MEASUREMENT OF THE BINDING OF TYROSYL PHOSPHOPEPTIDES TO SH2 DOMAINS - A REAPPRAISAL

被引:239
作者
LADBURY, JE
LEMMON, MA
ZHOU, M
GREEN, J
BOTFIELD, MC
SCHLESSINGER, J
机构
[1] NYU, MED CTR, DEPT PHARMACOL, NEW YORK, NY 10016 USA
[2] ARIAD PHARMACEUT INC, CAMBRIDGE 02139, ENGLAND
[3] ARIAD PHARMACEUT INC, CAMBRIDGE, MA 02139 USA
关键词
D O I
10.1073/pnas.92.8.3199
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 (K-d) 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 K-d of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a K-d of 4 mu M. 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.
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页码:3199 / 3203
页数:5
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