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. 1999 Nov 23;96(24):13813-8.
doi: 10.1073/pnas.96.24.13813.

An Eph receptor regulates integrin activity through R-Ras

J X Zou  1 , B Wang, M S Kalo, A H Zisch, E B Pasquale, E Ruoslahti
Affiliations

An Eph receptor regulates integrin activity through R-Ras

J X Zou et al. Proc Natl Acad Sci U S A. .

Abstract

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.

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Figures

Figure 1
Figure 1
Activation of the EphB2 receptor and its effects on cell adhesion. (A) Activation of EphB2 in transiently transfected 293 cells. EphB2 was immunoprecipitated from cells transfected with EphB2, kinase-inactive EphB2K669, or pcDNA3. The immunoprecipitates (IP) were probed by immunoblotting as indicated. (B) Morphological changes caused by expression of activated EphB2 in 293T cells. An enhanced green fluorescent protein (EGFP) vector was cotransfected to identify the transfected cells. The transfected cells were shown to be alive by trypan blue exclusion. EphB2K662R and pcDNA3 vector were used as controls. (Upper) Fluorescence microscopy. (Lower) Phase-contrast microscopy of same fields. (×200.) (C) Activation of EphB2 by ephrin-B1 ligand. EphB2 was immunoprecipitated from NIH 3T3 cells that had been stably transfected with EphB2 and treated with soluble ephrin-B1 Fc ligand or left untreated. The immunoprecipitates were probed by immunoblotting as indicated. (D) Ligand stimulation of EphB2 decreases adhesion to collagen I in NIH 3T3 cells stably transfected with EphB2.
Figure 2
Figure 2
R-Ras phosphorylation on tyrosine by EphB2 impairs binding to Raf-1. (A) Phosphorylation of R-Ras in cells expressing activated EphB2. Extracts of 293T cells cotransfected with EphB2, or EphB2K662R, and Myc-tagged R-Ras were subjected to immunoprecipitation (IP) with anti-Myc antibody. The immunoprecipitates, and cell lysates, were probed by immunoblotting as indicated. Anti-PY, anti-phosphotyrosine. (B) The labeling of R-Ras with anti-phosphotyrosine antibody is specific. R-Ras immunoprecipitated with anti-Myc antibody was probed with anti-phosphotyrosine antibody, or this antibody together with 1 mM phosphotyrosine, phosphoserine, or phosphothreonine. Only phosphotyrosine blocked antibody labeling of R-Ras. (C) Phosphorylation of R-Ras by EphB2 in vitro. Equal amounts of purified GST-R-Ras, GST-H-Ras, or GST alone were incubated with equal amounts of immunoprecipitated EphB2 in the presence of [γ-32P]ATP. Phosphorylation of R-Ras was analyzed by SDS/PAGE followed by autoradiography. The lower molecular weight phosphorylated band in the R-Ras lane is presumably a degradation product of GST-R-Ras. Coomassie blue protein staining shows the amount of each GST protein used in the assay. The arrows point to the Ras proteins. (D) Tyrosine-phosphorylated R-Ras lacks the ability to bind to Raf-1. Extracts of cells transfected with Myc-tagged R-Ras and EphB2 were incubated with the immobilized GST-Ras binding domain of Raf-1 (GST-RBD). Cell extracts and the supernatant of the GST-RBD incubation were immunoprecipitated with anti-Myc antibody. Immunoprecipitates (lanes 1 and 2) and proteins bound to GST-RBD (lane 3) were analyzed by immunoblotting as indicated. The large amount of GST-RBD in lane 3 interfered with the migration of the R-Ras band, making it appear of lower molecular weight than in the other lanes.
Figure 3
Figure 3
Tyrosine 66 of R-Ras is phosphorylated by EphB2 and is important for binding to Raf-1 but not for association with EphB2. (A) EphB2 phosphorylates R-Ras but not R-RasY66E in vitro. In vitro kinase reactions were carried out as described in the legend of Fig. 2. The faint phosphorylated band in the GST-R-RasY66E (YE) and GST lanes presumably represents phosphorylated IgG heavy chain. The amount of GST-R-Ras and GST-R-RasY66E proteins used for the assay is shown by Coomassie blue staining (Lower). (B) R-Ras phosphorylation in EphB2-transfected cells is Y66 dependent. EphB2 was cotransfected with Myc-tagged R-Ras or R-Ras variants into 293T cells. Equal amounts of cell lysates were immunoprecipitated with anti-Myc antibody. and the immunoprecipitates were probed by immunoblotting as indicated. R-RasY66F protein (YF), and to a lesser extent R-RasY66E (YE), migrated faster than the wild-type R-Ras (Wt), consistent with a lower level of phosphorylation.
Figure 4
Figure 4
Matrix-assisted laser desorption ionization (MALDI) mass spectra of R-Ras phosphopeptides. Tyrosine-phosphorylated peptides resulting from digestion with endoproteinase Asp-N alone (A) or endoproteinase Asp-N followed by endoproteinase Glu-C (B) were isolated from the peptide mixtures by using anti-phosphotyrosine antibodies conjugated to agarose and analyzed directly by MALDI mass spectrometry. Peaks representing peptides phosphorylated at Y66 are indicated by their respective masses and sequences. Peaks marked B are from background ions detected in control samples and unmarked peaks are unidentified. The peptide at m/z 1836.16 (Ox) has a mass consistent with its being an oxidized form of that at m/z 1819.21.
Figure 5
Figure 5
Association of EphB2 with R-Ras in cells. (A) EphB2 or EphB2K662R was cotransfected with wild-type or mutated Myc-tagged R-Ras, or pcDNA3. Equal amounts of cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were probed by immunoblotting with the antibodies indicated. (B) Raf-1 binding by R-RasY66 mutants. Extracts of cells transfected with wild-type R-Ras, R-RasY66F, and R-RasY66E were incubated with the immobilized GST fusion protein of the Ras-binding domain of Raf-1 (GST-RBD) or GST as a control. Bound proteins and cell extracts were analyzed by immunoblotting with anti-Myc antibody. The amounts of GST-RBD and GST were examined by Coomassie blue staining.
Figure 6
Figure 6
Phosphorylation of R-Ras mediates the effects of EphB2 on cell adhesion. (A) Effects of R-RasY66 mutants on cell adhesion. The 293T cells were transfected with various R-Ras vectors, together with EphB2, and plated on surfaces coated with fibronectin, collagen I, or BSA as a control. (B) R-Ras38V Y66F suppresses the effect of EphB2 on cell adhesion. The constitutively activated R-Ras38V and its Y66 mutants were cotransfected with EphB2 in 293T cells, and cell attachment was quantitated.
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