Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

Abstract

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Neoplastic transformation and growth require a massive increase in glucose uptake and glycolytic flux not only for energy production but also for the synthesis of nucleic acids, amino acids, and fatty acids. A central control point of glycolysis is the negative allosteric regulation of a rate-limiting enzyme, phosphofructokinase-1 (PFK-1), 2 by ATP (i.e. the Pasteur effect) (1, 2). When intracellular ATP production exceeds usage, ATP inhibits PFK-1 and glycolytic flux. Fructose 2,6-bisphosphate (Fru-2,6-BP) is a potent allosteric activator of PFK-1 that overrides this inhibitory influence of ATP on PFK-1, allowing forward flux of the entire pathway (3-5).The steady-state cellular concentration of Fru-2,6-BP is dependent on the activities of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four independent genes (PFKFB1-4) (6, 7). The PFKFB3 mRNA is distinguished by the presence of multiple copies of an AUUUA instability motif in its 3Ј-untranslated region and the PFKFB3 protein product has a high kinase:phosphatase activity ratio (740:1) (8). PFKFB3 mRNA is overexpressed by rapidly proliferating transformed cells and the PFKFB3 protein is high...

E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation

“…Our experiments have also confirmed the nuclear localization of PFKFB3 (16). The fact that Cdh1 is also localized in the nucleus (17) indicates that this is the site where both cyclins and PFKFB3 are targeted for degradation by APC/C-Cdh1.…”

Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

“…73 This is also the case for fructose-2,6-biphosphatase 3 (PFKFB3) 74 which, although not a direct participant in the glycolysis enzymatic cascade, has major effects on glycolysis through the allosteric regulator fructose 2,6-bisphosphate that apparently include variations in cellular energy source (glutamine vs glucose) as a function of the cell cycle in cancer cells. 74,75 There are scattered experimental reports of further functions of these and other glycolytic enzymes in the nucleus, mitochondria and elsewhere in cells (Supporting Information Table 5). Furthermore, GPI, HK1 and TKT have been detected in the nucleus in large scale antibody screening of subcellular protein locations 76 (Supporting Information Table 5).…”

Akt-dependent Activation of the Heart 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFKFB2) Isoenzyme by Amino Acids

“…PFKFB1 and PFKFB4 are specific isoenzymes of liver/kidney and testis tissues, respectively (1,25). In HeLa cells, predominant expression of PFKFB2 and PFKFB3 has been reported (22,26,27). To analyze whether the increase in Fru-2,6-P 2 concentrations caused by amino acids correlated with an increase in 6-phosphofructo-2-kinase (PFK-2) activity, we measured the activity of this enzyme.…”