Structural and Functional Analysis of the Post-translational Modifications of PTEN and MKP-1

摘要PTEN和MKP-1/DUSP 1通过表达/降解、突变 和/或翻译后修饰（PTM）有助于人类疾病的进展， 不同的癌症MKP-1（又名DUSP 1）和PTEN是细胞内主要的负调节磷酸酶。 MAPK和PI 3 K/AKT细胞信号通路，并且这两种酶都受到定位到其C- 终端机尾MKP-1在位置359和364处磷酸化两次，而PTEN是四磷酸化的 在位置380、382、383和385处，并且在位置402处乙酰化，但是在我们的序列中存在大的缺口。 了解这些PTM的结构和功能影响。因此，我们的目标是阐明 PTEN和MKP-1的PTM依赖性调节的分子基础。我们提案的关键是 通过表达蛋白连接促进位点特异性和化学计量插入的半合成酶 用于这三个具体目标的相应PTM及其模拟物：具体目标1将 确定磷酸化依赖的PTEN构象关闭的结构基础。直接 磷酸化的C-末端尾部与PTEN体的原子相互作用将通过UV诱导的免疫印迹来映射。 光交联，这将有助于定义C-末端尾部位置的计算模型 当处于闭合构象状态时。计算模型将通过生成站点定向的 突变体和评估他们与一系列的生化测定，包括酶活性，敏感性， 碱性磷酸酶的去磷酸化和脂质结合。特殊目标2利用了一系列的生化 和细胞技术来确定PTEN的C-末端（Lys 402）乙酰化的功能。这一目标涉及 通过阐明Lys 402乙酰化在调节PTEN生化和细胞功能中的作用 在调节酶活性、蛋白质-蛋白质相互作用、亚细胞定位、蛋白质稳定性和 调节不同的信号通路。一系列的生化和细胞学方法将被执行， 系统地定义该PTM对PTEN的细胞功能的调节作用。具体目标3 采用系统的方法来评估C-末端磷酸化对生物化学的作用， MKP-1的细胞功能这一目标旨在确定这些生物化学和/或细胞后果， 相对于酶活性的磷酸化事件，蛋白质-蛋白质相互作用，亚细胞定位， 蛋白质稳定性和不同信号通路的调节。一系列的生化和细胞分析将 用于定义C-末端磷酸化如何调节MKP-1功能。我们预计会发现 这些PTM的独特调控机制可以为更深入地了解细胞 信号和下一代疗法。

Summary. Dysregulation of PTEN and MKP-1/DUSP1 by changes in expression/degradation, mutation, and/or post-translational modifications (PTMs) contribute to the progression of human diseases such as different cancers. MKP-1 (aka DUSP1) and PTEN are major negative regulatory phosphatase enzymes in the MAPK and PI3K/AKT cell signaling pathways, and both enzymes are subject to PTMs mapped to their C- terminal tails. MKP-1 is phosphorylated twice at positions 359 and 364, whereas PTEN is tetra-phosphorylated at positions 380, 382, 383, and 385 and acetylated at position 402 but there are large gaps in our understanding of the structural and functional impacts of these PTMs. Therefore, our goal is to elucidate the molecular basis for the PTM-dependent regulation of PTEN and MKP-1. Key to our proposal is the generation of semi-synthetic enzymes by expressed protein ligation that facilitates site-specific and stoichiometric insertion of the corresponding PTM(s) and their mimics to be use in these three specific aims: Specific aim 1 will determine the structural basis for the phosphorylation-dependent conformational closure of PTEN. Direct atomic interactions for the phosphorylated C-terminal tail with the PTEN body will be mapped by UV-induced photocrosslinking, which will contribute to a computational model that defines the position of the C-terminal tail when in the closed conformational state. The computational model will be tested by generating site-directed mutants and evaluating them with a series of biochemical assays, including enzyme activity, sensitivity to dephosphorylation by alkaline phosphatase, and lipid binding. Specific aim 2 utilizes a series of biochemical and cellular techniques to define the function of C-terminal (Lys402) acetylation of PTEN. This aim concerns the role of Lys402 acetylation in modulating PTEN's biochemical and cellular function(s) by elucidating its role in regulating enzyme activity, protein-protein interactions, subcellular localization, protein stability, and regulation of different signaling pathways. A series of biochemical and cellular methods will be executed to systematically define the regulatory role that this PTM has on PTEN's cellular function. Specific aim 3 employs a systematic approach to evaluate the role C-terminal phosphorylation has on the biochemical and cellular function of MKP-1. This aim seeks to define the biochemical and/or cellular consequences for these phosphorylation events with respect to enzyme activity, protein-protein interactions, subcellular localization, protein stability, and regulation of different signaling pathways. A series of biochemical and cellular assays will be employed to define how C-terminal phosphorylation regulates MKP-1 function. We anticipate discovering unique regulatory mechanisms for these PTMs that can pave the way to a deeper understanding of cell signaling and next generation therapies.