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

Abstract

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.

Cancer Somatic Mutations Disrupt Functions of the EphA3 Receptor Tyrosine Kinase through Multiple Mechanisms

“…Phosphorylation of cytoplasmic tyrosine residues is a hallmark of Eph receptor activation and downstream signaling . Like other Eph receptors, wild-type EphA3 is constitutively tyrosine phosphorylated when overexpressed by transient transfection in HEK 293 cells (Figure A). − Interestingly, we found that four of the eight mutations examined in the EphA3 kinase domain [R728L, K761N, G766E, and D806N (Figure )] essentially abolish EphA3 tyrosine phosphorylation in HEK 293 cells (Figure A and Table ). This effect is similar to that of the K653R control mutation, which disrupts ATP binding .…”

“…The EphA3 receptor is widely expressed during embryonic development, with the highest levels occurring in the nervous system and heart. − EphA3 is expressed at lower levels in some adult tissues, such as the brain, lung, bladder, prostate, and colon. , The best characterized roles of EphA3 thus far are in the epithelial–mesenchymal transition in the developing heart , and in axon guidance in the developing nervous system, where this receptor plays a repulsive role that causes axons to avoid regions of strong ephrin ligand expression. − EphA3-dependent retraction of cellular processes has also been reported in non-neuronal cells . However, there is only limited information about the signal transduction pathways triggered by EphA3.…”

“…However, there is only limited information about the signal transduction pathways triggered by EphA3. Binding of the Nck and Crk adaptors and activation of RhoA have been implicated in the cellular repulsive effects. , Such repulsive signaling pathways could prevent the expansion of normal or tumor cells into ephrin-positive surrounding tissues, perhaps through a mechanism like contact inhibition of locomotion. − This repulsion may be overcome in tumor cells in which EphA3 is mutated, allowing tumor expansion. However, the role of EphA3 in cancer is not well established, and further studies are needed to more comprehensively elucidate its signaling pathways and biological effects in normal as well as cancer cells.…”

“…56−58 EphA3-dependent retraction of cellular processes has also been reported in nonneuronal cells. 28 However, there is only limited information about the signal transduction pathways triggered by EphA3. Binding of the Nck and Crk adaptors and activation of RhoA have been implicated in the cellular repulsive effects.…”

Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells

“…Activated RAS (RAS-GTP) regulates multiple cellular functions through effectors including Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide-dissociation stimulator (RALGDS) 38 – 41 . EFNA5 is the Eph receptor on the cell surface, a family of receptor tyrosine kinases, which plays an important role in migration and adhesion during the development of neurons, blood vessels and epithelium 42 , 43 . FGF3 and FGF8 are involved in the regulation of embryonic development, proliferation, differentiation and migration, and are required for normal brain, eyes, ears and limb development during embryogenesis 44 – 48 .…”