The p2.1 PF-00562271 and GalA assays revealed that double mutation of Pro-403/408 significantly reduced PKM2-mediated HIF-1 transactivation in HeLa cells (Figures 4F and 4G), despite the fact that PKM2(P403/408A)-V5 was detected in the nucleus at levels similar to WT PKM2-V5 (Figure S3C). demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O2 consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells. INTRODUCTION The glycolytic pathway involves conversion of glucose to lactate and the generation of ATP. Pyruvate kinase (PK), which catalyzes the reaction of phosphoenolpyruvate (PEP) + ADP pyruvate + ATP, is a key enzyme that determines glycolytic activity. PKM1 and PKM2 are alternatively spliced products of the Rabbit Polyclonal to FPRL2 primary RNA transcript that contain sequences encoded by exon 9 or exon 10, respectively, of the gene (Noguchi et al., 1986). Heterogeneous nuclear ribonucleoproteins (hnRNP) I, A1, and A2 bind to RNA sequences encoded by exon 9 and inhibit PKM1 mRNA splicing (David et al., 2010). The oncoprotein c-Myc activates transcription of hnRNPI, hnRNPA1, and hnRNPA2, resulting in preferential PKM2 isoform expression (David et al., 2010). Many cancer cells have increased glycolysis and lactate production and decreased O2 consumption compared to non-transformed cells, a phenomenon known as the Warburg effect (Gatenby and Gillies, 2004). PKM2 promotes the Warburg effect and tumorigenesis (Christofk et al., 2008; Hitosugi et al., 2009). Despite intensive studies, the mechanism by which PKM2 facilitates lactate production and blocks mitochondrial oxidative phosphorylation in cancer cells has remained a mystery. Activation of hypoxia-inducible factor 1 (HIF-1), which commonly occurs in human cancers either as a result of hypoxia or genetic alterations (Harris, 2002; Semenza, 2010), leads to a switch from oxidative to glycolytic metabolism (Seagroves et al., 2001; Wheaton and Chandel, 2011). HIF-1 is a transcription factor that consists of an O2-regulated HIF-1 subunit PF-00562271 and a constitutively expressed HIF-1 subunit (Wang et al., 1995). In well-oxygenated cells, HIF-1 is hydroxylated at proline (Pro) 402 and 564 (Kaelin and Ratcliffe, 2008). Three prolyl hydroxylases, PHD1-3, which require O2, Fe2+, 2-oxoglutarate, and ascorbate for their catalytic activity, have been shown to hydroxylate HIF-1 when overexpressed (Epstein et al., 2001). PHD2 is primarily responsible for regulating basal HIF-1 levels in cancer cells (Berra et al., 2003). Prolyl hydroxylated HIF-1 is bound by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C-Elongin B-Cullin 2-E3-ubiquitin-ligase complex, leading to proteasomal degradation of HIF-1. Under hypoxic conditions, HIF-1 prolyl hydroxylation is inhibited, thereby stabilizing HIF-1 protein (Kaelin and Ratcliffe, 2008). In the nucleus, HIF-1 dimerizes with HIF-1 and binds to the consensus nucleotide sequence 5-RCGTG-3, which is present within the hypoxia response element (HRE) of target genes (Semenza et al., 1996). Hydroxylation of HIF-1 at asparagine-803, which is catalyzed by the asparaginyl hydroxylase FIH-1 in normoxic cells, blocks the binding of the transcriptional coactivator p300 to HIF-1 (Lando et al., 2002). Under hypoxic conditions, p300 catalyzes the acetylation of lysine residues on the N-terminal tail of core histones at HIF-1 target genes, leading to changes in chromatin structure that promote HIF-1-dependent gene transcription (Arany et al., 1996). HIF-1 activates transcription of genes encoding proteins that are involved in key aspects of cancer biology, including angiogenesis, metabolism, cell survival, invasion, and metastasis (Harris, 2002; Melillo, 2007; Semenza, 2010). HIF-1 target genes include those encoding: the glucose transporter GLUT1, which increases glucose uptake; lactate dehydrogenase A (LDHA), which converts pyruvate to lactate; and pyruvate dehydrogenase kinase 1 (PDK1), which inactivates pyruvate dehydrogenase, thereby shunting pyruvate away from the mitochondria and inhibiting O2 consumption (Wheaton and Chandel, 2011). In the present study, we demonstrate that PKM2 functions as a coactivator that stimulates HIF-1 transactivation of target genes encoding GLUT1, LDHA, and PDK1 in cancer cells. PHD3 binds to PKM2 and stimulates its function as a HIF-1 coactivator. The effect of PHD3 on PKM2 is dependent upon its hydroxylase activity and the PF-00562271 presence of two Pro residues in PKM2. PHD3 knockdown reduces glucose uptake and lactate production and PF-00562271 increases O2 consumption in VHL-null renal cancer cells. HIF-1 activates transcription of the genes encoding PKM2 and PHD3, which provides a feedforward mechanism that amplifies HIF-1-dependent metabolic reprogramming, thus providing a molecular basis for the observed effects of PKM2 on tumor metabolism. RESULTS is a HIF-1 Target Gene Previous studies demonstrated that hypoxia induces PKM mRNA expression (Semenza et al., 1994). To determine whether mRNA encoding PKM1 or PKM2 is regulated by HIF-1, wild-type (WT) mouse embryonic fibroblasts (MEFs) and HIF-1-knockout (KO) MEFs were exposed to 20% or 1% O2 for 24 h. Quantitative real-time RT-PCR.