Details

Autor(en) / Beteiligte

• Ladbury, John E.
• Lemmon, Mark A.
• Zhou, Min
• Green, Jeremy
• Botfield, Martyn C.
• Schlessinger, Joseph

Titel

Measurement of the Binding of Tyrosyl Phosphopeptides to SH2 Domains: A Reappraisal

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 1995-04, Vol.92 (8), p.3199-3203

Ort / Verlag

United States: National Academy of Sciences of the United States of America

Erscheinungsjahr

1995

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 Kdof 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kdof 4 μM. 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.