Chemical Genetic Screen for AMPKα2 Substrates Uncovers a Network of Proteins Involved in Mitosis

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.National Institutes of Health (U.S.) (Grant R01-GM068762

AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

The Hedgehog (Hh) pathway regulates cell differen- tiation and proliferation during development by controlling the Gli transcription factors. Cell fate de- cisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP- activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthe- sis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhib- iting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcrip- tional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency

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

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