17A STRUCTURE OF ERK2/MAP KINASE

在本申请中，我们提出研究磷酸调节 MAP激酶ERK 2的作用机制和特异性 晶体X射线分析。蛋白激酶是细胞内的重要分子， 启动信号转导和调节， 细胞过程的整合。MAP激酶ERK 2已经被 广泛研究作为一种生长因子激活，酪氨酸 41 kDa的磷酸化酶。这种酶是由一种 在分化的细胞和生长中激素的显着变化 分裂细胞中的因子，表明它是一个多效性的 调节酶在激酶超家族的蛋白质中，ERK 2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， 专性双磷酸化，在一个单一的酪氨酸和一个单一的 苏氨酸残基它缺乏相关的调节蛋白，因此， 通过对单个多肽的研究， 机制可以理解。我们已经将ERK 2结晶化， 无活性的非磷酸化构象，并已完善了 分辨率为2.3埃。通过比较 具有PKA活性构象的未磷酸化酶的 （cAMP依赖性蛋白激酶），并通过突变分析， 已经有可能提出一个磷酸调节的部分模型 ERK2该模型将通过以下晶体学研究进行测试： 去磷酸化状态的突变ERK 2分子。双 磷酸化和单磷酸化形式已经或将要 通过细菌表达的MEK（MAP）的作用获得 激酶/ERK激酶）。这些磷酸化的酶将被研究 晶体学上阐明的结构基础， ERK 2的复杂磷酸化调节。

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.