Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity.
Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.
Tong
Jiefei
J
Programe in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5.
Elowe
Sabine
S
Nash
Piers
P
Pawson
Tony
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005910:Glioma; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma; D010446:Peptide Fragments; D010766:Phosphorylation; D036183:Receptor, EphB2 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D020703:ras GTPase-Activating Proteins / Q000378:metabolism; D018909:src Homology Domains
D010446:Peptide Fragments; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D036183:Receptor, EphB2