Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins
Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins
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DOI：
10.1074/jbc.271.24.14468
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发表时间：
1996-06-14
影响因子：
4.8
通讯作者：
Kurata, T
中科院分区：
文献类型：
--
作者：
Matsuda, M;Ota, S;Kurata, T
CRK是鸡v-Crk的人类同源物，鸡v-Crk是一种衔接蛋白。CRK的SH 2结构域结合几种酪氨酸磷酸化蛋白，包括表皮生长因子受体、p130（Cas）、She和桩蛋白。SH 3结构域进而结合135-145、160、180和220 kDa的胞质蛋白。我们筛选表达文库的远Western印迹，使用CRK SH 3作为探针，并确定了四个不同的蛋白质，包括C3 G，DOCK 180，EPS 15，和克隆ST 12的部分cDNA序列。CRK SH 3结合位点的保守序列为Pro（+3）-Pro（+2）-X（+1）-Leu（0）-Pro（-1）-X（-2）-Lys（-3）。CRK SH 3结构域与DOCK 180肽的相互作用用基于表面等离子体共振原理的光学生物传感器进行了检查。10（-7）数量级的低解离常数是由高缔合速率常数（k（asyndrome）= 3 × 10（4））和低解离速率常数（k（asyndrome）= 3 × 10（-3））引起的。除克隆ST 12外，所有CRK结合蛋白也与另一种衔接蛋白Grb 2结合。突变分析显示，ST 12的+1位的甘氨酸抑制与Grb 2的结合，同时保持与CRK SH 3的高亲和力结合。结果表明，+1位的氨基酸也有助于肽与Grb 2的SH 3结构域的高亲和力结合，但不与CRK的结合。
CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), She, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro(+3)-Pro(+2)-X(+1)-Leu(0)-Pro(-1)-X(-2)-Lys(-3). The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (k(assoc) = 3 x 10(4)) and low dissociation rate constant (k(assoc) = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.