Design of a Chemical Biology Toolkit to Monitor Protein Kinase Function 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，每一个生命过程都是由蛋白质完成的，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、组织或器官以形状和形式，最终形成整个身体。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，本提案将集中于所有动物和植物细胞中负责催化蛋白质磷酸化的细胞酶激酶。激酶在细胞调节中的广泛作用和重要性由两个重要发现说明。首先，每个细胞中都存在许多不同的激酶。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。其次，激酶通常在不同的生物体中执行完全相同的功能。例如，几种酵母激酶可以用另一种生物（如苍蝇、老鼠或人类）的激酶代替，而不会产生任何不良后果。尽管蛋白激酶在传递调控信息方面具有明显的重要性和广泛的影响，但我们对其在活细胞中的确切功能的了解仍然非常有限。事实上，我们目前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发展上，以定义活细胞中特定激酶的精确作用。最初的研究将使用一种称为CK2的激酶进行，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。一旦确定了CK2，我们希望我们的策略可以很容易地适用于其他激酶的研究。我们的第一个策略将涉及设计激酶突变体（指定的类似物敏感突变体），使它们对不影响任何正常细胞蛋白质的药物（称为atp类似物）敏感。因此，当我们将类似物敏感激酶引入细胞时，我们将能够使用atp类似物来调节类似物敏感激酶的活性，而不影响任何其他细胞成分。在这些研究中，我们还将利用激酶在酵母和人类细胞中的保守功能。酵母的初步研究结果将用于指导我们后续的人类细胞研究（使用广泛接受的人类细胞系）。这些研究将使我们能够识别-使用活细胞-由特定激酶直接调节的细胞事件。第二种策略将涉及荧光传感器的设计，这种荧光传感器可以被引入细胞中，以检测和监测活细胞内单个激酶的活性。使用配备数码相机的显微镜，我们将执行延时显微镜来监测细胞中蛋白激酶的时空动态(即。我们将确定活细胞内蛋白激酶何时何地活跃)。总的来说，这些研究将为活细胞内单个蛋白激酶的精确作用提供新的见解。考虑到蛋白质磷酸化作为一种调节机制的普遍作用，这一信息对于我们理解所有植物和动物如何对生物体和环境中的变化做出反应至关重要。

Our goal is to define how regulatory information is transmitted within cells that make up every living organism. Virtually every living process is carried out by 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 protein phosphorylation in the cells of all animals and plants. The widespread role and importance of kinases in cell regulation is illustrated by two important findings. Firstly, there are many distinct kinases present 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. Secondly, kinases often carry out exactly the same function in different organisms. For example, several yeast kinases can be replaced with a kinase from another organism (such as a fly, mouse or human) without any undesirable consequences. Despite the obvious importance and widespread impact of protein kinases in the transmission of regulatory information, our understanding of their precise functions in living cells remains very limited. In fact, much of our current 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 development of two complementary strategies to define the precise actions of specific kinases in living cells. Initial studies will be performed with a kinase known as CK2 where our lab has established a leadership position that enables us to perform studies not possible elsewhere. Once established for CK2, we expect that our strategies can be readily adapted to investigation of other kinases. Our first strategy will involve the design of kinase mutants (designated analog-sensitive mutants) to make them susceptible to drugs (known as ATP-analogs) that do not affect any normal cellular proteins. Therefore, when we introduce the analog-sensitive kinase into cells, we will be able to use the ATP-analogs to modulate the activity of the analog-sensitive kinase without affecting any other cellular constituents. For these studies, we will also take advantage of the conserved function of kinases in yeast and in human cells. Results from initial studies in yeast will be used to guide our subsequent studies in human cells (using widely accepted human cell lines). These studies will enable us to identify – using living cells – cellular events that are directly regulated by specific kinases. The second strategy will involve the design of fluorescent sensors that can be introduced into cells to detect and monitor the activity of individual kinases within living cells. Using microscopes equipped with digital cameras, we will perform time-lapse microscopy to monitor the spatiotemporal dynamics of protein kinases in cells (ie. we will determine when and where protein kinases are active within living cells). Collectively, these studies will provide new insights regarding the precise actions of individual protein kinases within living cells. Given the universal role of protein phosphorylation as a regulatory mechanism, this information is critical to our understanding of how all plants and animals respond to changes both within an organism and in the environment.