Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail.
Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail.
复制标题
Csk 催化 Src 尾酪氨酸磷酸化的分子决定因素。
DOI：
10.1021/bi002342n
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Philip A. Cole
中科院分区：
文献类型：
--
作者：
Dongxia Wang;Xin;Philip A. Cole
Src中一个关键的尾部酪氨酸残基的磷酸化调节其三维结构和蛋白酪氨酸激酶的活性。蛋白酪氨酸激酶CSK负责催化这一关键的Src酪氨酸残基的磷酸化，但对Src识别和催化的详细分子基础了解甚少。在这项研究中，我们使用纯化的重组CSK和Src蛋白和突变体来研究这种磷酸化事件。结果表明，CSK催化损伤的Src(Dsrc)磷酸化的表观K(Cat)和K(M)值与CSK催化的Src家族成员Lck的磷酸化参数相似。对于快速磷酸化而言，短链蛋白C的SH3(Src同源3)和SH2(Src同源2)结构域是完全可有可无的，这表明短链蛋白的催化结构域和尾部足以使其作为底物的高效率。在所研究的8个Src尾部残基中，只有丙氨酸扫描突变所研究的完全保守的Glu(Y-3位)和Gln(Y-1位)导致DSRC底物效率大幅下降(10-40倍)。对Y-3Glu的要求是严格的，因为用Asp或Gln保守的替代并不比Ala好，而用ILE替代Y-1Gln是很容易容忍的。有趣的是，用来自多肽文库分析的7个氨基酸的共有序列整体替换尾部并不比野生型序列好。令人惊讶的是，虽然不是CSK底物，但DSRC Y527F蛋白促进了CSK催化的DSRC的磷酸化。这些结果和其他数据表明，Src的二聚化(或更高阶齐聚)对于CSK催化的高效的Src尾部的磷酸化是重要的。
Phosphorylation of a critical tail tyrosine residue in Src modulates its three-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of this key Src tyrosine residue, but the detailed molecular basis for Src recognition and catalysis is poorly understood. In this study, we investigate this phosphorylation event using purified recombinant Csk and Src proteins and mutants. It was shown that the apparent k(cat) and K(m) values for Csk phosphorylation of catalytically impaired Src (dSrc) are similar to the parameters for Csk-catalyzed phosphorylation of the Src family member Lck. The SH3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dispensable with respect to rapid phosphorylation, indicating that the catalytic domain and tail of dSrc are sufficient for the high efficiency of dSrc as a substrate. Of the eight Src tail residues examined, only the fully conserved Glu (Y-3 position) and Gln (Y-1 position) investigated by alanine scanning mutagenesis caused large reductions (10--40-fold) in dSrc substrate efficiency. The Y-3 Glu requirement was stringent as conservative replacements with Asp or Gln were no better than Ala whereas replacement of the Y-1 Gln with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide library analysis was no better than the wild-type sequence. Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed phosphorylation of dSrc. These results and other data suggest that Src dimerization (or higher order oligomerization) is important for high-efficiency Csk-catalyzed phosphorylation of the Src tail.