Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction

• DOI: 10.1016/j.cmet.2015.09.009
• Publication Date: 2015-10-19T20:54:36Z
• Authors: Bethany E. Schaffer; Rebecca S. Levin; Nicholas T. Hertz; Travis J. Maures; Michael L. Schoof; Pablo E. Hollstein; Bérénice A. Benayoun; Max R. Banko; Reuben J. Shaw; Kevan M. Shokat; Anne Brunet
• Keywords: 570; Physiology; 1.1 Normal biological development and functioning; Medical Biochemistry and Metabolomics; AMP-Activated Protein Kinases; Substrate Specificity; Endocrinology & Metabolism; 03 medical and health sciences; Cell Movement; 616; Cell Adhesion; 2.1 Biological and endogenous factors; Medical biochemistry and metabolomics; Animals; Humans; Protein Interaction Maps; Phosphorylation; Molecular Biology; Oncogene Proteins; 0303 health sciences; Cell Biology; Biological Sciences; Extracellular Matrix; Biochemistry and cell biology; Biochemistry and Cell Biology; Generic health relevance; Single-Cell Analysis; Peptides

AMP-activated protein kinase (AMPK) is a central energy gauge that regulates metabolism and has been increasingly involved in non-metabolic processes and diseases. However, AMPK's direct substrates in non-metabolic contexts are largely unknown. To better understand the AMPK network, we use a chemical genetics screen coupled to a peptide capture approach in whole cells, resulting in identification of direct AMPK phosphorylation sites. Interestingly, the high-confidence AMPK substrates contain many proteins involved in cell motility, adhesion, and invasion. AMPK phosphorylation of the RHOA guanine nucleotide exchange factor NET1A inhibits extracellular matrix degradation, an early step in cell invasion. The identification of direct AMPK phosphorylation sites also facilitates large-scale prediction of AMPK substrates. We provide an AMPK motif matrix and a pipeline to predict additional AMPK substrates from quantitative phosphoproteomics datasets. As AMPK is emerging as a critical node in aging and pathological processes, our study identifies potential targets for therapeutic strategies.