Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.
Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.
Nishimura
R
R
Department of Pharmacology, New York University Medical Center, New York 10016.
Li
W
W
Kashishian
A
A
Mondino
A
A
Zhou
M
M
Cooper
J
J
Schlessinger
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000077214:Becaplermin; D001665:Binding Sites; D002478:Cells, Cultured; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D046911:Macromolecular Substances; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000077214:Becaplermin; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor