Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.
The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.
Ludwig Institute for Cancer Research, London, United Kingdom.
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Panayotou
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Gish
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Truong
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Dhand
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Fry
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Hiles
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Pawson
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Waterfield
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002417:Cattle; D016391:Genes, src; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D017479:Receptors, Platelet-Derived Growth Factor