Bu J Y, Shaw A S, Chan A C
Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Proc Natl Acad Sci U S A. 1995 May 23;92(11):5106-10. doi: 10.1073/pnas.92.11.5106.
Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.
17个氨基酸的免疫受体酪氨酸激活基序（ITAM）的酪氨酸磷酸化在T细胞抗原受体（TCR）的每个信号亚基中保守，介导ZAP-70和syk蛋白酪氨酸激酶（PTK）募集到活化的受体。通过实时测量动力学参数，研究了该家族PTK的两个串联排列的Src同源2（SH2）结构域与每个含磷酸酪氨酸的ITAM之间的相互作用。测定了ZAP-70和syk SH2结构域与CD3ε ITAM的结合速率和平衡结合常数。两种PTK的结合常数ka和Kd值分别为5×10⁶ M⁻¹·s⁻¹和约25 nM。与其他TCR ITAM（ζ1、ζ2、γ和δ ITAM）的结合情况相当，尽管ζ3 ITAM的结合能力约低2.5倍。对ZAP-70单个功能性SH2结构域亲和力的研究为双SH2结构域结合的协同性质提供了证据。任一单个SH2结构域的突变使Kd降低>100倍。最后，发现ITAM与syk结合的关键特征与ZAP-70结合所需的特征相似。这些数据为多亚基TCR与下游效应分子相互作用的机制提供了见解。
