Recruitment of Eph receptors into signaling clusters does not require ephrin contact

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions

Ephrin-A5 induces rounding, blebbing and deadhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response

Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B

Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells

Signaling by Eph receptors and cell-surface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact–dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5–induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors. Copyright © 2008 by American Society of Hematolog

Cytoplasmic Relaxation of Active Eph Controls Ephrin Shedding by ADAM10

Release of cell surface-bound ligands by A-Disintegrin-And-Metalloprotease (ADAM) transmembrane metalloproteases is essential for signalling by cytokine, cell adhesion, and tyrosine kinase receptors. For Eph receptor ligands, it provides the switch between cell-cell adhesion and repulsion. Ligand shedding is tightly controlled by intrinsic tyrosine kinase activity, which for Eph receptors relies on the release of an inhibitory interaction of the cytoplasmic juxtamembrane segment with the kinase domain. However, a mechanism linking kinase and sheddase activities had remained elusive. We demonstrate that it is a membrane-proximal localisation of the latent kinase domain that prevents ephrin ligand shedding in trans. Fluorescence lifetime imaging microscopy and electron tomography reveal that activation extends the Eph receptor tyrosine kinase intracellular domain away from the cell membrane into a conformation that facilitates productive association with ADAM10. Accordingly, EphA3 mutants with constitutively-released kinase domains efficiently support shedding, even when their kinase is disabled. Our data suggest that this phosphorylation-activated conformational switch of EphA3 directly controls ADAM-mediated shedding.National Health and Medical Research Council (grants 234707 and 384242