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 tandem SH2 (src homology 2) domains and a C-terminal catalytic domain. Glutathione S-transferase fusions of the SH2 domains alone and of a catalytically inactive full-length mutant were made, and binding assays were developed using the purified fusion proteins to directly determine what residues are involved in the recognition of binding targets by the SH2 domains. The binding kinetics of the SH2 domains to a phosphotyrosyl-containing peptide of the sequence surrounding Tyr1009 of the platelet-derived growth factor receptor (PDGFR) beta subunit [DTSSVL(pY)TAVQPN] were determined by surface plasmon resonance, confirming that this is a high-affinity binding ligand. Using various N- and C-terminal 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 indicated that in addition to the phosphotyrosine (pY), the critical residues for recognition and binding are at pY + 1 and pY + 3 as previously reported, 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. Peptides that bind to the SH2 domains in the binding assays were also found to stimulate the phosphatase activity of SH-PTP2.
Huyer
G
G
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Québec, Canada.
Li
Z M
ZM
Adam
M
M
Huckle
W R
WR
Ramachandran
C
C
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004789:Enzyme Activation; D066298:In Vitro Techniques; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011485:Protein Binding; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin; D017479:Receptors, Platelet-Derived Growth Factor; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases