Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.
Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.
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DOI：
10.1016/j.jmb.2005.05.042
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发表时间：
2005-08
影响因子：
5.6
通讯作者：
L. Wong;Scot A Lieser;O. Miyashita;Meghan T. Miller;K. Taskén;J. Onuchic;J. Adams;Virgil L. Woods;P. Jennings
中科院分区：
文献类型：
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作者：
L. Wong;Scot A Lieser;O. Miyashita;Meghan T. Miller;K. Taskén;J. Onuchic;J. Adams;Virgil L. Woods;P. Jennings
C-末端的Src激酶(CSK)可以磷酸化和下调Src家族的酪氨酸激酶。CSK结合蛋白(CBP)将CSK定位在其质膜底物附近，并增加了该激酶的比活性。为了研究这种长程催化作用，我们研究了CBP衍生的高亲和力磷酸肽对Src的磷酸化和CSK构象的影响。这种多肽与SH2结构域紧密结合，并通过增加CSK活性部位的表观磷酸转移速率来增强Src的识别(降低Km)，这是在快速猝灭流动实验中检测到的现象。以前的研究表明，CSK活性的调节与酶的构象变化有关，这种变化可以用氢-氚交换方法来探测。我们发现CBP多肽影响氘掺入其结合伙伴(SH2结构域)、SH2-激酶连接子和激活域的几个序列，包括活性部位的富含甘氨酸的环。这些发现以及正常模式分析的计算数据表明，SH2结构域相对于激活域的小叶以悬臂式方式移动，排列了催化活性部位。CBP衍生的小肽与CSK的SH2结构域的结合改变了这些运动，增强了Src的识别。
The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers Km) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen–deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.