DIRECT DETERMINATION OF THE SEQUENCE RECOGNITION REQUIREMENTS OF THE SH2 DOMAINS OF SH-PTP2

SH-PTP2 is a widely-expressed protein tyrosine phosphatase with two ta ndem SH2 (src homology 2) domains and a C-terminal catalytic domain. G lutathione S-transferase fusions of the SH2 domains alone and of a cat alytically inactive full-length mutant were made, and binding assays w ere developed using the purified fusion proteins to directly determine what residues are involved in the recognition of binding targets by t he SH2 domains. The binding kinetics of the SH2 domains to a phosphoty rosyl-containing peptide of the sequence surrounding Tyr(1009) of the platelet-derived growth factor receptor (PDGFR) beta subunit [DTSSVL(p Y)TAVQPN] were determined by surface plasmon resonance, confirming tha t this is a high-affinity binding ligand. Using various N- and C-termi nal truncations of this peptide as competitors in the binding assays, the minimum peptide that served as a high-affinity binding ligand was found to be VL(pY)TAV. Systematic Ala substitutions of this peptide in dicated that in addition to the phosphotyrosine (pY), the critical res idues for recognition and binding are at pY+1 and pY+3 as previously r eported, and notably at pY-2 as well. Binding competition results with these and other PDGFR, IRP, and IRS-1 peptides suggested some general rules for sequence recognition by the SH2 domains of SH-PTP2. Peptide s that bind to the SH2 domains in the binding assays were also found t o stimulate the phosphatase activity of SH-PTP2.