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.
Chan
P M
PM
The 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.
Nestler
H P
HP
Miller
W T
WT
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007502:Iron Chelating Agents; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000821:virology; D013379:Substrate Specificity
D007502:Iron Chelating Agents; D019151:Peptide Library; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases