Interactions and Dual Phosphorylation in MAP Kinase Cascades

MAP 激酶级联中的相互作用和双重磷酸化

• 批准号: 7224889
• 负责人: ELIZABETH J. GOLDSMITH
• 金额: $ 30.49万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7224889
• 关键词: Interactions; Dual; Phosphorylation; MAP; Kinase

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.

描述（由申请人提供）：MAP激酶模块是由MAPK、MAP 2K和MAP 3 K组成的三激酶酶开关。以前的研究主要集中在这些模块的组件的结构和它们的激活。在这里，我们将解决级联组件之间的相互作用的性质和MAP 2Ks催化的双磷酸化的化学，中央的MAP激酶级联的行动和保留的过程。在几个实验室中定义的MAP 2Ks和MAPKs之间的对接相互作用在低活性形式的MAPKs ERK 2和p38 α中进行了晶体学研究。这些数据表明，对接相互作用诱导的MAPK，30 A从对接槽的激活环的长程构象变化。由于在ERK 2和p38 α中观察到类似的偶联变化，因此构象变化可能具有功能意义，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是否从MAPK p38 α和ERK 2的活性形式获得相同的构象。活性ERK 2和p38 α的肽结合形式将用现有晶体解决。全长MEK 6-p38 α复合物的晶体将得到改进。最近已经证明了MAP 3 K和MAP 2 K之间的对接相互作用。MAP 3 K的结合位点在哪里？与MAP 2K-MAPK对接相互作用一样，MAP 3 K-MAP 2K相互作用是否涉及构象变化？我们已经结晶的MAP 3 K TAO 2将在MEK 6衍生肽的存在下结晶。这里解决的第二个问题是如何MAP 2Ks能够进行两种化学，丝氨酸/苏氨酸和酪氨酸磷酸化？激活环构象的变化是否也参与了这一过程？已经建立了通过MEK 6对p38 α的酪氨酸相对于苏氨酸磷酸化的定量测定，其将用于确定用突变和部分磷酸化的MEK建模的MEK 6上的两个磷酸化位点是否在酪氨酸相对于丝氨酸/苏氨酸磷酸化中具有独特的作用。已经合成了双底物抑制剂，ATP-pTyr-肽和ATP-pThr-肽模拟物（通过已发表的化学），其将用于与MAP 2K MEK 6结合的晶体学分析。