Peptide-Based Inhibitors of Neu Tyrosine Kinase.

This project focuses on the product of the HER2Neu oncogene, a receptor tyrosine kinase that is amplified in 25-30 of human primary breast tumors. The goal of this project is to characterize the substrate specificity of the Neu tyrosine kinase using combinatorial peptide libraries. This report covers the first year of the project. We have isolated the Neu tyrosine kinase from two sources 1 from SKBR3 breast cancer cells 2 from Sf9 cells using a baculovirus expression vector. We report here an initial study of the enzymes substrate specificity. Neu, unlike nonreceptor tyrosine kinases, appears to require acidic amino acids N-terminal to the site of phosphorylation. We have also demonstrated the feasibility of using a novel solid-phase kinase assay to screen E. coli expression libraries for Neu substrates. However, the Neu protein that we isolated is very unstable, and we have had problems producing large quantities of Neu. Thus, we are currently working on two new expression systems. We will optimize Neu expression and purification, then move to the library screening procedures. The final phase of the project will be to use this information to generate inhibitors.