背景:AMP激活的蛋白激酶(AMPK)是一种异三聚体丝氨酸/苏氨酸蛋白激酶,由与ATP消耗或应激相关的细胞扰动激活。虽然AMPK调节含有特定磷酸化共有序列的多种靶标的活性,目前认为AMPK靶标的数量及其对细胞过程的影响有限.
结果:我们向人和小鼠蛋白质组查询了含有AMPK磷酸化共有序列的蛋白质。将该数据库集成到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。进行了体外激酶测定,以初步验证各种细胞功能类别的12种新型AMPK靶标。包括转录,翻译,细胞迁移,蛋白质运输,和能量稳态。初始验证后,假设并实验测试了包括NAD合成酶1(NADSYN1)和蛋白激酶B(AKT2)的途径,以为分解代谢过程中AMPK调节细胞迁移和维持细胞NAD()浓度提供了机制基础。
结论:本研究描述了一种方法,该方法包括计算机模拟程序和体外实验,以产生AMPK调节细胞过程的可测试假设。
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.