Related Experiment Videos
Investigating the substrate specificity of the HER2/Neu tyrosine kinase using peptide libraries
P M Chan1, H P Nestler, W T Miller
1The Department of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.
Abstract:
The product of the HER2/Neu oncogene is a receptor tyrosine kinase that is amplified in 25-30% of human primary breast tumors. In this project, we have isolated the HER2/Neu kinase from Sf9 cells infected with a baculovirus expression vector. We probed the substrate specificity of the HER2/Neu kinase using two peptide libraries: (1) a soluble peptide library containing three degenerate positions N-terminal to tyrosine; and (2) a bead-supported combinatorial library possessing six degenerate positions at P-1, P-2, P-3, P+1, P+2, and P+3. We identified four novel substrate sequences for HER2/Neu from the two peptide libraries. We synthesized these peptides as individual sequences and measured steady-state kinetic properties for phosphorylation by HER2/Neu. One of the peptides, AAEEIYAARRG, is the best synthetic peptide substrate reported to date for HER2/Neu. All of the sequences bear a resemblance to sites of autophosphorylation on HER2/Neu and related epidermal growth factor (EGF) receptor family tyrosine kinases.
Insights
Researchers identified novel substrates for the HER2/Neu tyrosine kinase, a key protein in breast cancer. One synthetic peptide, AAEEIYAARRG, is the best substrate found to date for HER2/Neu kinase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- HER2/Neu oncogene product is a receptor tyrosine kinase amplified in 25-30% of human breast tumors.
- Understanding HER2/Neu substrate specificity is crucial for targeting this oncogene in cancer therapy.
Purpose of the Study:
- To isolate and characterize the HER2/Neu kinase.
- To identify novel substrate sequences for HER2/Neu kinase activity.
- To determine the kinetic properties of identified substrates.
Main Methods:
- HER2/Neu kinase isolated from Sf9 cells using a baculovirus expression vector.
- Substrate specificity probed using soluble and bead-supported peptide libraries.
- Kinetic properties of synthesized peptides measured for HER2/Neu phosphorylation.
Main Results:
- Four novel substrate sequences for HER2/Neu were identified.
- One peptide, AAEEIYAARRG, demonstrated superior substrate activity for HER2/Neu.
- Identified sequences resemble autophosphorylation sites on HER2/Neu and EGF receptor family kinases.
Conclusions:
- Novel substrates for HER2/Neu kinase have been identified.
- The peptide AAEEIYAARRG represents the best synthetic substrate for HER2/Neu reported.
- Findings contribute to understanding HER2/Neu kinase function and potential therapeutic targets.