IN SITU MEASUREMENT OF PROTEIN KINASE ACTIVITY

蛋白激酶活性的原位测量

• 批准号: 2662360
• 负责人: DONALD K BLUMENTHAL
• 金额: $ 10万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2662360
• 关键词: enzyme activity; enzyme substrate; green fluorescent proteins; mitogen activated protein kinase; protein kinase; protein kinase A; protein kinase C; tissue /cell culture

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.

蛋白质的丝氨酸、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。 蛋白质磷酸化是已知的重要手段 调节多种细胞过程，包括细胞分裂 和分化、细胞运动、肌肉收缩、蛋白质合成、 和细胞代谢。 现在已知许多疾病过程是 蛋白质磷酸化失调的结果，包括多种形式 癌症和心脏病、糖尿病和其他代谢疾病，以及 肌肉疾病，例如肌营养不良症。 于是，详细的 了解催化酶的生化特性 蛋白质的磷酸化，即蛋白激酶，对于 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。 了解蛋白激酶如何在活细胞中发挥作用的关键是 是为了确定它们的生化特性。 对于大多数蛋白质 然而，目前不可能或很难测量激酶 它们在活细胞中的活动。 拟议研究的总体目标 是将最近的实验结果应用于开发新的 使用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 确定了以下具体目标： 具体目标 1：开发荧光 三种蛋白激酶（蛋白激酶 C）的磷酸盐受体底物 (PKC)、cAMP 依赖性蛋白激酶 (PKA) 和丝裂原激活 蛋白 (MAP) 激酶称为 ERK。 这三种蛋白激酶是关键 参与调节细胞生长和分化的酶。 具体的 目标 2：使用单细胞显微注射技术，将这些 将荧光激酶底物注入培养细胞并进行原位跟踪 响应适当的细胞刺激而激活蛋白激酶。 这项技术有很多应用，包括确定 特定的蛋白激酶是否参与调节特定的 细胞功能，测试潜在治疗剂的作用 对特定蛋白激酶的影响，并确定其可能的作用 各种疾病过程中的特定蛋白激酶。 因此，一般 测定培养细胞中蛋白激酶活性的方法可以是 应用于各种实验问题 了解特定蛋白激酶在生理和功能中的作用 病理生理过程。