PerspectiveCancer

PKM2 Tyrosine Phosphorylation and Glutamine Metabolism Signal a Different View of the Warburg Effect

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Science Signaling  17 Nov 2009:
Vol. 2, Issue 97, pp. pe75
DOI: 10.1126/scisignal.297pe75

Abstract

New evidence suggests that the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) directly phosphorylates pyruvate kinase M2 (PKM2), resulting in reduced conversion of phosphoenolpyruvate to pyruvate, which is further catabolized to lactate by lactate dehydrogenase A. Mutation of the critical tyrosine Tyr105 to Phe rendered PKM2 more active but was associated with decreased cellular lactate production, increased oxygen consumption, and decreased hypoxic cell proliferation relative to wild-type PKM2. The apparent paradoxical effect of growth signaling through tyrosine phosphorylation, which decreases rather than increases PKM2 activity, stimulates a revised perspective of the Warburg effect. This effect, which describes the propensity for cancer cells to convert glucose to lactate at a high rate, must now accommodate links among glycolysis, the tricarboxylic acid cycle, and glutamine metabolism in cancer cells.

The Warburg effect, which is also known as aerobic glycolysis and is currently described as the avid conversion of glucose to lactate (by way of pyruvate) in cancer cells, has a convoluted history with a prevailing view that distorts its origin. Careful inspection of Warburg and his associates’ original work suggests that cancer cells and tissue undergo not only aerobic glycolysis, but also oxidative phosphorylation (1). In Warburg’s book published in 1930, Minami measured lactate production and oxygen consumption in tissue sections from liver tumors and from normal rat liver (2). He reported that, relative to normal liver tissue, carcinoma tissue glucose consumption rates were higher by a factor of 9 and lactate production was increased by a factor of 7, although oxygen consumption rates were similar. In addition to a sizable increase in glycolysis, many cancer cells still have ongoing tricarboxylic acid (TCA) cycle activity and oxidative phosphorylation (3, 4). The increase in aerobic glycolysis in cancer cells has been linked to hypoxia-inducible factor (HIF), to loss of tumor suppressors (such as p53 and VHL), or to activation of oncogenes (such as those encoding RAS, MYC, AKT, and phosphatidylinositol 3-kinase) (59). This link is mediated by increased transcription of genes encoding glucose transporters and glycolytic enzymes or modifications of these proteins that suppress mitochondrial oxidation, thereby favoring the conversion of glucose to lactate. Many of these studies, however, did not directly assess oxidative phosphorylation or activity of the TCA cycle.

With the use of hematopoietic cells expressing a constitutively active fusion protein that contains the FGFR1 tyrosine kinase domain and is associated with a myeloproliferative disorder, Hitosugi et al. identified a group of tyrosine-phosphorylated metabolic enzymes, including PKM2, lactate dehydrogenase A (LDHA), glucose-6-phosphate dehydrogenase (G6PD), and malate dehydrogenase 2 (MDH2) (10). These observations suggest that tyrosine phosphorylation of glycolytic enzymes, which was first reported by Cooper et al. in 1983, might confer the Warburg effect and provide a proliferative advantage to cancer cells (11).

