Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase.

BACKGROUND: Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions. AIM: The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis. METHODS: Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells. RESULTS & CONCLUSIONS: AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca2+. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 is essential for p53-null cancer cells.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 downregulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53-deficient cancer cells increased levels of the allosteric regulator fructose-2,6-bisphosphate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose-phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53-deficient cancer cells. Moreover, depletion of PFKFB4-attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4-induced apoptosis in p53-deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.