17A STRUCTURE OF ERK2/MAP KINASE

17A ERK2/MAP 激酶的结构

• 批准号: 2146317
• 负责人: ELIZABETH J. GOLDSMITH
• 金额: $ 14.99万
• 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 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146317
• 关键词: X ray crystallography; cell cycle proteins; conformation; crystallization; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; isozymes; mutant; nucleotide analog; phosphorylation; protein kinase; site directed mutagenesis; stereochemistry; structural biology; synthetic enzyme

In this application we propose to study the phosphoregulatory mechanism and specificity of the MAP kinase ERK2 using single crystal x-ray analysis. Protein kinases are essential molecules in both initiation of signal transduction and in the regulation and integration of cellular processes. The MAP kinase ERK2 has been widely studied as a growth-factor activated, tyrosine phosphorylated enzyme of 41 kDa. This enzyme is activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells, indicating that it is a pleiotropic regulatory enzyme. Of proteins in the kinase superfamily, ERK2 is an especially appropriate target for crystallographic studies. ERK2 has a complex and interesting mechanism of regulation that involves obligate dual phosphorylations, on a single tyrosine and a single threonine residue. It lacks associated regulatory proteins, so that through the study of the single polypeptide the regulatory mechanism can be understood. We have crystallized ERK2 in its inactive unphosphorylated conformation and have refined the structure at 2.3 Angstroms resolution. By comparing the structure of the unphosphorylated enzyme with the active conformation of PKA (cAMP-dependent protein kinase) and by mutational analysis, it has been possible to propose a partial model for the phosphoregulation of ERK2. This model will be tested with crystallographic studies of mutant ERK2 molecules in their dephosphorylated states. Doubly phosphorylated and singly phosphorylated forms have been or will be obtained through the action of bacterially expressed MEK (MAP kinase/ERK kinase). These phosphorylated enzymes will be studied crystallographically to elucidate the structural basis for the complex phosphoregulation of ERK2.

在此应用中，我们建议研究磷酸盐调节 使用单个的MAP激酶ERK2的机理和特异性 晶体X射线分析。蛋白激酶是必不可少的分子 信号转导的启动和调节和 细胞过程的整合。地图激酶ERK2是 被广泛研究为生长因子激活的酪氨酸 41 kDa的磷酸化酶。该酶被A激活 分化细胞和生长中的多种激素种类繁多 分裂细胞的因素，表明它是多效性的 调节酶。激酶超家族中的蛋白质的ERK2为 晶体学研究的特别合适的目标。 ERK2 具有复杂而有趣的调节机制，涉及 在单个酪氨酸和一个单一的双重磷酸上 苏氨酸残留物。它缺乏相关的调节蛋白，因此 通过研究单多肽调节 可以理解机制。我们在它的Erk2中结晶 非活性的无磷酸化构象，并完善了 结构为2.3埃斯特姆斯分辨率。通过比较结构 具有PKA的活性构象的未磷酸化酶 （依赖CAMP的蛋白激酶）和突变分析，它具有 可以提出磷化调节的部分模型 Erk2。该模型将通过晶体学研究进行测试 其去磷酸化状态中的突变ERK2分子。双重 磷酸化和单一磷酸化形式已经或将是 通过细菌表达的MEK的作用获得（地图 激酶/ERK激酶）。将研究这些磷酸化酶 从晶体学上阐明 ERK2的复杂磷化调节。