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

Isobel D. Lawrenson^1,*, Sabine H. Wimmer-Kleikamp^1,*, Peter Lock¹, Simone M. Schoenwaelder², Michelle Down³, Andrew W. Boyd³, Paul F. Alewood⁴ and Martin Lackmann^1,

¹ Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia
² The Australian Centre for Blood Diseases, Monash Department of Medicine, Box Hill Hospital, Victoria 3128, Australia
³ Queensland Institute for Medical Research, 300 Herston Rd, Herston, Queensland 4029, Australia
⁴ Centre for Drug Design and Development, IMB, St Lucia, Queensland 4068, Australia

Author for correspondence (e-mail: martin.lackmann{at}ludwig.edu.au )

Accepted 3 December 2001

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.

Key words: Cell adhesion, Actin cytoskeleton, Eph receptor protein-tyrosine kinases, Melanoma, Metastasis

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