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

Abstract

SUMMARY 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.

Generation and characterization of an analog-sensitive PERK allele

“…For screening purposes, this should reduce the number of false positives. In vivo labeling techniques have recently been used to successfully identify novel substrates of Erk2 and AMPK, 28,30 giving us confidence in identifying PERK substrates in a similar fashion. Such a screen would provide significant information for understanding PERK signaling -/-cells expressing either PeRK WT or M886A were permeabilized, then incubated with thapsigargin and N 6 -furfuryl ATPγs for substrate labeling.…”

“…[28][29][30][31] With this approach, the analog-sensitive kinase uses N 6 -alkylated ATPγS to transfer a thiophosphoryl group to its substrates. The bulky group again confers specificity for the analog-sensitive kinase, and the thiophosphoryl group acts as a label for distinguishing direct substrates from all other phosphorylated proteins in the cell.…”

Serine79-phosphorylated acetyl-CoA carboxylase, a downstream target of AMPK, localizes to the mitotic spindle poles and the cytokinesis furrow

“…Threonine 172 -phosphorylated AMPKα transiently associates with several mitotic structures, including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis; 4,5 other studies have further identified a network of proteins involved in mitosis that are substrates of AMPK. 6 Indeed, it has been unambiguously confirmed that threonine 172 -phosphorylated AMPKα localizes to the mitotic spindle poles and increases when cells enter mitosis; 7 the mitotic AMPK activity appears to be essential for normal spindle orientation, and when it is defective, mitosis does not proceed efficiently. In this scenario, we envisioned that the mitosis-associated phosphorylated status of ACACA, a downstream target of AMPK, could also be explained in terms of a previously unrecognized ability of phospho-ACACA to directly associate with the mitotic/cytokinetic apparatus during cell division.…”

Sestrins Link Tumor Suppressors with the AMPK-TOR Signaling Network

“…Activated AMPK phosphorylates many targets involved in the regulation of glucose metabolism: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), glycogen synthase-1 (GYS1), glutamine:fructose-6-phosphate amidotransferase (GFAT1), TBC1D1; lipid metabolism: acetyl-CoA Carboxylase-1 and -2 (ACC1 and ACC2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), PLD1; polarity: cytoplasmic linker protein-170 (CLIP170), golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), kinesin light chain-2 (KLC2); transcription: (PGC1, sterol regulatory elementbinding protein-1 (SREBP1), FoxOs, Histone-2B, p300 and HDAC4,5,6; and mitosis: protein phosphatase-1 regulatory subunit 12C (PPP1R12C), p21-activated protein kinase (PAK2) [4,39]. AMPK also contributes to mitochondrial function stimulating mitochondrial biogenesis via phosphorylation of PGC1 co-activator and regulating expression of mitochondrial genes [4].…”