Investigating the substrate specificity of the HER2/Neu tyrosine kinase using peptide libraries

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. (C) 2000 Elsevier Science Ireland Ltd.