Dentification of GIYWHHY as a novel peptide substrate for human p60^c-src protein tyrosine kinase

We have recently determined that -Ile-Tyr-were the two critical residues as a peptide substrate for p60^c-src protein tyrosine kinase (Lou, Q. et al., Lett. Peptide Sci., 1995, 2, 289). Here, we report on the design and synthesis of a secondary 'one-bead, one-compound' combinatorial peptide library based on this dipeptide motif (XIYXXXX, where X = all 19 eukaryotic amino acids except for cysteine). This secondary library was screened for its ability to be phosphorylated by p60^c-src PTK using [γ³²P]ATP as a tracer. Five of the strongest [³²P]-labeled peptide-beads were identified and microsequenced: GIYWHHY, KIYDDYE, EIYEENG, EIYEEYE, and YIYEEED. A solid-phase phosphorylation assay was used to evaluate the structure-activity relationship of GIYWHHY. It was determined that Ile², Tyr³, His⁵, and His⁶ were crucial for its activity as a substrate.