IN SITU MEASUREMENT OF PROTEIN KINASE ACTIVITY

丝氨酸、苏氨酸和酪氨酸残基上的蛋白质磷酸化是 真核生物中最常见的可逆蛋白质修饰形式 细胞 已知蛋白质磷酸化是一种重要的 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，包括许多形式的 癌症和心脏病，糖尿病和其他代谢疾病，以及 肌肉疾病，如肌肉萎缩症。 因此， 了解催化这些酶的生化特性， 蛋白质的磷酸化，即蛋白激酶， 了解许多重要的疾病过程， 开发针对特定蛋白激酶的治疗策略。 理解蛋白激酶在活细胞中如何发挥作用的关键将是 来确定它们的生物化学特性。 对于大多数蛋白质 然而，激酶，目前不可能或很难测量 它们在活细胞中的活性。 拟议研究的总体目标 是将最近的实验发现应用于开发一种新的 用于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 明确了以下具体目标：具体目标1： 三种蛋白激酶的磷酸受体底物，蛋白激酶C (PKC)cAMP依赖性蛋白激酶（PKA）和丝裂原活化的 蛋白激酶（MAP），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 目的2：使用单细胞显微注射技术，将这些 荧光激酶底物进入培养的细胞， 响应于适当的细胞刺激而激活蛋白激酶。 这项技术有许多应用，包括确定 一个特定的蛋白激酶是否参与调节一个特定的 细胞功能，测试潜在的治疗剂的作用 对特定的蛋白激酶，并确定可能的作用 在各种疾病过程中的特定蛋白激酶。 因此，一位将军 测定培养细胞中蛋白激酶活性的方法， 应用于各种实验问题， 了解特定蛋白激酶在生理和 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.