Orthogonally Gated Kinases and Phosphatases

 描述(由申请人提供)：许多蛋白质的时间和位置特定的活性由无数的翻译后修饰控制。在这些精细的化学修饰中，磷酸化和去磷酸化分别由蛋白激酶和磷酸酶催化，调节从细胞分裂到细胞死亡的各种细胞事件。&gt；500人类蛋白激酶和147蛋白磷酸酶串联在一起对各种细胞内外环境信号做出反应，据估计，至少有三分之一的蛋白质组能够被磷酸化。不足为奇的是，磷酸化的放松会导致包括癌症在内的多种人类疾病。尽管磷酸化驱动的信号转导很重要，但破译特定的激酶或磷酸酶的作用仍然具有巨大的挑战性。目前，几乎没有唯一的选择性小分子来对天然的激酶或磷酸酶进行药理扰动。目前流行的siRNA和其他基因敲除方法提供了对特定酶功能的洞察，无法提供时间控制，机制细节被补偿细胞机制所掩盖。一些优雅而强大的方法已经被开发出来，以提供对单个激酶的暂时关闭或开启控制，以研究细胞生物学。然而，没有一种方法可以控制细胞信号通路中经常涉及的多个用户定义的激酶和磷酸酶。我们假设，这一知识差距可以通过开发一种方法来解决，该方法允许对分裂激酶(目标1)以及分裂磷酸酶(目标2)进行正交小分子控制，这是我们最近设计的。提出的三个目标旨在为研究信令提供具有可互换部分的有效方法(目标3)。我们建议展示一般性，量化动力学和底物特异性，并为研究活细胞中特定的激酶(目标1)和磷酸酶(目标2)的时间方面提供有效的方法。这些方法可能对研究细胞运动和细胞黏附的动力学特别相关，这是基本的兴趣，也是与合作者一起理解癌症迁移和转移途径的核心。

 DESCRIPTION (provided by applicant): The temporal and location specific activity of numerous proteins is controlled by myriad posttranslational modifications. Of these exquisite chemical modifications, phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, regulate a diversity of cellular events from cell division to cell death. The >500 human protein kinases and 147 protein phosphatases in tandem respond to a variety of both intra- and extra-cellular environmental cues and it has been estimated that at least a third of the proteome is capable of being phosphorylated. Not surprisingly, the deregulation of phosphorylation leads to a variety of human diseases including cancer. Despite the importance of phosphorylation driven signaling, decrypting the role of a specific kinase or phosphatase remains enormously challenging. Currently there are almost no uniquely selective small molecules for pharmacological perturbation of native kinases or phosphatases. Prevailing siRNA and other genetic knockdown methods, which provide insight regarding the function of a specific enzyme, cannot afford temporal control and mechanistic details are obscured by compensatory cellular mechanisms. A few elegant and powerful methods have been developed to provide temporal turn-off or turn-on control over a single kinase to study cell biology. However, there are no methods that allow for controlling multiple user-defined kinases and phosphatases that are often implicated in a cell signaling pathway. We hypothesize that this knowledge gap can be addressed by developing an approach that allows for orthogonal small molecule control over split-kinases (Aim 1) as well as split-phosphatases (Aim 2), which we have recently designed. The three proposed aims are designed to provide validated methods with interchangeable parts (Aim 3) for studying signaling. We propose to demonstrate generality, quantify kinetics and substrate specificity, and provide validate methods for studying temporal aspects of specific kinases (Aim 1) and phosphatases (Aim 2) in live cells. These methods will perhaps be particularly relevant for studying the dynamics of cell motility and cell adhesion, which are of fundamental interest as well as central to understanding migration and metastatic pathways in cancer with collaborators.