Shc Phosphotyrosine-Binding Domain Dominantly Interacts with Epidermal Growth Factor Receptors and Mediates Ras Activation in Intact Cells

Abstract

The adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.

Graphene Enhances Cellular Proliferation through Activating the Epidermal Growth Factor Receptor

“…As reported, the association of ShC to EGFR may lead to its tyrosine phosphorylation and it is the main step in EGF-dependent induction of the MAPK pathway. 40 Thus, the phosphorylation of ShC in this result indicated the induction of the MAPK pathway upon treatment of graphene.…”

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

“…7B), which employed bacterially expressed GST fusion protein containing the PTB domain of Shc, indicate that the age-related difference in hepatocytes is in the activated EGF receptor protein. The PTB domain recognizes the tyrosine phosphoryl- ation at position 1148 of the EGF receptor (27,32,57) and position 1173 (32,57). We also observed a similar age-related decrease in the association of the EGF receptor with the SH2 domain of the Shc protein, which recognizes tyrosine 1173, although the magnitude of the association was not as great as with the Shc-PTB domain (data not shown).…”

“…The amount of decreased association between the proteins ranged from 2-to 3-fold in three independent experiments. Tyrosine 1148 has been identified to be a recognition site for binding of the Shc PTB domain (27,32), and tyrosine 1173 has been reported to be recognized by the Shc-SH2 domain (26,32) as well as the PTB domain (32). Therefore, it is possible that the EGF receptor may be less phosphorylated specifically at tyrosine 1148 and/or 1173 with age.…”

EGFR and NF-κB: partners in cancer

“…Activated RAS binds to RAF, and this interaction leads to mitogen-activated protein kinase kinase 1 (MEK1) followed by extracellular signalregulated kinases 1/2 (ERK1/2) phosphorylations [5,6]. RAS activation also relies on the recruitment of the SRC homology domain-containing adaptor protein C (SHC) to phosphorylated EGFR [7]. The p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K), the kinase SRC, and protein tyrosine phosphatases such as PTP1B, SHP1, and SHP2 also associate with distinct phosphorylated EGFR residues ( Figure 1) [8].…”