Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

External signals that control cellular behavior in metazoan organisms are often transduced at the cell surface by receptor tyrosine kinases (RTKs). Eph receptors comprise the largest family of mammalian RTKs, with 14 members. The family has apparently undergone a striking expansion during the evolution of multicellular animals, since only a single Eph receptor has been identified in Caenorhabditis elegans (30) or Drosophila (65), suggesting that these receptors might be involved in controlling complex cellular interactions. The ligands for Eph receptors, termed ephrins, are themselves anchored to the plasma membrane, either via a glycosylphosphatidylinositol linkage (A class) or through a transmembrane sequence (B class) (21, 28, 39). Consequently, signaling generally requires direct contact between ephrin- and Eph receptor-expressing cells. The Eph receptors are also classified into A and B groups on the basis of sequence homology and ephrin-binding ability (27). Although the binding of receptors to ephrins is generally nonselective within a given class, different combinations of receptors and ligands interact with distinct affinities, while EphA4 can bind both classes of ephrins (28). In C. elegans, the VAB-1 Eph receptor and corresponding ephrins regulate a series of morphogenetic cell movements important for development (14, 30, 72). In mammals, Eph receptors and ephrins are expressed in reciprocal compartments of the developing embryo (28, 33) and are important for axon guidance and topographic map formation in the central nervous system (7, 19, 25, 34, 74), neural crest cell migration (18), patterning of the hindbrain and paraxial mesoderm (28), and vascular network assembly (1, 29, 31, 71). For both invertebrates and vertebrates, there are data to suggest that Eph receptors have both kinase-dependent and kinase-independent functions, with the latter potentially reflecting either an ability of Eph-ephrin interactions to mediate cell adhesion or an intrinsic ephrin-signaling activity (13, 22, 23, 37). In the guidance of axons in the nervous system, and in cell migrations, ephrin-Eph receptor signaling commonly has a repulsive effect on cell movement (11, 53, 54, 57). In vitro, the activation of Eph receptors in neuronal cells induces deadhesive responses and collapse of neural growth cones (6), correlating with axon and neural crest cell repulsion from ephrins displayed on cells or isolated membranes (53, 54). Although ephrins and Eph receptors clearly activate repellant responses in many cells, there is increasing evidence that specific ligand-receptor pairs can also initiate an attractive response in some cell types, for example by eliciting endothelial cell sprouting (1), increased cellular adhesion (8, 22, 23, 41), neural tube closure (40), and projection of vomeronasal axons (47). This resembles the ability of several other guidance molecules to induce either attraction or repulsion (56). The intracellular signaling pathways that mediate the biological effects of Eph receptors and ephrins are only starting to emerge. Activated receptors become autophosphorylated at multiple sites, including two absolutely conserved tyrosine residues in the juxtamembrane region and a tyrosine within the activation segment of the kinase domain (6, 44). Interestingly, prior to phosphorylation, the juxtamembrane tyrosines (Y604 and Y610 in EphB2) repress receptor kinase activity, but following phosphorylation they are released to serve as docking sites for SH2 domain proteins (6). RTKs commonly signal through cytoplasmic proteins with SH2 domains, which bind either directly to phosphotyrosine (pTyr) sites on the activated receptor or to phosphorylated docking proteins. Both mechanisms may be used by Eph receptors. A variety of SH2 proteins have been identified as potential Eph receptor-binding partners, including the Fyn and Src tyrosine kinases (15, 26, 35, 75), the p120-Ras GTPase-activating protein (p120-RasGAP) (see interaction ID:123 at www.BIND.ca [35, 38]), the Nck and Crk adaptors (35, 69), SHEP1 (24), the Ras-binding protein AF6 (36), and the Src-like adaptor protein SLAP (58). Which of these targets are relevant to the biological functions of Eph receptors remain uncertain. In addition, we and others have found that activated Eph receptors preferentially phosphorylate the p62dok-1 docking protein in neuronal and endothelial cells (4, 38). p62dok-1 has an N-terminal pleckstrin homology (PH) domain followed by a phosphotyrosine-binding (PTB) domain and multiple tyrosine phosphorylation sites which engage the SH2 domains of p120-RasGAP and Nck (73). In NG108 neuronal cells expressing EphB2 and stimulated with clustered ephrin-B1, p62dok-1 is the most prominently tyrosine-phosphorylated protein other than the receptor itself (38). The Ras-mitogen-activated protein kinase (MAPK) pathway is commonly activated by RTKs, and indeed is viewed as a hallmark of RTK signaling (16). Autophosphorylation of RTKs such as the epidermal growth factor, platelet-derived growth factor (PDGF), or insulin receptors leads to the recruitment (either directly or indirectly) of the Grb2-Sos1 complex, which in turn induces the exchange of GDP for GTP on Ras proteins, and the association of Ras with the Raf serine/threonine protein kinase (59). Raf phosphorylates the dual-specificity protein kinases MEK1 and MEK2, which consequently activate the MAPKs extracellular signal-related kinases 1 and 2 (ERK1/2). This core biochemical pathway is regulated by many different signals in numerous cell types, raising the issue of how such a widespread signaling pathway generates distinct biological responses in different cells or following stimulation by different ligands. In metazoans, each cell is simultaneously exposed to multiple extracellular signals and must integrate these inputs to initiate the appropriate outcome. The combined nature of these external signals, together with regulators expressed within the target cell, may therefore determine the extent and duration of Ras-MAPK activation, which in turn can determine how the cell responds (51). Unlike other RTKs, Eph receptors appear inefficient at stimulating cell proliferation in fibroblasts or epithelial cells (10, 12), and the role of MAPK signaling downstream of activated Eph receptors remains unclear. Here, we show that EphB2 tyrosine kinase activity down regulates Ras and ERK1/2 MAPKs in a neuronal cell culture system. Our data suggest that p120-RasGAP contributes to Ras inhibition by Eph receptors and indicate that Ras activation interferes with neurite retraction induced by ephrin-B1.