IN SITU MEASUREMENT OF PROTEIN KINASE ACTIVITY

蛋白质在丝氨酸、苏氨酸和酪氨酸残基上的磷酸化是 真核生物中最常见的可逆蛋白质修饰形式 细胞。蛋白质的磷酸化是已知的一种重要手段 调节各种各样的细胞过程，包括细胞分裂 分化，细胞运动，肌肉收缩，蛋白质合成， 和细胞新陈代谢。现在已知许多疾病的过程是 蛋白磷酸化失调的结果，包括多种形式的 癌症、心脏病、糖尿病和其他代谢性疾病，以及 肌肉疾病，如肌肉营养不良症。因此，一个详细的 对催化酶的生化性质的了解 蛋白质的磷酸化，即蛋白激酶，对 了解许多重要的疾病过程，对于 开发针对特定蛋白激酶的治疗策略。 了解蛋白激酶在活细胞中如何发挥作用的关键是 在原位测定它们的生化特性。对于大多数蛋白质来说 然而，目前不可能或很难测量肌动蛋白 它们在活细胞中的活动。拟议研究的总体目标 是将最新的实验结果应用于开发一种新的 测量活细胞中蛋白激酶活性的技术 荧光蛋白激酶底物。为了实现这一目标，我们有 明确了以下具体目标：具体目标1：发展荧光 三种蛋白激酶--蛋白激酶C的磷酸受体底物 (PKC)、cAMP依赖的蛋白激酶(PKA)和丝裂原激活的 蛋白(MAP)激酶，又称ERK。这三种蛋白激酶是关键 参与调节细胞生长和分化的酶。特定的 目标2：使用单细胞显微注射技术，将这些 将荧光蛋白激酶底物导入培养细胞并原位追踪 对适当的细胞刺激作出反应的蛋白激酶的激活。 这项技术有许多应用，包括确定 一种特定的蛋白激酶是否参与调节特定的 细胞功能，测试潜在的治疗剂的作用 关于特定的蛋白激酶，并确定其可能的作用 不同疾病过程中的特定蛋白激酶。因此，一位将军 检测培养细胞中蛋白激酶活性的方法可能是 应用于针对各种实验问题的 了解特定蛋白激酶在生理学和生物化学中的作用 病理生理过程。

Phosphorylation of proteins on serine, threonine, and tyrosine residues is the most common form of reversible protein modification in eukaryotic cells. Protein phosphorylation is known to be an important means of regulating a wide variety of cellular processes, including cell division and differentiation, cell motility, muscle contraction, protein synthesis, and cellular metabolism. Many disease processes are now known to be the result of dysregulated protein phosphorylation including many forms of cancer and heart disease, diabetes and other metabolic diseases, and diseases of muscle such as muscular dystrophy. Thus, a detailed understanding of the biochemical properties of the enzymes that catalyze the phosphorylation of proteins, the protein kinases, is critical for understanding many important disease processes and will be critical to developing therapeutic strategies that target specific protein kinases. A key to understanding how protein kinases function in living cells will be to determine their biochemical properties in situ. For most protein kinases, however, it is currently impossible or quite difficult to measure their activity in living cells. The overall goal of the proposed research is to apply recent experimental findings to the development of a new technique for measuring protein kinase activities in living cells using fluorescent protein kinase substrates. To achieve this goal, we have defined the following specific aims: Specific Aim 1: Develop fluorescent phosphate-acceptor substrates for three protein kinases, protein kinase C (PKC), the cAMP-dependent protein kinase (PKA), and the mitogen-activated protein (MAP) kinase known as ERK. These three protein kinases are key enzymes involved in regulating cell growth and differentiation. Specific Aim 2: Using single cell microinjection techniques, incorporate these fluorescent kinase substrates into cultured cells and follow in situ activation of the protein kinases in response to appropriate cell stimuli. There are many applications of this technology including determining whether a specific protein kinase is involved in regulating a particular cellular functions, testing potential therapeutic agents for their action on specific protein kinases, and determining the possible roles of specific protein kinases in various disease processes. Thus, a general method for assaying protein kinase activities in cultured cells could be applied to a variety of experimental problems directed towards understanding the role of specific protein kinases in physiological and pathophysiological processes.