EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118.
Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.
Vindis
Cécile
C
Division of Nephrology and Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland.
Teli
Thalia
T
Cerretti
Douglas P
DP
Turner
Christopher E
CE
Huynh-Do
Uyen
U
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002465:Cell Movement; D006224:Cricetinae; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D005799:Genes, Dominant; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D036225:Receptor, EphB1 / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
D003598:Cytoskeletal Proteins; D008024:Ligands; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D014443:Tyrosine; D036225:Receptor, EphB1