The EphA8 receptor regulates integrin activity through p110gamma phosphatidylinositol-3 kinase in a tyrosine kinase activity-independent manner.
Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via alpha(5)beta(1)- or beta(3) integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110gamma isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110gamma. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110gamma mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110gamma PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion.
Gu
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Institute of Environment and Life Science, Hallym University, Chuncheon 200-702, Korea.
Park
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D016475:3T3 Cells; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D036386:Ephrin-A5; D005091:Exons; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D036143:Receptor, EphA8; D017469:Receptors, Fibronectin / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D015703:Antigens, CD; D036386:Ephrin-A5; D005353:Fibronectins; D039661:Integrin beta3; D008024:Ligands; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D017469:Receptors, Fibronectin; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D036143:Receptor, EphA8