Waas W F, Lo H H, Dalby K N
Division of Medicinal Chemistry, University of Texas, Austin, Texas 78712, USA.
J Biol Chem. 2001 Feb 23;276(8):5676-84. doi: 10.1074/jbc.M008787200. Epub 2000 Nov 7.
The mitogen-activated protein kinases (MAPKs) are a family of enzymes conserved among eukaryotes that regulate cellular activities in response to numerous external signals. They are the terminal component of a three-kinase cascade that is evolutionarily conserved and whose arrangement appears to offer considerable flexibility in encompassing the diverse biological situations for which they are employed. Although multistep protein phosphorylation within mitogen-activated protein kinase (MAPK) cascades can dramatically influence the sensitivity of signal propagation, an investigation of the mechanism of multisite phosphorylation by a MAPK has not been reported. Here we report a kinetic examination of the phosphorylation of Thr-69 and Thr-71 of the glutathione S-transferase fusion protein of the trans-activation domain of activating transcription factor-2 (GST-ATF2-(1-115)) by p38 MAPKalpha (p38alpha) as a model system for the phosphorylation of ATF2 by p38alpha. Our experiments demonstrated that GST-ATF2-(1-115) is phosphorylated in a two-step distributive mechanism, where p38alpha dissociates from GST-ATF2-(1-115) after the initial phosphorylation of either Thr-69 or Thr-71. Whereas p38alpha showed similar specificity for Thr-71 and Thr-69 in the unphosphorylated protein, it displayed a marked difference in specificity toward the mono-phosphoisomers. Phosphorylation of Thr-71 had no significant effect on the rate of Thr-69 phosphorylation, but Thr-69 phosphorylation reduced the specificity, k(cat)/K(M), of p38alpha for Thr-71 by approximately 40-fold. Computer simulation of the mechanism suggests that the activation of ATF2 by p38alpha in vivo is essentially Michaelian and provides insight into how the kinetics of a two-step distributive mechanism can be adapted to modulate effectively the sensitivity of a signal transduction pathway. This work also suggests that whereas MAPKs utilize docking interactions to bind substrates, they can be weak and transient in nature, providing just enough binding energy to promote the phosphorylation of a specific substrate.
丝裂原活化蛋白激酶（MAPK）是一类在真核生物中保守的酶家族，可响应众多外部信号调节细胞活动。它们是三激酶级联反应的终端组分，该级联反应在进化上是保守的，其排列方式似乎在涵盖它们所应用的各种生物学情况方面具有相当大的灵活性。尽管丝裂原活化蛋白激酶（MAPK）级联反应中的多步蛋白质磷酸化可显著影响信号传播的敏感性，但尚未有关于MAPK多位点磷酸化机制的研究报道。在此，我们报道了以p38 MAPKα（p38α）磷酸化激活转录因子2（GST-ATF2-(1-115)）反式激活结构域的谷胱甘肽S-转移酶融合蛋白的苏氨酸-69和苏氨酸-71为模型系统，对p38α磷酸化ATF2的动力学研究。我们的实验表明，GST-ATF2-(1-115)以两步分布机制被磷酸化，其中在苏氨酸-69或苏氨酸-71初始磷酸化后，p38α从GST-ATF2-(1-115)解离。虽然p38α对未磷酸化蛋白中的苏氨酸-71和苏氨酸-69表现出相似的特异性，但它对单磷酸异构体的特异性存在显著差异。苏氨酸-71的磷酸化对苏氨酸-69的磷酸化速率没有显著影响，但苏氨酸-69的磷酸化使p38α对苏氨酸-71的特异性（kcat/KM）降低了约40倍。该机制的计算机模拟表明，p38α在体内对ATF2的激活本质上是米氏动力学的，并为两步分布机制的动力学如何有效调节信号转导途径的敏感性提供了见解。这项工作还表明，虽然MAPK利用对接相互作用结合底物，但它们本质上可能是弱的和短暂的，仅提供足够的结合能量来促进特定底物的磷酸化。
