Interactions and Dual Phosphorylation in MAP Kinase Cascades

描述（由申请人提供）：MAP激酶模块是由MAPK、MAP2K和MAP3K组成的三激酶酶开关。先前的研究主要集中在这些模块的组件结构及其激活上。在这里，我们将探讨级联成分之间相互作用的本质以及 MAP2K 催化的双磷酸化的化学性质，这是 MAP 激酶级联的作用和保留的核心过程。多个实验室定义了 MAP2K 和 MAPK 之间的对接相互作用，并在低活性形式的 MAPK ERK2 和 p38α 中进行了晶体学研究。这些数据表明，对接相互作用诱导 MAPK 激活环（距对接槽 30 A）的长程构象变化。由于在 ERK2 和 p38α 中观察到类似的耦合变化，因此构象变化可能具有重要的功能，可能使磷酸化位点可用于加工。这里要解决的第一个问题是 MAPK p38alpha 和 ERK2 的活性形式是否具有相同的构象。活性 ERK2 和 p38alpha 的肽结合形式将用现有晶体来解决。全长 MEK6-p38alpha 复合物的晶体将得到改进。 MAP3K 和 MAP2K 之间的对接相互作用最近已得到证实。 MAP3K 中的结合位点在哪里？与 MAP2K-MAPK 对接相互作用一样，MAP3K-MAP2K 相互作用是否涉及构象变化？我们已经结晶的 MAP3K TAO2 将在 MEK6 衍生肽存在的情况下结晶。这里要解决的第二个问题是 MAP2K 如何能够进行两种化学反应：丝氨酸/苏氨酸和酪氨酸磷酸化？该过程是否也涉及激活环构象变化？已经建立了 MEK6 对 p38α 的酪氨酸与苏氨酸磷酸化的定量测定，该测定将用于确定以突变和部分磷酸化 MEK 为模型的 MEK6 上的两个磷酸化位点是否在酪氨酸与丝氨酸/苏氨酸磷酸化中具有独特的作用。双底物抑制剂、ATP-pTyr-肽和 ATP-pThr-肽模拟物已经合成（通过已发表的化学方法），将用于与 MAP2K MEK6 结合的晶体学分析。

DESCRIPTION (provided by applicant): MAP kinase modules are three-kinase enzyme switches comprised of a MAPK, a MAP2K, and a MAP3K. Prior studies have focused primarily on the structures of components of these modules and their activation. Here we will address the nature of the interactions between cascade components and the chemistry of the double phosphorylations catalyzed by MAP2Ks, processes central to the action and retention of MAP kinase cascades. Docking interactions between MAP2Ks and MAPKs, defined in several labs, were studied crystallographically in the low activity forms of MAPKs ERK2 and p38alpha. These data reveal that the docking interactions induce long range conformational changes in the activation loop of the MAPK, 30 A from the docking groove. Since similar coupled changes were observed in both ERK2 and p38alpha, the conformational changes are likely functionally significant, possibly to make the phosphorylation sites available for processing. The first question to be addressed here is whether the same conformation is accessed from the active forms of the MAPKs p38alpha and ERK2. Peptide bound forms of active ERK2 and p38alpha will be solved with existing crystals. Crystals of full-length MEK6-p38alpha complexes will be improved. Docking interactions between MAP3Ks and MAP2Ks have been demonstrated recently. Where is the binding site in the MAP3K? As with the MAP2K- MAPK docking interaction, does the MAP3K-MAP2K interaction involve conformational changes? The MAP3K TAO2, which we have crystallized, will be crystallized in the presence of MEK6-derived peptides. The second question addressed here is how are MAP2Ks capable of carrying out two chemistries, serine/threonine and tyrosine phosphorylation? Are activation loop conformational changes involved in this process as well? A quantitative assay for tyrosine versus threonine phosphorylation of p38alpha by MEK6 has been established that will be used to determine whether the two phosphorylation sites on MEK6, modeled with mutant and partially phosphorylated MEKs, have unique roles in tyrosine versus serine/threonine phosphorylation. Bisubstrate inhibitors, ATP-pTyr- peptide and ATP-pThr-peptide mimetics have been synthesized (by published chemistry) that will be used for crystallographic analysis bound to the MAP2K MEK6.