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Involvement of pp60c-src with two major signaling pathways in human breast cancer
D K Luttrell1, A Lee, T J Lansing
1Department of Cell Biology, Glaxo Research Institute, Research Triangle Park, NC 27709.
Abstract:
The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.
Insights
The SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu in human breast cancer cells. This interaction suggests a role for SRC kinase in receptor tyrosine kinase signaling and potential contribution to cancer development.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncology
Background:
- Receptor tyrosine kinases (RTKs) are crucial for intracellular signaling, with their phosphotyrosine residues acting as binding sites for proteins with SRC homology 2 (SH2) domains.
- Overexpression or activation of pp60c-src kinase is implicated in various human tumors, including breast carcinomas.
- Understanding the interactions of SRC domains with target proteins is vital for deciphering cancer signaling pathways.
Purpose of the Study:
- To investigate the binding interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines.
- To determine if SRC kinase associates with activated receptor tyrosine kinases in breast cancer.
- To explore the potential role of SRC-RTK association in malignant transformation.
Main Methods:
- Affinity purification of tyrosine-phosphorylated proteins using glutathione S-transferase (GST) fusion proteins of human SRC SH2, SH3, or SH3/SH2 domains.
- Analysis of SRC SH2 domain binding to activated epidermal growth factor receptor (EGFR) and p185HER2/neu in human breast carcinoma cell lines.
- Investigation of endogenous pp60c-src association with tyrosine-phosphorylated EGFR.
- Blocking of SRC SH2-EGFR association using specific tyrosyl phosphopeptides.
Main Results:
- The SRC SH2 domain demonstrated binding to activated EGFR and p185HER2/neu in human breast carcinoma cell lines.
- SRC SH2 binding to EGFR was also observed in a non-tumorigenic cell line following hormone stimulation.
- Endogenous pp60c-src was found to associate tightly with tyrosine-phosphorylated EGFR.
- Specific tyrosyl phosphopeptides successfully blocked the association between SRC SH2 and EGFR.
Conclusions:
- The SRC SH2 domain plays a significant role in binding to activated receptor tyrosine kinases like EGFR and HER2/neu.
- The tight association between endogenous pp60c-src and tyrosine-phosphorylated EGFR suggests a functional link in cancer cells.
- These findings indicate that the association of pp60c-src with RTKs may be integral to signaling pathways mediating malignant transformation.