Jain Ravi, Garg Swati, Singh Shailja
Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, India.
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
MethodsX. 2019 Jan 18;6:162-168. doi: 10.1016/j.mex.2018.12.003. eCollection 2019.
Protein kinases play a crucial role in cellular functions by adding phosphate group to the protein substrates. It is an indispensable post-translational modification that regulates intracellular signaling and key cellular processes. They thus serve as an excellent target for chemotherapeutic interventions. A vast repertoire of protein kinases is present in a cell with diverse substrates as well as phosphorylation sites. To study full kinome for its activity, there is an urgent need of designing a comprehensive, assay which itself is an impractical task. However, in this study, we have attempted to develop a robust assay that not only mimics the nature of the kinases but can also be used in a high throughput drug-screening platform. Herein, the parasites are lysed and the total protein content is extracted. This extracted proteome is further sub divided into two parts: one active fraction containing cellular kinases and the substrate is heat-denatured fraction that loses all the enzymatic activity but retains the potential phosphorylation sites. These fractions are then co-incubated in the presence of ATP to initiate the kinase reaction and the total kinase activity is measured using ADP-glo kinase assay. Overall, this method •Presents a simple and robust approach to understand the participation of kinases in signaling networks.•Presents a high-throughput platform for ex-vivo drug screening.
蛋白激酶通过向蛋白质底物添加磷酸基团在细胞功能中发挥关键作用。它是一种不可或缺的翻译后修饰，可调节细胞内信号传导和关键细胞过程。因此，它们是化疗干预的理想靶点。细胞中存在大量具有不同底物和磷酸化位点的蛋白激酶。为了研究整个激酶组的活性，迫切需要设计一种全面的检测方法，而这本身就是一项不切实际的任务。然而，在本研究中，我们试图开发一种强大的检测方法，该方法不仅能模拟激酶的性质，还可用于高通量药物筛选平台。在此，将寄生虫裂解并提取总蛋白含量。提取的蛋白质组进一步分为两部分：一部分是含有细胞激酶的活性部分，另一部分是热变性部分，其失去了所有酶活性但保留了潜在的磷酸化位点。然后将这些部分在ATP存在下共同孵育以启动激酶反应，并使用ADP - glo激酶检测法测量总激酶活性。总体而言，该方法•提供了一种简单而强大的方法来了解激酶在信号网络中的参与情况。•提供了一个用于体外药物筛选的高通量平台。
