EphB1-mediated Cell Migration Requires the Phosphorylation of Paxillin at Tyr-31/Tyr-118

Abstract

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 ⌬LD4) 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.The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are key regulators of several developmental processes, including cell migration, boundary formation, axonal guidance, synaptogenesis, and angiogenesis. Both receptor-and ligand-initiated signals mediate repulsion, adhesion, and de-adhesion mechanisms involved in the motility of adherent cells. After activation by their cognate ligands, Eph receptors are phosphorylated at specific tyrosine residues in the cytoplasmic domain, thus displaying new docking sites for known signaling molecules. Indeed, a variety of Src homology domain (SH2/SH3)-containing proteins have been identified as Eph receptor-binding partners, including the adaptor proteins Nck, Grb10, Grb7, SHEP-1, SLAP, the low molecular weight phosphotyrosine phosphatase LMW-PTP, the tyrosine kinases Crk, Fyn, and Src, the phosphatidylinositol-3 kinase, and the Ras GTPase-activating protein (reviewed in Ref. 1). Furthermore, PDZ-binding motifs located at the C terminus of the Eph receptors bind PDZ domain containing proteins such as AF6, Ryk (an RTK-interacting protein), Pick1, syntenin, and the two glutamate receptor-interacting proteins, Grip1 and Grip2 (2).Importantly, many of the proteins identified in the Eph signaling pathways have been implicated in the regulation of cell morphology, adhesion, and motility. Activation of Eph receptors and ephrins has been shown to affect cell attachment by means of integrin and focal adhesion protein-dependent mechanisms, but contradictory results have been described for the interplay between these systems. In previous works, we showed that EphB1 receptor regulates integrin-de...

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

“…Paxillin Y31 phosphorylation was shown to regulate the assembly and formation of cell-matrix adhesions, and p130Cas Y249 phosphorylation by Src provides a docking site for the Crk cytoskeleton adaptor protein (37), whereas Erk phosphorylation on T185/Y187 is necessary for its kinase activity. Importantly, the phosphorylation of these proteins at these specific amino acid residues are known to play important roles in modulation of the cytoskeleton and cell migration (17,18,30,(38)(39)(40). In contrast, depletion of endogenous PEAK1 using a specific siRNA (siPEAK1) decreased the phosphorylation of paxillin (Y31) and Erk under these conditions (Fig.…”

The EphB4 Receptor-tyrosine Kinase Promotes the Migration of Melanoma Cells through Rho-mediated Actin Cytoskeleton Reorganization

“…EphB receptors have been shown to promote cell migration through recruitment of Src, Shc, and components of the focal adhesion complex (26,49,53,54). The inhibiting or activating effects of EphB receptors on cell migration have also been attributed to their ability to phosphorylate and activate the cytoskeletal protein paxillin (54) or to inhibit the ability of small GTPase R-Ras to support integrin-mediated adhesion (25).…”

“…EphB receptors have been shown to promote cell migration through recruitment of Src, Shc, and components of the focal adhesion complex (26,49,53,54). The inhibiting or activating effects of EphB receptors on cell migration have also been attributed to their ability to phosphorylate and activate the cytoskeletal protein paxillin (54) or to inhibit the ability of small GTPase R-Ras to support integrin-mediated adhesion (25). Our data show that the effects of EphB4 on melanoma cell migration require its kinase activity because inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 with a single K647R mutation in its kinase domain resulted in slower cell migration and significantly altered cell morphology and actin cytoskeleton organization.…”

Paxillin: Adapting to Change

“…Furthermore, a recent study of ephrin B1-stimulated cell migration in a variety of cell types including renal vascular endothelial cells demonstrated a requirement for paxillin tyrosine Y31/118 phosphorylation and the LD4 motif. In this example, the LD4 motif was required for Nckmediated recruitment to the activated receptor (291). A model is emerging in which growth factor and integrinmediated phosphorylation of paxillin stimulates the formation of a paxillin-Crk complex at focal adhesions where Crk-DOCK180 can then locally activate Rac to produce lamellipodial extension and enhance migration (Figs.…”