SIRT3-dependent GOT2 acetylation status affects the malate-aspartate NADH shuttle activity and pancreatic tumor growth

The malate–aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate–aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD+ redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate–aspartate NADH shuttle activity and oxidative protection

WT1 Recruits TET2 to Regulate Its Target Gene Expression and Suppress Leukemia Cell Proliferation

The TET2 DNA dioxygenase regulates cell identity and suppresses tumorigenesis by modulating DNA methylation and expression of a large number of genes. How TET2, like most other chromatin modifying enzymes, is recruited to specific genomic sites is unknown. Here we report that WT1, a sequence-specific transcription factor, is mutated in a mutually exclusive manner with TET2, IDH1 and IDH2 in acute myeloid leukemia (AML). WT1 physically interacts with and recruits TET2 to its target genes to activate their expression. The interaction between WT1 and TET2 is disrupted by multiple AML-derived TET2 mutations. TET2 suppresses leukemia cell proliferation and colony formation in a manner dependent on WT1. These results provide a mechanism for targeting TET2 to specific DNA sequence in the genome. Our results also provide an explanation for the mutual exclusivity of WT1 and TET2 mutations in AML and suggest an IDH1/2-TET2-WT1 pathway in suppressing AML

Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1.

Phosphoglycerate kinase 1 (PGK1) catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP, producing 3-phosphoglycerate (3-PG) and ATP. PGK1 plays a key role in coordinating glycolytic energy production with one-carbon metabolism, serine biosynthesis, and cellular redox regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220), which inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the potential acetyltransferase and deacetylase, respectively, for PGK1. Insulin promotes K220 deacetylation to stimulate PGK1 activity. We show that the PI3K/AKT/mTOR pathway regulates HDAC3 S424 phosphorylation, which promotes HDAC3-PGK1 interaction and PGK1 K220 deacetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulation of glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation

Correction: Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1.

[This corrects the article DOI: 10.1371/journal.pbio.1002243.]

Abstract 58

Phosphoglycerate kinase 1 (PGK1) catalyzes the reversible transfer of a phosphoryl group from 1, 3-bisphosphoglycerate (1, 3-BPG) to ADP, producing 3-phosphoglycerate (3-PG) and ATP. PGK1 plays a key role in coordinating glycolytic energy production with one-carbon metabolism, serine biosynthesis, and cellular redox regulation. Here, we report that PGK1 is acetylated at lysine 220 (K220), which inhibits PGK1 activity by disrupting the binding with its substrate, ADP. We have identified KAT9 and HDAC3 as the potential acetyltransferase and deacetylase, respectively, for PGK1. Insulin promotes K220 deacetylation to stimulate PGK1 activity. We show that the PI3K/AKT/mTOR pathway regulates HDAC3 S424 phosphorylation, which promotes HDAC3-PGK1 interaction and PGK1 K220 deacetylation. Our study uncovers a previously unknown mechanism for the insulin and mTOR pathway in regulation of glycolytic ATP production and cellular redox potential via HDAC3-mediated PGK1 deacetylation

Correction: Insulin and mTOR Pathway Regulate HDAC3-Mediated Deacetylation and Activation of PGK1

[This corrects the article DOI: 10.1371/journal.pbio.1002243.]

Insulin promotes K220 acetylation to stimulate PGK1 activity.

<p>(<b>A</b>) TSA treatment blocks the effect of insulin on changing PGK1 K220 acetylation and enzyme activity. Flag-tagged PGK1 was ectopically expressed in HEK293T cells treated with increased concentrations of insulin as indicated, and these cells were co-treated with or without TSA (5 μM for 12 hr). PGK1 proteins were purified by Flag beads. The K220 acetylation levels and enzyme activity were determined by western blot analysis and enzyme assay, respectively. Relative K220 acetylation levels were normalized against Flag. (<b>B</b>) K220R mutant PGK1 displays a negligible response in changing K220 acetylation and enzyme activity upon insulin treatment. Flag-tagged wild-type or K220R mutant PGK1 were transiently overexpressed in HEK293T cells treated with increased concentrations of insulin as indicated. PGK1 proteins were purified by Flag beads. The K220 acetylation levels and enzyme activity were determined by western blot analysis and enzyme assay, respectively. Relative K220 acetylation levels were normalized against Flag. (<b>C</b>) Quantification of the percentage of K220 acetylated endogenous PGK1 in HEK293T cells. Recombinant fully K220 acetylated PGK1 was loaded onto the same gel, together with endogenous PGK1 from HEK293T cells treated without or with insulin (25 nM, 2 hr). PGK1 protein and K220 acetylation were detected by western blot. Relative PGK1 K220 acetylation levels were normalized against endogenous PGK1. (<b>D, E</b>) Quantification of the percentage of K220 acetylated endogenous Pgk1 in mouse livers. Insulin (5 U/kg) was intraperitoneally injected into wild-type mice, and blood glucose levels were measured at the indicated time points after injection. Mouse livers were harvested at the indicated time points post injection, and the K220 acetylation levels of endogenous Pgk1 were determined by western blot. Relative Pgk1 K220 acetylation levels were normalized against endogenous Pgk1. Shown are average values with standard deviation (S.D.) of triplicated experiments. * denotes <i>p</i> < 0.05 and ** denotes <i>p</i> < 0.01 versus t = 0 (before injection); n.s. = not significant. The numerical data and statistical analysis used in the figures are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002243#pbio.1002243.s001" target="_blank">S1 Data</a>. Supporting information can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002243#pbio.1002243.s007" target="_blank">S6 Fig</a>.</p