Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells.
Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.
Holland
S J
SJ
Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Gale
N W
NW
Gish
G D
GD
Roth
R A
RA
Songyang
Z
Z
Cantley
L C
LC
Henkemeyer
M
M
Yancopoulos
G D
GD
Pawson
T
T
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins; D036387:Ephrin-B1; D020690:GTPase-Activating Proteins; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D015513:Oncogene Proteins / Q000378:metabolism / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D016601:RNA-Binding Proteins; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D036183:Receptor, EphB2; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C489561:Dok1 protein, mouse; D036387:Ephrin-B1; C074561:GAP-associated protein p62; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D010649:Phenylalanine; D020794:Receptor Protein-Tyrosine Kinases; D036183:Receptor, EphB2