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A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase
W Li1, R Nishimura, A Kashishian
1Department of Pharmacology, New York University Medical Center, New York 10016.
Abstract:
Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.
Insights
Platelet-derived growth factor (PDGF) receptor signaling differs from EGF receptor signaling. The protein tyrosine phosphatase Syp acts as an adapter, linking PDGF receptor to GRB2-Sos complex for Ras pathway activation.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Protein-protein interactions
Background:
- Growth factor receptors, like EGF and PDGF receptors, initiate intracellular signaling cascades upon ligand binding.
- Autophosphorylated receptors serve as docking sites for signaling molecules with Src homology 2 domains.
- Epidermal growth factor (EGF) receptor directly recruits GRB2-Sos complex, activating the Ras pathway.
Purpose of the Study:
- To investigate if the platelet-derived growth factor (PDGF) receptor recruits the GRB2-Sos complex.
- To elucidate the mechanism of GRB2-Sos interaction with the PDGF receptor.
Main Methods:
- Utilized PDGF-beta receptor mutants to identify critical tyrosine phosphorylation sites for GRB2 binding.
- Employed co-immunoprecipitation assays to detect protein complex formation in PDGF-stimulated cells.
- Performed in vitro binding assays with purified proteins to confirm direct interactions.
Main Results:
- GRB2 does not directly bind to the PDGF receptor; instead, it binds to a 70-kDa protein.
- Tyrosine-1009 of the PDGF receptor is essential for GRB2 complex formation.
- The 70-kDa protein was identified as the protein tyrosine phosphatase Syp, which binds to the PDGF receptor via Tyr-1009 and directly to GRB2.
- GRB2 and Sos co-immunoprecipitated with Syp in PDGF-stimulated cells.
Conclusions:
- The GRB2-Sos complex interacts with the PDGF receptor through the adapter protein Syp.
- Syp acts as a crucial link between the PDGF receptor and the GRB2-Sos complex, facilitating Ras pathway activation.
- This mechanism highlights differential recruitment strategies of GRB2-Sos by various growth factor receptors.