Table 2.

Reported AMPK substrates and their roles in regulating cellular metabolism and functions

AMPK substrate	Functional consequence
Glucose metabolism (glucose uptake↑; glycolysis↑; glycogen biosynthesis↓)
PFK2	PFK2 is a bifunctional enzyme central to glycolytic flux. AMPK-mediated PFK-2 activation via phosphorylation at Ser-466 is involved in the stimulation of glycolysis in heart cells during ischemia [59] or in neuronal cells in response to nitric oxide [57]. In cancer cells, PFKBP3, the major form of PFK2, is targeted by AMPK for phosphorylation to increase glycolysis [60].
TCB1D1	TCB1D1 functions as a regulator of fuel homeostasis by regulating GLUT4 translocation, and is activated by AMPK through multi-site phosphorylation as part of the insulin- and contraction-stimulated signaling [56].
Glycogen synthase (GYS)	Phosphorylation of GYS1/2 by AMPK led to enzyme inactivation by attenuating the affinity for its substrates, UDP-Glc and Glc-6-P [61].
Lipid metabolism (fatty acid oxidation↑; fatty acid and cholesterol biosynthesis↓)
ACC	AMPK phosphorylates and inactivates ACC [2], the key enzyme governing the rate-limiting step of fatty acid biosynthesis.
HMG-CoA reductase	AMPK phosphorylates and inactivates HMG-CoA reductase [3], leading to the inhibition of cholesterol biosynthesis.
HSL	HSL facilitates triacylglycerol degradation by hydrolyzing diacylglycerols to monoacylglycerols in adipose and skeletal muscles. AMPK phosphorylates HSL at Ser-565, thereby preventing β-adrenergic agonist-induced HSL activation by blocking protein kinase A-mediated Ser-660 phosphorylation [62].
ATGL (aka, desnutrin /iPLA2ζ)	ATGL is phosphorylated and activated by AMPK to increase lipolysis in brown adipose tissues, which stimulates fatty acid oxidation and UCP-1 induction for thermogenesis [63].
Phospholipase D1 (PLD1)	AMPK activates PLD1 through phosphorylation at Ser-505, and this AMPK-induced PLD1 activation is required for increased glucose uptake in muscle cells under glucose deprivation conditions [64].
Nucleoside metabolism
Nucleoside diphosphate kinase (NDPK)	NDPK maintains pools of nucleoside and deoxynucleoside triphosphates for processes central to energy utilization; for example, DNA synthesis and translation [65]. During nutritional stress, AMPK switches off NDPK through phosphorylation at Ser-120, thereby conserving energy [66].
Insulin signaling
Insulin receptor substrate-1 (IRS-1)	IRS-1 represents the most upstream component of the insulin-signaling cascade. AMPK phosphorylates IRS-1 at Ser-789 in cell-free assays, as well as in mouse C2C12 myotubes, leading to increases in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity [67].
mTORC1 signaling (cell growth and protein biosynthesis ↓)
Tuberous sclerosis protein 2 (TSC2) Raptor	AMPK negatively regulates mTORC1 activity by targeting two key proteins for phosphorylation, the TSC2 tumor suppressor and the mTORC1 scaffold subunits raptor [68]. In light of the pivotal role of mTORC1 in regulating cell growth, cell cycle progression, autophagy, and macromolecule biosynthesis [69], suppression of mTORC1 signaling represents a major mechanism by which AMPK activators inhibit cancer cell proliferation.
Autophagy ↑
ULK1	ULK1 (hATG1), a mammalian ortholog of the yeast protein kinase Atg1, plays a crucial role in autophagy and mitochondrial homeostasis. Evidence suggests that AMPK triggers autophagy through two ULK1-dependent mechanisms [29, 70]: (a) AMPK overrides the suppressive effect of mTORC1 on ULK1, and (b) AMPK directly phosphorylates and activates ULK1.
Activity of transcription factors that regulate cell metabolism
Forkhead box type O3a (Foxo3a)	Foxo3a is a transcriptional factor with known tumor suppressive activity [71]. It was reported that AMPK phosphorylates Foxo3a, leading to the activation of Foxo3a transcriptional activity without affecting Foxo3a subcellular localization [72]. This AMPK-Foxo3a signaling axis promotes the transcription of target genes involved in cell metabolism, cell cycle arrest, cell death, autophagy, and stress resistance [73].
cAMP-response element-binding protein (CREB)	Atypical protein kinase C (PKC)ι/λ	AMPK activates aPKCι/λ by increasing its phosphorylation at Thr403 and Thr555, leading to the phosphorylation of the transcriptional coactivator CREB binding protein (CBP) at Ser-436 [74]. This event triggers the dissociation of the CREB-CBP-CRTC2 (CREB-regulated transcription coactivator 2) transcription complex and reduces gluconeogenic enzyme gene expression, including genes encoding peroxisome-proliferator-activated receptor γ coactivator 1α (PGC-1α) and its downstream targets including phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase).
