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成部分的结构和它们的激活。在这里，我们将讨论级联组分之间相互作用的性质和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持的核心过程。在几个实验室中定义的map2k和MAPKs之间的对接相互作用，在低活性形式的MAPKs ERK2和p38alpha中进行了晶体学研究。这些数据表明，对接相互作用在对接槽的MAPK， 30a激活回路中诱导了长距离构象变化。由于在ERK2和p38alpha中观察到类似的偶联变化，因此构象变化可能在功能上很重要，可能是为了使磷酸化位点可用于加工。这里要解决的第一个问题是，MAPKs p38alpha和ERK2的活性形式是否可以获得相同的构象。活性ERK2和p38 α的肽结合形式将用现有的晶体解决。全长mek6 -p38 α配合物的结晶将得到改善。MAP3Ks和MAP2Ks之间的对接相互作用最近得到了证实。MAP3K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？激活环的构象变化也参与了这个过程吗？MEK6对p38α的酪氨酸和苏氨酸磷酸化的定量分析已经建立，将用于确定MEK6上的两个磷酸化位点是否在酪氨酸和丝氨酸/苏氨酸磷酸化中具有独特的作用，这些磷酸化位点是用突变体和部分磷酸化的mek模拟的。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），将用于结合MAP2K MEK6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.