Glucose taken up by cells is phosphorylated by hexokinase and subsequently catabolized to trioses, which provide the glycerol backbone for lipids, and then to phosphoenolpyruvate (PEP). PEP is converted to pyruvate by pyruvate kinase (PK) (Fig. 1). PK is a tetrameric enzyme that comprises four isoenzymes (M1, M2, L, and R) differing in their kinetic properties and distribution pattern among cells and tissues. PKM1 is found in the vast majority of normal cells and tissues, whereas PKM2, which is abundant during embryogenesis and in select tissues such as adipose tissue and pancreatic islets, is the predominant form found in cancer cells (12). Mazurek, Eigenbrodt, and co-workers documented a role for PKM2 in tumor metabolism by demonstrating that the more active tetrameric PKM2 favors glycolysis and lactate production, whereas the less active dimeric form predominantly found in cancer cells favors the diversion of trioses toward synthetic processes such as lipid and amino acid biosynthesis (Fig. 1) (12). They also noted that PKM is part of a large glycolytic enzyme complex that could provide supramolecular organization of glycolysis for substrate channeling, thereby permitting the efficient transfer of metabolites from one enzyme to another. Christofk, Cantley, and co-workers reported that replacement of PKM2 by PKM1 in tumor cell lines rendered them less glycolytically active and diminished tumor xenograft growth, which suggests that PKM2 is responsible for the Warburg effect (13). By solving the crystal structure of PKM2, Cantley and co-workers further demonstrated that fructose bisphosphate (FBP), a positive allosteric regulator of PKM2, could be released from tetrameric PKM2 by interaction with specific tyrosine-phosphorylated peptides, including that of LDHA, resulting in the less active dimeric form of PKM2 (14). Hence, regulation of PKM2 seems to be pivotal for regulating glycolysis in proliferating cells.

Fig. 1

(A) Schematic depiction of tyrosine kinase phosphorylation of active PKM2 tetramer to a less active dimer of PKM2. The associated cellular changes with increased oxygen consumption by cells with the active PKM2 tetramer may be linked to glutamine or pyruvate oxidation (or both). The decreased pyruvate production by the less active tyrosine-phosphorylated (P) PKM2 dimer may permit channeling of glucose carbons upstream of phosphoenolpyruvate (PEP) for biosynthetic purposes. Concurrent glutaminolysis provides a lactate source as well as a carbon source for anapleurosis in the TCA cycle, which supports biosynthesis. (B) The key substrates, glucose and glutamine, and the associated metabolic pathways. AcCoA, acetyl-CoA; αKG, α-ketoglutarate; ASCT2, glutamine transporter; GLS, glutaminase; GPT, glutamate pyruvate transaminase; GLUD, glutamate dehydrogenase; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; ME, malate enzyme; PDH, pyruvate dehydrogenase. Dashed lines represent multistep pathways.

Hitosugi et al. found that PKM2 is tyrosine-phosphorylated at six positions by FGFR1, and mutation of Tyr105 to Phe (Y105F) was the most potent in elevating PKM2 activity (10). Furthermore, a peptide containing the phosphorylated Tyr105 site in PKM2 could release FBP from PKM2 and convert PKM2 to the less active dimeric form, which suggests that FGFR1-mediated phosphorylation of PKM2 decreases its activity. In this regard, the Y105F PKM2 mutant did not stimulate lung cancer H1299 cell proliferation as much as wild-type PKM2 did. H1299 cells expressing the Y105F mutant had increased oxygen consumption and decreased lactate production, and produced tumor xenografts that had diminished tumorigenic potential relative to H1299 cells expressing wild-type PKM2. These observations were construed to suggest that tyrosine phosphorylation of wild-type PKM2, which decreases its enzymatic activity, somehow promotes the Warburg effect or the high rate of glucose conversion to lactate. Hence, the conundrum is how the decrease in activity of wild-type PKM2 in a critical step of glycolysis promotes the Warburg effect or an increase in lactate production, whereas the more active Y105F PKM2 mutant is not as effective (Fig. 1).

A decrease in wild-type PKM2 activity through tyrosine phosphorylation should be accompanied by a decrease in the conversion of glucose to pyruvate and subsequently to lactate, unless there is an alternative pathway for additional pyruvate production (Fig. 1). The putative decrease in pyruvate concentrations generated by wild-type PKM2, an effect that is attenuated by tyrosine phosphorylation, is expected to result in lower rather than higher lactate production. The converse, however, was observed by Hitosugi et al. when wild-type PKM2 was compared to the Y105F mutant (10). A possible interpretation of these observations is that the putative high concentrations of pyruvate produced by the mutant Y105F PKM2 somehow favor oxidative phosphorylation rather than lactate production. But what is the mechanism that controls this switch, whereby the less active wild-type PKM2 favors lactate production but the Y105F mutant induces higher cellular oxygen consumption? Mazurek et al. proposed that the less active phosphorylated PKM2 diverts glucose carbons into synthesis of lipids and amino acids rather than into production of pyruvate, which is converted to lactate (12). This mechanism could explain the increased proliferation of cancer cells and tumorigenic phenotypes having high biosynthetic requirements, but it is inconsistent with the increase in lactate production in cancer cells. Some tumors exhibit increased abundance of malic enzyme and glutaminase, both of which could participate in the conversion of glutamine to lactate, thereby providing additional fuel and anabolic carbons (Fig. 1) (15). The work of Hitosugi et al., however, did not address whether glutamine oxidation or glutaminolysis is operative in H1299 cells and contributes to lactate production.

