Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase

The ephrins, ligands of Eph receptor tyrosine kinases, have been shown to act as repulsive guidance molecules and to induce collapse of neuronal growth cones. For the first time, we show that the ephrin-A5 collapse is mediated by activation of the small GTPase Rho and its downstream effector Rho kinase. In ephrin-A5-treated retinal ganglion cell cultures, Rho was activated and Rac was downregulated. Pretreatment of ganglion cell axons with C3-transferase, a specific inhibitor of the Rho GTPase, or with Y-27632, a specific inhibitor of the Rho kinase, strongly reduced the collapse rate of retinal growth cones. These results suggest that activation of Rho and its downstream effector Rho kinase are important elements of the ephrin-A5 signal transduction pathway.     

Coexpressed EphA receptors and ephrin-A ligands mediate opposing actions on growth cone navigation from distinct membrane domains

Contact-dependent signaling between membrane-linked ligands and receptors such as the ephrins and Eph receptor tyrosine kinases controls a wide range of developmental and pathological processes. Paradoxically, many cell types coexpress both ligands and receptors, raising the question of how specific signaling readouts are achieved under these conditions. Here, we studied the signaling activities exerted by coexpressed EphA receptors and GPI-linked ephrin-A ligands in spinal motor neuron growth cones. We demonstrate that coexpressed Eph and ephrin proteins segregate laterally into distinct membrane domains from which they signal opposing effects on the growth cone: EphAs direct growth cone collapse/repulsion and ephrin-As signal motor axon growth/attraction. This subcellular arrangement of Eph-ephrin proteins enables axons to discriminate between cis- versus trans-configurations of ligand/receptor proteins, thereby allowing the utilization of both Ephs and ephrins as functional guidance receptors within the same neuronal growth cone.     

On the turning of Xenopus retinal axons induced by ephrin-A5

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-Kinase-dependent Rac1 activation

Endocytosis of Eph receptors is critical for a number of biological processes, including modulating axon growth cone collapse response and regulating cell surface levels of receptor in epithelial cells. In particular, ephrin-A ligand stimulation of tumor cells induces EphA2 receptor internalization and degradation, a process that has been explored as a means to reduce tumor malignancy. However, the mechanism and regulation of ligand-induced Eph receptor internalization are not well understood. Here we show that SHIP2 (Src homology 2 domain-containing phosphoinositide 5-phosphatase 2) is recruited to activated EphA2 via a heterotypic sterile alpha motif (SAM)-SAM domain interaction, leading to regulation of EphA2 internalization. Overexpression of SHIP2 inhibits EphA2 receptor endocytosis, whereas suppression of SHIP2 expression by small interfering RNA-mediated gene silencing promotes ligand-induced EphA2 internalization and degradation. SHIP2 regulates EphA2 endocytosis via phosphatidylinositol 3-kinase-dependent Rac1 activation. Phosphatidylinositol 3,4,5-trisphosphate levels are significantly elevated in SHIP2 knockdown cells, phosphatidylinositol 3-kinase inhibitor decreases phosphatidylinositol 3,4,5-trisphosphate levels and suppresses increased EphA2 endocytosis. Ephrin-A1 stimulation activates Rac1 GTPase, and the Rac1-GTP levels are further increased in SHIP2 knockdown cells. A dominant negative Rac1 GTPase effectively inhibited ephrin-A1-induced EphA2 endocytosis. Together, our findings provide evidence that recruitment of SHIP2 to EphA2 attenuates a positive signal to receptor endocytosis mediated by phosphatidylinositol 3-kinase and Rac1 GTPase.     

Rho与中枢神经系统轴突再生

Rho是小分子质量GTP酶Rho家族成员,在细胞的一些信号转导途径中起着分子开关的作用.Rho能通过作用于肌动蛋白骨架系统引起轴突生长锥塌陷,从而抑制轴突生长.研究表明,Nogo-A、MAG、OMgp等髓鞘源性的轴突再生抑制分子均可通过激活Rho介导的信号转导途径抑制轴突再生.     

Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse.

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.     

