Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells.
A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr766, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by "far-Western" analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway.
Klint
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P
Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Kanda
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Claesson-Welsh
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L
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020664:Son of Sevenless Proteins; D014443:Tyrosine / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495950:Grb2 protein, rat; D008565:Membrane Proteins; D011506:Proteins; D017468:Receptors, Fibroblast Growth Factor; D020664:Son of Sevenless Proteins; D016222:Fibroblast Growth Factor 2; D014443:Tyrosine; C496348:FGFR1 protein, human; C496350:Fgfr1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D018631:ras Proteins