INHIBITION OF BINDING OF PHOSPHOLIPASE-C-GAMMA-1 SH2 DOMAINS TO PHOSPHORYLATED EPIDERMAL GROWTH-FACTOR RECEPTOR BY PHOSPHORYLATED PEPTIDES

A series of tyrosine-containing peptides 1-12: [GRAPHICS] (six pairs w ith and without the tyrosine phosphorylated) has been synthesized. The peptides were derived from tyrosine autophosphorylation sites in the epidermal growth factor receptor (EGFR): Tyr 992, 1068, 1148 and 1173. Peptide 1, derived from the Tyr 992 site, inhibited binding of a S-35 -labelled fusion protein containing both of the SH2 domains from PLC71 to the phosphorylated EGFR with an IC50 Of 8 mum. All of the phosphor ylated peptides except 11 (1, 3, 5, 7 and 9) inhibited this binding to some degree (20-55%) at 10 muM. The nonphosphorylated peptides were i nactive in this assay. The nonphosphorylated peptides 2, 4, 6, 8, 10 a nd 12 were obtained by standard solid-phase synthetic methodologies us ing both Boc/benzyl and Fmoc/tert-butyl strategies. The phosphorylated peptides 1, 3, 5, 7, 9 and 11 were similarly obtained using a Fmoc/te rt-butyl strategy incorporating unprotected N(alpha)-Fmoc-Tyr, followe d by phosphitylation and oxidation of the tyrosine in the resin-bound peptide. In addition, -Glu-Phe992(4-CH2PO3H2)-Leu-Ile-Pro-Gln-Gln-Gly- OH (15), an analog of 1 incorporating an enzymatically stable phosphot yrosine mimic, 4-phosphonomethyl-L-phenylalanine, was synthesized and found to be inactive. (C) Munksgaard 1993.