kinase is also inhibited by XLPTP1 morpholino antisense oligonucleotides; however, introduction of RNA encoding XLPTP1 is able to rescue morphological and biochemical effects of antisense inhibition. Inhibition of FGF-induced MAP kinase activity due to loss of XLPTP1 is also rescued by an active Ras, implying that XLPTP1 may act upstream of or parallel to Ras. Finally, XLPTP1 physically associates only with an activated FGFR1, and this interaction requires the presence of SNT1/FRS-2 (FGFR substrate 2). Although LMW-PTP1 has been shown to participate in other receptor systems, the data presented here also reveal XLPTP1 as a new and important component of the FGF signaling pathway.

Contribution of JNK, Mek, Mos and PI-3K signaling to GVBD in Xenopus oocytes.

In Xenopus oocytes, induction of the G2/M transition by progesterone is a complex process that is promoted by a network of signaling molecules whose cumulative effect results in the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). We examined the role of Mos, Mek, PI-3 kinase and c-Jun N-terminal kinase (JNK) in progesterone stimulation of GVBD. Expression of an activated form of JNK neither induced nor enhanced progesterone-mediated GVBD in oocytes, suggesting a limited role in cell-cycle progression. We blocked Mek, Mos and PI-3 kinase activities by a variety of means that included expression of dominant-negative kinase suppressor of Ras (DnKSR), expression of a dominant-negative PI-3 kinase (DnPI3K), treatment of oocytes with a Mek inhibitor (U1026) or PI-3 kinase (LY294002) inhibitor, and introduction of Mos antisense morpholinos. Inhibition of any one pathway alone failed to block GVBD induced by either high or low concentrations of progesterone. In contrast, inhibiting Mos or Mek function in addition to abrogating PI-3 kinase activity effectively blocked oocyte maturation. Furthermore, by expressing suboptimal amounts of Mos in conjunction with an activated form of Mek and an activated form of the p110 catalytic subunit of PI-3 kinase, we show cooperation among these signaling molecules toward the induction of GVBD. Moreover, expression of optimal amounts of these three proteins in conjunction with inhibitors of Mos, Mek or PI-3 kinase demonstrated that activated Mek-induced GVBD is independent of Mos or PI-3 kinase activity. In addition, Mos-induced GVBD is dependent upon Mek activity, but does not require PI-3 kinase activity. Finally, Mos appears to be a major contributor to GVBD induced by activated PI-3 kinase, while Mek is a minor contributor to this process.

Tyr-298 in ephrinB1 is critical for an interaction with the Grb4 adaptor protein.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.

EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.

The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold 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.

EphrinB1 interacts with the transcriptional co-repressor Groucho/xTLE4.

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.

Fibroblast growth factor receptor-induced phosphorylation of ephrinB1 modulates its interaction with Dishevelled.

The Eph 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 ephrinB1 protein is a bidirectional 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). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here, we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity pathway. These results provide mechanistic insight into how fibroblast growth factor signaling modulates ephrinB1 control of retinal progenitor movement within the eye field.

EphrinB1 controls cell-cell junctions through the Par polarity complex.

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 signalling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. 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 association with Par-6. This competition causes inactivation of the Par complex, resulting in the loss of tight junctions. Moreover, the interaction between ephrinB1 and Par-6 is disrupted by tyrosine phosphorylation of the intracellular domain of ephrinB1. Thus, we have identified a mechanism by which ephrinB1 signalling regulates cell-cell junctions in epithelial cells, and this may influence how we devise therapeutic interventions regarding these molecules in metastatic disease.

ephrinB1 signals from the cell surface to the nucleus by recruitment of STAT3.

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 ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal 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.

Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling.

Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell-cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal-binding partners and downstream signaling pathways in cell-cycle regulation. We show that cell-cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr-Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C-terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell-cycle progression and activation of MAPK and JNK, while the binding of phospholipase-Cgamma or phosphatidylinositol 3-kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3-kinase, phospholipase-Cgamma, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK-dependent and independent signaling contribute to Met receptor-mediated cell-cycle progression, but phospholipase-Cgamma or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3-kinase signaling are required for cell-cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors.

SNT1/FRS2 mediates germinal vesicle breakdown induced by an activated FGF receptor1 in Xenopus oocytes.

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from the fibroblast growth factor receptor (FGFR), which plays vital roles during embryogenesis. Activating FGFR mutations cause several craniosynostoses and dwarfism syndromes in humans. Here we show that the Xenopus homolog of mammalian FRS-2 (XFRS2) is essential for the induction of oocyte maturation by an XFGFR1 harboring an activating mutation (XFGFR1act). Using a dominant-negative form of kinase suppressor of Ras, we show the Mek activity is required for germinal vesicle breakdown (GVBD) induced by co-expression of XFGFR1act and XFRS2, but this activity is not required for progesterone-induced GVBD. Furthermore, Mek/MAPK activity is critical for the induction and/or maintenance of H1 kinase activity at metaphase of meiosis II in progesterone-treated oocytes. An activated XFGFR1 containing a mutation in the phospholipase Cgamma binding site (XFGFR1actY672F) displayed a reduced ability to induce cell-cycle progression in oocytes, suggesting phospholipase Cgamma may not be necessary but that it augments XFGFR signaling in this system. Oocytes co-expressing XFGFR1act and XFRS2 showed substantial H1 kinase activity, but this activity was blocked when the oocytes were treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Although phosphatidylinositol 3-kinase activity is essential for XFGFR1act/XFRS2-induced oocyte maturation, this activity is not required for maturation induced by progesterone. Finally, ectopic expression of Xspry2, a negative regulator of XFGFR signaling, greatly reduced MAPK activation and GVBD induced by the expression of either XFGFR1act plus XFRS2 or activated Ras (H-RasV12). In contrast, Xspry2 did not prevent GVBD induced by an activated form of Raf1, suggesting that Xspry2 exerts its inhibitory function upstream or parallel to Raf and downstream of Ras.

Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.

Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos.

Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.