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Polymerases

Molecular pharmacology

Molecular pharmacology. energy. Normal cells produce ATP in the mitochondria through oxidative phosphorylation (OXPHOS), whereas under hypoxia, LDE225 Diphosphate glucose is converted to lactate LDE225 Diphosphate through glycolysis to produce ATP (Cairns et al., 2011; Kroemer and Pouyssegur, 2008). Glucose oxidation starts from your irreversible decarboxylation of glycolytic intermediate pyruvate to acetyl-CoA in mitochondria by pyruvate dehydrogenase complex (PDC), a large complex of three functional enzymes: E1, E2 and E3. PDC is organized around a 60-meric dodecahedral core created by dihydrolipoyl transacetylase (E2) and E3-binding protein (E3BP) (Hiromasa et al., 2004), which binds pyruvate dehydrogenase (PDH; E1), dihydrolipoamide dehydrogenase (E3) as well as pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) (Read, 2001). PDH is the first and most important enzyme component of PDC that converts pyruvate to acetyl-CoA, which, along with the acetyl-CoA from your fatty acid -oxidation, enters the Krebs cycle to produce ATP and electron donors including NADH. Thus, PDC links glycolysis to the Krebs cycle and thus plays a central role in glucose homeostasis in mammals (Harris et al., 2002). Since PDH catalyzes the rate-limiting step during the pyruvate Bmp3 decarboxylation, activity of PDH determines the LDE225 Diphosphate rate of PDC flux. The current understanding of PDC regulation involves the cyclic phosphorylation/dephosphorylation of PDH catalyzed by specific PDKs and PDPs, respectively (Holness and Sugden, 2003). PDK1 is a Ser/Thr kinase that inactivates PDC by phosphorylating at least one of three specific serine residues (Sites 1, 2 and 3 are S293, S300, and S232, respectively) of PDHA1 while dephosphorylation of PDHA1 by PDP1 restores PDHA1 and subsequently PDC activity (Roche et al., 2001). The Warburg effect describes the observation that cancer cells take up more glucose than normal tissue and favor aerobic glycolysis more than mitochondrial oxidation of pyruvate (Kroemer and Pouyssegur, 2008; Vander Heiden et al., 2009; Warburg, 1956). An emerging concept suggests that the metabolic change in cancer cells to reply more on glycolysis may be due in part to attenuated mitochondrial function through inhibition of PDC. In consonance with this concept, gene expression of PDK1, in addition to diverse glycolytic enzymes, is upregulated by Myc and HIF-1 LDE225 Diphosphate in cancer cells (Kim et al., 2007; Kim et al., 2006a; Papandreou et al., 2006). Moreover, we recently also reported that diverse oncogenic tyrosine kinases (TKs), including FGFR1, are localized to different mitochondrial compartments in cancer cells, where they phosphorylate and activate PDK1 to inhibit PDH and consequently PDC, providing a metabolic advantage to tumor growth (Hitosugi et al., 2011). Here we report a mechanism where lysine acetylation of PDHA1 and PDP1 contributes to inhibitory regulation of PDC, providing complementary insight into the current understanding of PDHA1 regulation through the phosphorylation/dephosphorylation cycle. RESULTS K321 and K202 acetylation inhibits PDHA1 and PDP1, respectively Our recent finding that tyrosine phosphorylation activates PDK1 (Hitosugi et al., 2011) suggests an important role for post-translational modifications in PDC regulation. To examine the potential effect of lysine acetylation on PDC activity, we treated lung cancer H1299 cells that overexpress FGFR1 (Marek et al., 2009) with deacetylase inhibitors nicotinamide (NAM) and Trichostatin A (TSA) for 16 hours, which led to increased global lysine acetylation in cells without affecting cell viability (Figure S1A). NAM+TSA treatment resulted in decreased PDC flux rate in isolated mitochondria from H1299 cells (Figure 1A), suggesting alteration of global lysine acetylation levels leads to PDC inhibition in human cancer cells. Interestingly, multiple proteomics-based studies performed by our collaborators at Cell Signaling Technology (CST) identified key components of PDC including PDHA1 (http://www.phosphosite.org/proteinAction.do?id=1271&showAllSites=true) and PDP1 (http://www.phosphosite.org/proteinAction.do?id=19516&showAllSites=true), but not PDK1 (http://www.phosphosite.org/proteinAction.do?id=2352&showAllSites=true), as acetylated at a group of lysine residues in human cancer cells. To test the hypothesis that lysine acetylation might directly affect PDHA1 and PDP1 activity, we incubated recombinant FLAG-tagged PDHA1 and PDP1 with cell lysates from NAM+TSA treated H1299 cells. Such treatment results in increased lysine acetylation of PDHA1 (Figure 1B; test. The error bars represent mean.