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

基本信息

批准号：
8000154
负责人：
ELIZABETH J. GOLDSMITH
金额：
$ 6万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8000154
关键词：
Active Sites Address Binding Binding Sites Biochemical Biological Assay Catalysis Cell Nucleus Cells Chemistry Cocrystallography Complex Coupled Data Docking Enzymes Event Hand Individual Length MAP Kinase Gene MAP Kinase Kinase Kinase MAP Kinase Modules MAP2K6 gene MAPK1 gene MAPK14 gene MEKs Measures Mitogen-Activated Protein Kinase Kinases Mitogen-Activated Protein Kinases Modeling Molecular Conformation Nature Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphothreonine Phosphotransferases Phosphotyrosine Process Protein Kinase Publishing Reaction Reagent Research Personnel Role Serine Serine/Threonine Phosphorylation Shapes Signal Transduction Site Specificity Structure Tertiary Protein Structure Threonine Time Tyrosine Tyrosine Phosphorylation Work extracellular improved inhibitor/antagonist member mimetics mutant programs research study

项目摘要

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，MAP2K和MAP3K组成的三激酶开关。先前的研究主要集中在这些模块的组成部分及其激活上。在这里，我们将解决级联成分之间相互作用的性质与MAP2K催化的双磷酸化的化学之间的相互作用，这是MAP激酶级联反应的作用和保留的核心。在几个实验室中定义的MAP2K和MAPKS之间的对接相互作用，以MAPKS ERK2和P38Alpha的低活性形式进行了结晶研究。这些数据表明，对接相互作用会引起MAPK激活环的长距离构象变化，从扩展坞凹槽30 a。由于在ERK2和p38alpha中都观察到了类似的耦合变化，因此构象变化可能在功能上很重要，可能使磷酸化位点可用于处理。这里要解决的第一个问题是是否从MAPKS p38alpha和Erk2的活动形式访问了相同的构象。活性ERK2和P38Alpha的肽结合形式将用现有晶体解决。全长MEK6-P38Alpha复合物的晶体将得到改善。最近已经证明了MAP3KS和MAP2K之间的对接相互作用。 MAP3K中的绑定位点在哪里？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衍生肽的存在下结晶。这里解决的第二个问题是MAP2K如何能够进行两种化学分配，丝氨酸/苏氨酸和酪氨酸磷酸化？激活环构象也是否也涉及此过程？已经确定了MEK6对p38alpha酪氨酸与苏氨酸磷酸化的定量测定，该测定将用于确定MEK6上的两个磷酸化位点是否以突变体建模并部分磷酸化的MEK并在酪氨酸与丝氨酸/硫氨酸磷酸化中具有独特的作用。已经合成了（通过已发表的化学）合成双底物抑制剂，ATP-PTYR-肽和ATP-PTHR肽Mimetics（通过已发表的化学）合成，用于与MAP2K MEK6结合的晶体学分析。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Crystal structure of the TAO2 kinase domain: activation and specificity of a Ste20p MAP3K.

DOI：
10.1016/j.str.2004.07.021
发表时间：
2004-10
期刊：
Structure
影响因子：
5.7
作者：
Tianjun Zhou;Malavika Raman;Yan Gao;Svetlana Earnest;Zhu Chen;M. Machius;M. Cobb;E. Goldsmith
通讯作者：
Tianjun Zhou;Malavika Raman;Yan Gao;Svetlana Earnest;Zhu Chen;M. Machius;M. Cobb;E. Goldsmith

MAP kinases and their roles in pancreatic beta-cells.

MAP 激酶及其在胰腺 β 细胞中的作用。

DOI：
10.1385/cbb:40:3:191
发表时间：
2004
期刊：
Cell biochemistry and biophysics
影响因子：
2.6
作者：
Khoo,Shih;Gibson,TaraBeers;Arnette,Don;Lawrence,Michael;January,Bridgette;McGlynn,Kathleen;Vanderbilt,ColleenA;Griffen,StevenC;German,MichaelS;Cobb,MelanieH
通讯作者：
Cobb,MelanieH

Crystal structure of the MAP3K TAO2 kinase domain bound by an inhibitor staurosporine.

抑制剂十字孢菌素结合的 MAP3K TAO2 激酶结构域的晶体结构。

DOI：
10.1111/j.1745-7270.2006.00173.x
发表时间：
2006
期刊：
Acta biochimica et biophysica Sinica
影响因子：
3.7
作者：
Zhou,Tian-Jun;Sun,Li-Guang;Gao,Yan;Goldsmith,ElizabethJ
通讯作者：
Goldsmith,ElizabethJ

Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK.

DOI：
10.1021/acs.biochem.5b00593
发表时间：
2015-08-18
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Taylor, Clinton A.;Juang, Yu-Chi;Earnest, Svetlana;Sengupta, Samarpita;Goldsmith, Elizabeth J.;Cobb, Melanie H.
通讯作者：
Cobb, Melanie H.

MAP kinase modules: the excursion model and the steps that count.