Erk5 Participates in Neuregulin Signal Transduction and Is Constitutively Active in Breast Cancer Cells Overexpressing ErbB2

The four receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes and have also been implicated in tumor generation and/or progression. Activation of ErbB1/EGFR is mainly triggered by epidermal growth factor (EGF) and other related ligands, while activation of ErbB2, ErbB3, and ErbB4 receptors occurs by binding to another set of EGF-like ligands termed neuregulins (NRGs). Here we show that the Erk5 mitogen-activated protein kinase (MAPK) pathway participates in NRG signal transduction. In MCF7 cells, NRG activated Erk5 in a time- and dose-dependent fashion. The action of NRG on Erk5 was dependent on the kinase activity of ErbB receptors but was independent of Ras. Expression in MCF7 cells of a dominant negative form of Erk5 resulted in a significant decrease in NRG-induced proliferation of MCF7 cells. Analysis of Erk5 in several human tumor cell lines indicated that a constitutively active form of this kinase was present in the BT474 and SKBR3 cell lines, which also expressed activated forms of ErbB2, ErbB3, and ErbB4. Treatments aimed at decreasing the activity of these receptors caused Erk5 inactivation, indicating that the active form of Erk5 present in BT474 and SKBR3 cells was due to a persistent positive stimulus originating at the ErbB receptors. In BT474 cells expression of the dominant negative form of Erk5 resulted in reduced proliferation, indicating that in these cells Erk5 was also involved in the control of proliferation. Taken together, these results suggest that Erk5 may play a role in the regulation of cell proliferation by NRG receptors and indicate that constitutively active NRG receptors may induce proliferative responses in cancer cells through this MAPK pathway.     

Transient activation of the MAP kinase signaling pathway by the forward signaling of EphA4 in PC12 cells

In the present study, we demonstrate that ephrin-A5 is able to induce a transient increase of MAP kinase activity in PC12 cells. However, the effects of ephrin-A5 on the MAP kinase signaling pathway are about three-fold less than that of EGF. In addition, we demonstrate that EphA4 is the only Eph member expressed in PC12 cells, and that tyrosine phosphorylation induced by ephrin-A5 treatment is consistent with the magnitude and longevity of MAP kinase activation. Experiments using the Ras dominant negative mutant N17Ras reveal that Ras plays a pivotal role in ephrin-A5-induced MAP kinase activation in PC12 cells. Importantly, we found that the EphA4 receptor is rapidly internalized by endocytosis upon engagement of ephrin-A5, leading to a subsequent reduction in the MAP kinase activation. Together, these data suggest a novel regulatory mechanism of differential Ras-MAP kinase signaling kinetics exhibited by the forward signaling of EphA4 in PC12 cells.     

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.     

Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Granulocyte colony-stimulating factor induces ERK5 activation, which is differentially regulated by protein-tyrosine kinases and protein kinase C. Regulation of cell proliferation and survival

Granulocyte colony-stimulating factor (G-CSF) plays a major role in the regulation of granulopoiesis. Treatment of cells with G-CSF has been shown to activate multiple signal transduction pathways. We show here that Erk5, a novel member of the MAPK family, and its specific upstream activator MEK5 were activated in response to incubation of cells with G-CSF. Different from other members of the MAPK family including Erk1/2, JNK, and p38, maximal activation of Erk5 by G-CSF required the C-terminal region of the G-CSF receptor. Genistein, a specific inhibitor of protein-tyrosine kinases, blocked G-CSF-induced Erk5 activation. In contrast, inhibition of protein kinase C activity increased G-CSF-mediated activation of Erk5 and MEK5, whereas stimulation of protein kinase C activity inhibited activation of the two kinases by G-CSF. The proliferation of BAF3 cells in response to G-CSF was inhibited by expression of a dominant-negative MEK5 but potentiated by expression of a constitutively active MEK5. Expression of the constitutively active MEK5 also increased the survival of BAF3 cells cultured in the absence of or in low concentrations of G-CSF. Together, these data implicate Erk5 as an important signaling component in the biological actions of G-CSF.     

EphA kinase activation regulates HGF-induced epithelial branching morphogenesis

Eph kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.     

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.     

PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk

Although it is appreciated that canonical signal‐transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross‐talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross‐talk mechanisms in the platelet‐derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3‐kinase (PI3K) and Ras/extracellular signal‐regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross‐talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation.