INTERACTION OF PHOSPHORYLATED FC-EPSILON-RI-GAMMA IMMUNOGLOBULIN RECEPTOR TYROSINE ACTIVATION MOTIF-BASED PEPTIDES WITH DUAL AND SINGLE SH2DOMAINS OF P72(SYK) - ASSESSMENT OF BINDING PARAMETERS AND REAL, TIMEBINDING-KINETICS
T. Chen et al., INTERACTION OF PHOSPHORYLATED FC-EPSILON-RI-GAMMA IMMUNOGLOBULIN RECEPTOR TYROSINE ACTIVATION MOTIF-BASED PEPTIDES WITH DUAL AND SINGLE SH2DOMAINS OF P72(SYK) - ASSESSMENT OF BINDING PARAMETERS AND REAL, TIMEBINDING-KINETICS, The Journal of biological chemistry, 271(41), 1996, pp. 25308-25315
To examine the characteristics of the interaction of the Fc epsilon RI
gamma ITAM with the SH2 domains of p72(syk), the binding of an I-125-
labeled dual phosphorylated Fc epsilon RI gamma ITEM-based peptide to
the p72(syk) SH2 domains was monitored utilizing a novel scintillation
proximity based assay. The K-d for this interaction, determined from
the saturation binding isotherm, was 1.4 nM. This high affinity bindin
g was reflected in the rapid rate of association for the peptide bindi
ng to the SH2 domains. Competition studies utilizing a soluble C-termi
nal SH2 domain knockout and N-terminal SH2 domain knockouts revealed t
hat both domains contribute cooperatively to the high affinity binding
. I-125-labeled dual phosphorylated peptide competed with the I-125-la
beled peptide for binding to the dual p72(syk) SH2 domains with an IC5
0 value of 4.8 nM. Monophosphorylated 24-mer Fc epsilon RI gamma ITAM
peptides, and phosphotyrosine also competed for binding, but with subs
tantially higher IC50 values. This, and other data discussed, suggest
that high affinity binding requires both tyrosine residues to be phosp
horylated and that the preferred binding orientation of the ITAM is su
ch that the N-terminal phosphotyrosine occupies the C-terminal SH2 dom
ain and the C-terminal phosphotyrosine occupies the N-terminal SH2 dom
ain.