Related Experiment Videos
Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells
P Klint1, S Kanda, L Claesson-Welsh
1Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden.
Abstract:
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.
Insights
Fibroblast growth factor-2 (FGF-2) activates Ras signaling indirectly via Shc and a novel p89 protein, which binds Grb2 to mediate mitogenicity. This pathway is specific to FGF-2, highlighting distinct growth factor signaling mechanisms.
Area of Science:
- Cellular Biology
- Molecular Biology
- Signal Transduction
Background:
- Mitogenicity is often regulated by the Ras GTP-binding protein.
- Receptor tyrosine kinases link to Ras via the Grb2 protein, which contains an SH2 domain.
Purpose of the Study:
- To investigate the mechanism of fibroblast growth factor receptor-1 (FGFR-1) signaling in L6 myoblasts.
- To elucidate the role of the adaptor protein Shc and identify novel components in FGF-2-mediated Ras pathway activation.
Main Methods:
- Utilized L6 myoblasts expressing wild-type and mutant FGFR-1.
- Employed tyrosine phosphorylation assays, co-immunoprecipitation, and far-Western analysis.
- Investigated protein-protein interactions and subcellular localization.
Main Results:
- Activated FGFR-1 did not directly bind Grb2 but did so indirectly through tyrosine-phosphorylated Shc.
- A mutant FGFR-1 with altered Tyr766 showed reduced Shc phosphorylation but still supported mitogenic signaling.
- FGF-2 stimulation induced tyrosine phosphorylation of an 89-kDa protein (p89) that directly binds the Grb2 SH2 domain.
- p89 was localized to the membrane fraction, facilitating Grb2 and Sos recruitment.
Conclusions:
- FGF-2 signaling involves Shc and a novel membrane-associated p89 protein for Grb2 recruitment.
- This pathway represents a growth factor-specific mechanism for coupling to the Ras pathway.
- The findings provide insights into the specificity of growth factor signaling in cellular processes like mitogenicity.