Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses

Abstract

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

SHEP1 Function in Cell Migration Is Impaired by a Single Amino Acid Mutation That Disrupts Association with the Scaffolding Protein Cas but Not with Ras GTPases

“…The EGF receptor can also promote neurite extension through a pathway involving Src, the Cas family protein Sin, and Crk (52). In contrast, the EphB2 receptor promotes repulsive migratory responses and neurite retraction (34,53,54) and, as we found, causes dissociation of SHEP1 from Cas. An intriguing possibility is that differential regulation of the SHEP1-Cas complex may contrib- FIG.…”

“…evaluate the importance of tyrosine 635 in the association of SHEP1 with Cas, we used site-directed mutagenesis to replace this tyrosine with glutamic acid (Y635E mutation), which introduces a negative charge like that of a phosphate group (34). We also generated a SHEP1 Y635F mutant, in which the phenylalanine cannot be phosphorylated.…”

The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization

“…Eph receptor signaling has been proposed to in¯uence the movement of neuronal growth cones and the segregation of subpopulations of cells, including endothelial cells (Adams et al, 1999;Flanagan and Vanderhaeghen, 1998;Wang et al, 1998). Eph receptor signaling in tumors may promote the migration of endothelial cells and their assembly into capillary structures by regulating cytoskeletal plasticity, matrix attachment, and/or intercellular adhesion (Huynh-Do et al, 1999;Pandey et al, 1995;Stein et al, 1998;Zisch et al, 2000). Accordingly, in vitro ephrin-A1 acts as a chemoattractant for endothelial cells (Pandey et al, 1995), consistent with a stimualtory role in vascular remodeling.…”

In Vivo Tyrosine Phosphorylation Sites of Activated Ephrin-B1 and EphB2 from Neural Tissue

“…The juxtamembrane tyrosines 605 and 611, in particular, appear to be prominent phosphorylation sites in both retinal tissue and cultured cells. Phosphorylation of these tyrosines regulates both binding of many SH2 domain-containing proteins and kinase activity (39,40). Thus, tyrosines 605 and 611 could play a particularly important functional role in EphB2 signaling in vivo.…”