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Identification of autophosphorylation sites of HER2/neu
R Hazan1, B Margolis, M Dombalagian
1Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406.
Abstract:
HER2 or c-erbB-2 is a putative growth factor receptor with sequence homology to the epidermal growth factor receptor. It is the human homologue of the rat protooncogene neu and may have an important role in human malignancies such as breast and ovarian cancers. Like other growth factor receptors, HER2 has intrinsic protein tyrosine kinase activity and undergoes autophosphorylation. Recently, we have demonstrated that, similar to the epidermal growth factor receptor, all autophosphorylation sites of HER2 are localized in the carboxyl terminus of this protein. In the present study, immunopurified HER2 was allowed to autophosphorylate, and tryptic phosphopeptides were generated. After purification of these phosphopeptides by high performance liquid chromatography, microsequencing was performed. Utilizing this approach, two autophosphorylation sites were unequivocally identified at Y1023 and Y1248. The sequences of two other tyrosine phosphorylated tryptic peptides were determined, but the exact site of autophosphorylation could not be determined because multiple tyrosines were located on each peptide. However, each of these peptides contains tyrosines that correspond to major autophosphorylation sites of the epidermal growth factor receptor, suggesting that, in addition to Y1023 and Y1248, Y1139 and Y1222 also serve as autophosphorylation sites of HER2.
Insights
Researchers identified key autophosphorylation sites (Y1023, Y1248) on the HER2 receptor, crucial for its role in cancers like breast and ovarian malignancies. Additional potential sites (Y1139, Y1222) were also suggested.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- HER2 (Human Epidermal growth factor Receptor 2), also known as c-erbB-2, is a growth factor receptor.
- It shares sequence homology with the epidermal growth factor receptor and is the human homologue of the rat protooncogene neu.
- HER2 plays a significant role in human malignancies, particularly breast and ovarian cancers.
Purpose of the Study:
- To identify the specific autophosphorylation sites on the HER2 protein.
- To further elucidate the molecular mechanisms of HER2 signaling in cancer.
Main Methods:
- Immunopurification of HER2 protein.
- Induction of HER2 autophosphorylation.
- Generation and purification of tryptic phosphopeptides using high-performance liquid chromatography (HPLC).
- Microsequencing of purified phosphopeptides.
Main Results:
- Two definitive autophosphorylation sites were identified at tyrosine residues Y1023 and Y1248.
- Two additional tyrosine phosphopeptides were sequenced, suggesting Y1139 and Y1222 as potential autophosphorylation sites.
- These findings indicate that HER2 autophosphorylation sites are located in the carboxyl terminus, similar to the epidermal growth factor receptor.
Conclusions:
- The study unequivocally identified Y1023 and Y1248 as HER2 autophosphorylation sites.
- Y1139 and Y1222 are proposed as additional autophosphorylation sites, contributing to HER2's function in cancer.
- Understanding these sites is critical for targeted cancer therapies.