Marin Traci L, Gongol Brendan, Martin Marcy, King Stephanie J, Smith Lemar, Johnson David A, Subramaniam Shankar, Chien Shu, Shyy John Y-J
Divisions of Biochemistry and Molecular Biology and Biomedical Sciences, University of California, Riverside, CA, 92521-0121, USA.
Department of Cardiopulmonary Sciences and Anatomy, Schools of Allied Health and Medicine, Loma Linda University, Loma Linda, CA, 92350, USA.
BMC Syst Biol. 2015 Mar 11;9:13. doi: 10.1186/s12918-015-0156-0.
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that is activated by cellular perturbations associated with ATP depletion or stress. While AMPK modulates the activity of a variety of targets containing a specific phosphorylation consensus sequence, the number of AMPK targets and their influence over cellular processes is currently thought to be limited.
We queried the human and the mouse proteomes for proteins containing AMPK phosphorylation consensus sequences. Integration of this database into Gaggle software facilitated the construction of probable AMPK-regulated networks based on known and predicted molecular associations. In vitro kinase assays were conducted for preliminary validation of 12 novel AMPK targets across a variety of cellular functional categories, including transcription, translation, cell migration, protein transport, and energy homeostasis. Following initial validation, pathways that include NAD synthetase 1 (NADSYN1) and protein kinase B (AKT2) were hypothesized and experimentally tested to provide a mechanistic basis for AMPK regulation of cell migration and maintenance of cellular NAD(+) concentrations during catabolic processes.
This study delineates an approach that encompasses both in silico procedures and in vitro experiments to produce testable hypotheses for AMPK regulation of cellular processes.
AMP 激活的蛋白激酶（AMPK）是一种异源三聚体丝氨酸/苏氨酸蛋白激酶，可被与 ATP 耗竭或应激相关的细胞扰动激活。虽然 AMPK 可调节多种含有特定磷酸化共有序列的靶标的活性，但目前认为 AMPK 靶标的数量及其对细胞过程的影响是有限的。
我们在人类和小鼠蛋白质组中查询含有 AMPK 磷酸化共有序列的蛋白质。将该数据库整合到 Gaggle 软件中有助于基于已知和预测的分子关联构建可能的 AMPK 调节网络。针对包括转录、翻译、细胞迁移、蛋白质转运和能量稳态在内的多种细胞功能类别中的 12 个新型 AMPK 靶标进行了体外激酶测定以进行初步验证。在初步验证之后，对包括 NAD 合成酶 1（NADSYN1）和蛋白激酶 B（AKT2）的通路进行了假设并进行了实验测试，以为 AMPK 在分解代谢过程中调节细胞迁移和维持细胞 NAD(+)浓度提供机制基础。
本研究描述了一种方法，该方法包括计算机程序和体外实验，以产生关于 AMPK 调节细胞过程的可测试假设。
