Identification of AMP-activated protein kinase targets by a consensus sequence search of the proteome.

Marcy Martin,Traci L Marin,Shankar Subramaniam,Stephanie J King,Lemar Smith,David A Johnson,Shu Chien,Brendan Gongol,John Y-J Shyy

doi:10.1186/s12918-015-0156-0

Abstract

BackgroundAMP-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.ResultsWe 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.ConclusionsThis study delineates an approach that encompasses both in silico procedures and in vitro experiments to produce testable hypotheses for AMPK regulation of cellular processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0156-0) contains supplementary material, which is available to authorized users.

Highlights

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that is activated by cellular perturbations associated with ATP depletion or stress
Bioinformatics and systems biology approach Initially, we performed an in silico search of the AMPK phosphorylation consensus sequence to the human and mouse proteomes
AMPK phosphorylation of NAD synthetase 1 (NADSYN1) and transactivation through forkhead box O3a (FOXO3a) We focused on an array of cellular processes that appear to be regulated by AMPK for validation and partial pathway delineation

Summary

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. 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

Background

Results and discussion

Conclusion and perspective

Methods