Glutamine metabolism is increased by MYC, partly through the repression of the microRNAs miR-23a and miR-23b (1618). Furthermore, glucose deprivation results in increased glutamine use through glutamate dehydrogenase, which converts glutamate to α-ketoglutarate in a glioblastoma cell line (Fig. 1) (19). In the presence of glucose, however, glutamate can be converted to α-ketoglutarate by glutamate pyruvate transaminase (GPT) concurrently with the conversion of pyruvate to alanine (Fig. 1) (20). α-Ketoglutarate is further oxidized through the TCA cycle or catabolized to malate, shunted out of the TCA cycle, and then converted to lactate (21). It is possible that GPT-mediated glutamine oxidation, a reaction that consumes pyruvate, is increased by the putative high concentrations of pyruvate associated with the more active Y105F PKM2 mutant, thereby explaining the increased oxygen consumption by cells expressing the PKM2 mutant compared to those expressing wild-type PKM2 (Fig. 1). Pyruvate itself can also be converted to acetyl–coenzyme A (CoA) for oxidative phosphorylation. With tyrosine phosphorylation of the wild-type PKM2 by FGFR1 resulting in decreased pyruvate concentrations, it is possible that GPT-mediated glutamine oxidation is diminished. In this case, the alternative pathway for glutamine oxidation is augmented, with glutamate dehydrogenase converting glutamine to α-ketoglutarate. α-Ketoglutarate can proceed through the TCA cycle to be incorporated into adenosine triphosphate and biosynthetic precursors for growth and proliferation, or can exit the cycle as malate to be converted into pyruvate and then lactate (Fig. 1). This scenario would allow lactate production to be fueled by glutamine rather than glucose carbons, particularly when PKM2 activity is suppressed.

With the increasing evidence for the role of glutamine metabolism within the network of cancer metabolism, studies of glucose metabolism must account for all processes that potentially contribute to the production of lactate, which is ultimately produced from pyruvate by LDHA (5, 1518). Because Hitosugi et al. and Cooper et al. found that LDHA was tyrosine-phosphorylated, the roles of these phosphorylations downstream of tyrosine kinase signaling will be critically important to fully understand how receptor signaling contributes to cancer cell metabolism in general and to the Warburg effect in particular (10, 11). Furthermore, if the phosphorylation of MDH2 reported by Hitosugi et al. reduces its conversion of malate to oxaloacetate or affects metabolite channeling, then glutamine carbons converted to α-ketoglutarate could be further shunted out of the mitochondria as malate and then converted to lactate. It is expected that further studies in cancer metabolism, using modern tools and a different perspective, will provide a new view of the Warburg effect in which signaling pathways intersect with metabolic enzymes and aerobic glycolysis is associated with a concurrent increase in the use of glutamine by certain cancer cells. Not only is glutamine a substrate for anabolic carbon, nitrogen, nucleotide, and carbohydrate metabolism, redox homeostasis, and energy production, but its use also affects glucose uptake and glycolysis (22). Understanding the interplay between glucose and glutamine metabolism is hence critical to our full appreciation of cancer metabolism (19, 20, 22).

Acknowledgments

I thank A. Le, L. Gardner, and R. DeBerardinis for comments. Supported by the Leukemia and Lymphoma Society, NIH grants CA51497 and CA57341, and the AACR Stand Up to Cancer (SU2C) initiative. C.V.D. is a consultant for Agios Pharmaceuticals Inc.

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