Application of a Chemical Biology Toolkit to Decipher Kinase Activity in Time and Space.

我们的目标是定义构成每一个生物体的细胞是如何传递调控信息的。实际上，每一个生命过程都涉及到蛋白质，这些蛋白质的功能就像酶一样催化化学反应或传递调节信息，就像机器或马达一样产生力量或使运动成为可能，或者就像结构成分一样赋予细胞、组织或器官以至整个身体形状和形态。几乎所有的细胞蛋白质都受到磷酸化过程的调节，磷酸化过程涉及蛋白质上特定位点的化学修饰。因此，本提案将重点关注在所有动物和植物细胞中负责催化调节蛋白磷酸化事件的细胞酶，即激酶。激酶在细胞调节中的广泛作用可以通过在每个细胞中存在许多不同的激酶来说明。事实上，在面包酵母中有超过100种不同的激酶在人类中，有超过500种不同的激酶。值得注意的是，激酶通常在不同的生物体中执行完全相同的功能。尽管它们的重要性显而易见，但我们对活细胞中激酶的确切功能的了解仍然非常有限。事实上，到目前为止，我们对激酶的大部分知识都是通过在细胞提取物或组织匀浆中进行生化测量获得的，这些细胞提取物或组织匀浆没有保留活细胞的许多特征。为了克服这一限制，本建议将重点放在两种互补策略的应用上，以定义活细胞中特定激酶的精确作用。我们将继续研究一种名为CK2的激酶，我们的实验室已经建立了领导地位，使我们能够进行其他地方不可能进行的研究。我们的第一个策略将利用我们设计的传感器，当它们被CK2磷酸化时，它们会经历荧光的变化。通过将这些传感器引入活细胞，我们将使用延时荧光显微镜来监测细胞生命不同阶段（即CK2）活性的时空动态。我们将确定CK2在活细胞中的活跃时间和位置)。为了补充这些研究，我们还设计了CK2突变体，这些突变体可以被精确设计的化学物质选择性靶向，以便这些化学物质可以用于研究CK2在特定细胞过程中的精确作用。总的来说，我们的研究将使我们能够利用活细胞识别由CK2直接调节的细胞事件。鉴于CK2与激酶家族其他成员的相似性，我们的方法可以很容易地翻译为其他激酶的研究。由于很明显，蛋白质磷酸化是所有活细胞的普遍调节机制，这一信息对于我们理解所有植物和动物如何应对环境变化或自身适应性的变化至关重要。

Our goal is to define how regulatory information is transmitted within cells that make up every living organism. Virtually every living process involves proteins that function as enzymes to catalyze chemical reactions or transmit regulatory information, as machines or motors that generate force or enable movement, or as structural constituents that give shape and form to cells, tissues or organs, and ultimately to the entire body. Almost all cellular proteins are regulated by a process known as phosphorylation that involves chemical modification at specific sites on the protein. Consequently, this proposal will focus on cellular enzymes known as kinases that are responsible for catalyzing regulatory protein phosphorylation events in the cells of all animals and plants. The widespread role of kinases in cell regulation is illustrated by the existence of many distinct kinases in every cell. In fact, there are more than 100 different kinases present in baker's yeast and in humans, there are more than 500 different kinases. Notably, kinases often carry out exactly the same function in different organisms. Despite their obvious importance, our understanding of the precise functions of kinases in living cells remains very limited. In fact, to date much of our knowledge of kinases has been obtained by performing biochemical measurements in cell extracts or tissue homogenates that do not retain many of the features of living cells. To overcome this limitation, this proposal will be focused on the application of two complementary strategies to define the precise actions of specific kinases in living cells. We will continue our studies with a kinase known as CK2 where our lab has established a leadership position that enables us to perform studies not possible elsewhere. Our first strategy will exploit sensors that we have designed to undergo changes in fluorescence when they are phosphorylated by CK2. By introducing these sensors into living cells, we will use time-lapse fluorescence microscopy to monitor the spatiotemporal dynamics of CK2 activity during different stages in the life of cells (ie. we will determine when and where CK2 is active within living cells). To complement these studies, we have also engineered mutants of CK2 that can be selectively targeted by precisely-designed chemicals so that these chemicals can be used to investigate the precise role(s) of CK2 in specific cellular processes. Collectively, our studies will enable us to identify - using living cells - cellular events that are directly regulated by CK2. Given the similarity of CK2 to other members of the kinase family, our approaches can be readily translated to investigation of other kinases. Since it is evident that protein phosphorylation is a universal regulatory mechanism in all living cells, this information is critical to our understanding of how all plants and animals respond to changes in the environment or in their own fitness.