Ephrin-independent regulation of cell substrate adhesion by the EphB4 receptor.
Receptor tyrosine kinases of the Eph family become tyrosine phosphorylated and initiate signalling events upon binding of their ligands, the ephrins. Eph receptors such as EphA2 and EphB4 are highly expressed but poorly tyrosine phosphorylated in many types of cancer cells, suggesting a limited interaction with ephrin ligands. Nevertheless, decreasing the expression of these receptors affects the malignant properties of cancer cells, suggesting that Eph receptors may influence cancer cells independently of ephrin stimulation. Ligand-independent activities of Eph receptors in cancer, however, have not been demonstrated. By using siRNA (small interfering RNA) to downregulate EphB4 in MCF7 and MDA-MB-435 cancer cells, we found that EphB4 inhibits integrin-mediated cell substrate adhesion, spreading and migration, and reduces beta1-integrin protein levels. Low expression of the EphB4 preferred ligand, ephrin-B2, and minimal contact between cells in these assays suggest that cell contact-dependent stimulation of EphB4 by the transmembrane ephrin-B2 ligand does not play a role in these effects. Indeed, inhibitors of ephrin-B2 binding to endogenous EphB4 did not influence cell substrate adhesion. Increasing EphB4 expression by transient transfection inhibited cell substrate adhesion, and this effect was also independent of ephrin stimulation because it was not affected by single amino acid mutations in EphB4 that impair ephrin binding. The overexpressed EphB4 was tyrosine phosphorylated, and we found that EphB4 kinase activity is important for inhibition of integrin-mediated adhesion, although several EphB4 tyrosine phosphorylation sites are dispensable. These findings demonstrate that EphB4 can affect cancer cell behaviour in an ephrin-independent manner.
Noren
Nicole K
NK
The Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
Yang
Nai-Ying
NY
Silldorff
Morgan
M
Mutyala
Ravi
R
Pasquale
Elena B
EB
D002448:Cell Adhesion; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D036388:Ephrin-B2 / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D034741:RNA, Small Interfering; D036224:Receptor, EphB4 / Q000235:genetics / Q000378:metabolism*
D036388:Ephrin-B2; D034741:RNA, Small Interfering; D036224:Receptor, EphB4