	CREB-regulated transcriptional co-activator 2 (CRTC2)	AMPK attenuates the gluconeogenic program by promoting the phosphorylation of CTRC2, a transcriptional co-activator for CREB, which blocks its nuclear accumulation through cytoplasmic sequestration by binding to 143-3 scaffold proteins [75].
	Glycogen synthase kinase (GSK)3β	AMPK phosphorylates GSK3β at Ser-9, leading to reductions in Ser-129 CREB phosphorylation and its transcriptional activation of PEPCK expression [76].
PGC-1α	PGC-1α is a master regulator of mitochondrial biogenesis, and has been shown to mediate certain AMPK effects on cell metabolism, particularly in fatty acid oxidation [77]. AMPK binds to and activates PGC-1α in muscle by direct phosphorylation on two critical residues, threonine-177 and serine-538 [78]
Sterol regulatory element binding protein-1c (SREBP-1c)	AMPK phosphorylates SREBP-1c at Ser-372, and thereby suppresses its cleavage and nuclear translocation, and represses the expression of SREBP-1c target genes, including those encoding ACC1 and fatty acid synthase, leading to reduced lipogenesis and lipid accumulation [79].
Hepatic nuclear factor 4α (HNF4α)	HNF4α is an orphan nuclear receptor that regulates the expression of genes involved in energy metabolism in the liver, intestine, and endocrine pancreas. AMPK phosphorylates HNF4α at Ser-304, thereby repressing its transcriptional activity by reducing the ability of the transcription factor to form homodimers and bind DNA and increasing its degradation rate [80].
AICAR responsive element binding protein (AREBP)	AREBP is a zinc finger transcription factor. AMPK-mediated phosphorylation of AREBP at Ser-470 results in loss of DNA-binding activity, leading to transcriptional repression of PEPCK [81].
Testicular nuclear receptor 4 (TR4)	TR4 is a nuclear receptor that might play a role in lipid metabolism. AMPK phosphorylates TR4 at Ser-351 in hepatocytes, leading to the suppression of the target gene stearoyl-CoA desaturase (SCD)1 [82]
Carbohydrate-response-element-binding protein (ChREBP)	The glucose-responsive transcription factor ChREBP binds to the carbohydrate-responsive element of the L-type pyruvate kinase and fatty acid synthase genes, thereby redirecting glucose metabolism toward lipogenesis in hepatocytes [83]. AMPK phosphorylates ChREBP at Ser-568, resulting in the inactivation of its transcriptional activity by reducing DNA binding [84].
p53	AMPK activation has been reported to induce phosphorylation of the cell cycle regulator p53 at Ser-15 in primary mouse embryonic fibroblasts (MEFs), which is required for glucose deprivation-induced cell cycle arrest [85]. In human osteosarcoma-derived cells, this glucose starvation-induced AMPK activation, however, facilitates p53 phosphorylation at Ser-46, but not Ser-15 [86]. Alternatively, a recent paper indicates that AMPK enhances the acetylation and stability of p53 by phosphorylating inactivation of the NAD+-dependent class III HDAC Sirt1 in HCC cells [22].
Histone-modifying enzymes
Class II HDACs (HDAC4, 5, and 7)	Class II HDACs are targeted by AMPK family kinases for phosphorylation, which lead to cytoplasmic sequestration by 14-3-3 binding [87] (Fig. 1B). In response to glucagon, these HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of genes encoding gluconeogenic enzymes, which stimulates the transcriptional induction of these genes.
Histone acetyl-transferase (HAT) p300	Phosphorylation of p300 at Ser-89 by AMPK and its related kinases inhibits the HAT activity of p300, which, in turn, decreases the acetylation and activity of ChREBP in mediating lipogenesis [88].
Sirt1	Two contradictory mechanisms have been reported for the modulatory effect of AMPK on Sirt1, which might underlie differences between nonmalignant versus cancer cells. In C2C12 myotubes, pharmacological activation of AMPK enhances Sirt1 deacetylase activity indirectly by increasing cellular NAD+ levels [89] (Fig. 1B), while in liver cancer cells, activated AMPK phosphorylated Sirt1 at Thr-344, leading to its inactivation [22].
Nuclear import of the mRNA-binding protein HuR
Importin α1	Importin α1 functions as an adaptor that, in association with importin β, transports cargo proteins, such as HuR, through the nuclear pore complex into the nucleus. AMPK phosphorylates importin α1 at Ser-105, which promotes the shuttling of HuR from the cytoplasm to nucleus [90]. As HuR controls the stability and translation of mRNA, this AMPK-facilitated nuclear sequestration of HuR leads to decreased stability/translation of target mRNAs encoding critical cell cycle regulators such as cyclin A, cyclin B1, and p21 in cancer cells [91].
Angiogenesis
Endothelial nitric oxide synthase (eNOS)	AMPK is one of the three kinases, besides Akt and protein kinase A, that phosphorylate eNOS at multiple sites, leading to eNOS activation, in endothelial cells in response to various stimuli [92].