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.

在本申请中，我们建议研究磷酸调节 MAP 激酶 ERK2 的机制和特异性 晶体X射线分析。蛋白激酶是体内必需的分子 信号转导的启动和调节 细胞过程的整合。 MAP 激酶 ERK2 已被 作为生长因子激活的酪氨酸被广泛研究 41 kDa 的磷酸化酶。这种酶被激活 分化细胞和生长中的激素种类繁多 细胞分裂因子，表明它是多效性的 调节酶。在激酶超家族的蛋白质中，ERK2 是 一个特别适合晶体学研究的目标。 ERK2 有一个复杂而有趣的调节机制，涉及 专性双磷酸化，在单个酪氨酸和单个 苏氨酸残基。它缺乏相关的调节蛋白，因此 通过研究单一多肽的调控 机制可以理解。我们已将 ERK2 结晶化 无活性的非磷酸化构象并改进了 结构分辨率为 2.3 埃。通过结构比较 具有PKA活性构象的非磷酸化酶 （cAMP依赖性蛋白激酶）并且通过突变分析，它具有 可以提出磷酸调节的部分模型 ERK2 的。该模型将通过晶体学研究进行测试 处于去磷酸化状态的突变 ERK2 分子。双重 磷酸化和单磷酸化形式已经或将会是 通过细菌表达的MEK（MAP 激酶/ERK 激酶）。将研究这些磷酸化酶 从晶体学角度阐明其结构基础 ERK2 的复杂磷酸调节。