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.

FIGURE 1.

PFKFB3 localizes to the nucleus. A, HeLa cells were plated onto chamber slides and transfected with either empty vector (vector) or a construct carrying FLAG-tagged PFKFB3 (Flag-PFKFB3) and 24 h later, cells were fixed in paraformaldehyde and incubated with mouse anti-FLAG primary and then with Alexa Fluor® 488-labeled secondary antibodies. Slides were analyzed with a confocal laser microscope. α-Tubulin was used as cytoplasmic marker. B, cytoplasmic (C) and nuclear fractions (N) were prepared from the indicated cell lines and subjected to Western blot using a PFKFB3 carboxyl terminus specific antibody. Membranes were also probed with antibodies against PFK-1, PFKFB2, and PFKFB4. The purity of cytoplasmic and nuclear fractions was confirmed by assessing the expression of α-tubulin and Oct-1 proteins, respectively.

FIGURE 2.

Nuclear localization of PFKFB3 mRNA splice variant 5 requires lysines 472 and 473. A, stop codons were introduced at the indicated positions below (*) in the construct carrying the full-length, His-tagged PFKFB3 splice variant 5 using site-directed mutagenesis and the resultant constructs were transfected into HeLa cells, which were then fixed and stained with a fluorescein isothiocyanate-conjugated monoclonal antibody against the His₆. L502X, PFKFB3-DAKKGPNPLMRRNSVTPLASPEPTKKPRINSFEEHVASTSAALPSCLPPEVPTQ*; F479X, PFKFB3-DAKKGPNPLMRRNSVTPLASPEPTKKPRINS*; K472X, PFKFB3-DAKKGPNPLMRRNSVTPLASPEPT*; R458X, PFKFB3-DAKKGPNPLM*. B, site-directed mutagenesis was used to substitute codons encoding lysines at 472 and 473 with codons encoding alanines using the construct containing the full-length, FLAG-tagged PFKFB3 splice variant 5. HeLa cells were transfected with constructs that encode either WT or mutant (K472A/K473A) PFKFB3 constructs and 24 h later cells were fixed in paraformaldehyde and incubated with a FLAG primary and then with Alexa Fluor® 488-labeled secondary antibodies. Images were captured under a confocal laser microscope. α-Tubulin was used as cytoplasmic marker. * indicates that residues afterwards are deleted.

FIGURE 3.

Ectopic expression of wild type PFKFB3 variant 5 does not activate glycolysis. HeLa cells were transfected with either WT or cytoplasmic mutant (K472A/K473A) FLAG-tagged PFKFB3 constructs, or with empty vector (Vec) as control. 48 h later (A) cells were lysed and subjected to Western blot using antibody specific for FLAG epitope; B, cells were lysed in NaOH and analyzed for total Fru-2,6-BP levels; C, nuclei were isolated and incubated in a kinase buffer (see “Experimental Procedures”) for 10, 20, and 40 min, and supernatants were assayed for Fru-2,6-BP levels; D and E, media was collected and analyzed for glucose and lactate. Measurements were done in triplicate, and results are presented as mean ± S.D., p < 0.01; ***, p < 0.001. F, for one-dimensional NMR analysis, cells were transfected with the above constructs and grown in [¹³C]glucose containing medium and 48 h later, medium was trichloroacetic acid-extracted, lyophilized, and dissolved in 100% D₂O. NMR spectra were recorded at 800 MHz on a Varian Inova Spectrometer. Shown are the methyl regions of representative samples.

FIGURE 4.

Ectopic expression of a kinase-active PFKFB3 in the nucleus stimulates cell proliferation. HeLa cells were transfected with wild type (WT), cytoplasmic (K472/473A), kinase-inactive (R75/76A) FLAG-tagged PFKFB3 constructs, or with empty vector (Vec) as control and 48 h post-transfection (A), Western blot was performed to confirm the equal expression of PFKFB3 from FLAG-tagged constructs; B, the trypan blue-excluding live cells were counted; and C, [³H]thymidine incorporation into DNA was assessed. Error bars indicate mean ± S.D. of three independent experiments. Asterisks indicate p < 0.001.

FIGURE 5.

Ectopic expression of a kinase-active, nuclear PFKFB3 regulates the expression of key cell cycle proteins and phosphorylation of the cell cycle inhibitor p27. A, HeLa cells were transfected with either a construct carrying PFKFB3, or empty vector as control, and 36 h later, cells were lysed and subjected to antibody array. Portions of the array illustrating the differential expression of cyclin D3, Cdk1, and Cdc25 proteins between vector control (Vec) and PFKFB3-expressing (PFKFB3) cells are shown. Each panel contains six replicates of a specific antibody-protein reaction. B, densitometric quantitation of the average signal for each protein between samples. C and D, HeLa cells were transfected with WT, cytoplasmic (K472/473A), kinase-inactive (R75/76A) FLAG-tagged PFKFB3 constructs, or with empty vector (Vec) as control and 36 h post-transfection, cells were lysed and subjected to Western blot analysis using antibodies specific for cyclin D3, Cdk1, Cdc25C, and β-actin (C), and densitometric quantitation of the Western blot (D). E and F, HeLa cells were transfected as above and Western blot analysis was used to detect total and phosphorylated (threonine 187)-p27 levels (E) and densitometric quantitation of the Western blot (F). Error bars indicate mean ± S.D. of two experiments.

FIGURE 6.

Fru-2,6-BP increases the phosphorylation of p27 at the Cdk-specific Thr-187 site in HeLa cell lysates. Exponentially growing HeLa cells were lysed, and bacterially expressed GST-p27 (4 μg) and Fru-2,6-BP (200 μm) were added. p27 was pulled down and subjected to Western blot using total and Thr-187-phosphorylated p27 antibodies. Densitometric quantitation of the average signal for Thr-187-phosphorylated p27 relative to total p27 was conducted on protein preparations from three separate experiments.