title: Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.
abstract: Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.
authors.affiliation: Department of Pharmacology, New York University Medical Center, NY 10016, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Ladbury
authors.forename: M A
authors.initials: MA
authors.lastname: Lemmon
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Green
authors.forename: M C
authors.initials: MC
authors.lastname: Botfield
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D010446:Peptide Fragments; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017479:Receptors, Platelet-Derived Growth Factor; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D016392:Proto-Oncogene Proteins pp60(c-src)