title: 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.
authors.affiliation: The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, California, CA 92037, USA.
authors.forename: A H
authors.initials: AH
authors.lastname: Zisch
authors.forename: C
authors.initials: C
authors.lastname: Pazzagli
authors.forename: A L
authors.initials: AL
authors.lastname: Freeman
authors.forename: M
authors.initials: M
authors.lastname: Schneller
authors.forename: M
authors.initials: M
authors.lastname: Hadman
authors.forename: J W
authors.initials: JW
authors.lastname: Smith
authors.forename: E
authors.initials: E
authors.lastname: Ruoslahti
authors.forename: E B
authors.initials: EB
authors.lastname: Pasquale
mesh_terms: D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D048429:Cell Size / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016296:Mutagenesis; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D036183:Receptor, EphB2; D014443:Tyrosine / Q000235:genetics / Q000502:physiology*; D018909:src Homology Domains / Q000235:genetics; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D020794:Receptor Protein-Tyrosine Kinases; D036183:Receptor, EphB2; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases