Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle

Abstract

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-β-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.

Fig. 1.

Protein levels of the glycolysis-promoting enzyme PFKFB3 rise temporarily in mid- to late-G1 phase. (A) HeLa cells were released from double thymidine block (DTB) (Left) or DTB plus nocodazole (Right). Whole cell extracts from synchronized cells were subjected to immunoblotting at the indicated times after release. (B) The cell cycle profile of the cells at different times after release as determined by FACS analysis of DNA content. (C) Changes in Pfkfb3 (variants 1 and 2) mRNA levels at different times after release as determined by qRT-PCR. (A and C) Representative of three independent experiments. (B) Mean of three independent experiments.

Fig. 2.

Regulation of the stability of PFKFB3 during the cell cycle. (A) PFKFB3 is detectable by immunoblotting at 8 h in cells released from DTB and nocodazole (Left). Treatment with the proteasomal inhibitor MG 132 (2 h after release) resulted in detectable amounts of the protein at other time points. (B) Illustration of the WT PFKFB3 and various mutations carried out in the KEN box, the DSG box, and in both recognition sites. (C) Effect of overexpressed β-TrCP on PFKFB3 in synchronized (DTB plus nocodazole) cells. Immunoblotting was carried out 8 h after release in cells expressing only β-TrCP or in which the KEN box-mutated or the double-mutated forms of PFKFB3 were overexpressed in the presence or absence of overexpressed β-TrCP. (D) Time course of the appearance of PFKFB3 in synchronized cells overexpressing the WT form (PFKFB3 KEN^WT DSG^WT) or the mutated forms shown in Fig. 2B (representative of three independent experiments).

Fig. 3.

PFKFB3 can only promote glycolysis at a certain stage of cell proliferation. (A) The appearance of PFKFB3 was compared with the production of lactate and the cell cycle stage in nontransfected cells synchronized by DTB plus nocodazole. Lactate was measured as the difference from the previous hour in the amount detected. (B) The same procedure was carried out in cells transfected with PFKFB3 KEN^MUT DSG^MUT. Despite the constant presence of PFKFB3 in the double-mutant form during the cell cycle, the pattern of lactate generation was similar to that in cells that had not been transfected (representative of three to four independent experiments).

Fig. 4.

Silencing PFKFB3 in synchronously proliferating cells blocks G1-to-S progression. (A) Schematic diagram showing experimental design for PFKFB3 silencing in cells synchronized by DTB plus nocodazole. (B) At 8 h after release from nocodazole arrest, PFKFB3 protein was present in cells transfected with nontargeting (NT) control siRNA but was greatly reduced in PFKFB3-silenced cells. (C) Flow cytometry profiles of DNA content (Left) and derived cell cycle phase distribution (Right) of synchronized cells transfected with NT siRNA or with Pfkfb3 siRNA 12 h after release from nocodazole and (D) 18 h after release from nocodazole. (B; and C and D, Left) Representative of three independent experiments. (C and D, Right) Mean of three independent experiments.

Fig. 5.

PFKFB3 links glycolytic activity with S phase entry. (A) Schematic diagram showing design of experiment in which cells were deprived of glucose for 48 h, treated with Pfkfb3 siRNA (or nontargeting control siRNA), incubated in the absence of glucose for a further 24 h, and then incubated for 18 h in the presence of glucose. (B) qRT-PCR and immunoblots of cell lysates prepared 18 h after replacement of glucose showing that PFKFB3 mRNA and protein levels were significantly reduced in cells transfected with Pfkfb3 siRNA. (C) Flow cytometry profiles of DNA content (Left) and derived cell cycle phase distribution (Right) of control-transfected and PFKFB3-silenced cells incubated in glucose-free medium for 72 h (i.e., at 0 h) (Upper) and 18 h (Lower) after readdition of glucose. (D) Increase in lactate production in control-transfected but not PFKFB3-silenced cells 18 h after readdition of glucose. (B, Left, and D), mean ± SEM of three independent experiments. (B, Right, and C, Left), representative of three independent experiments. (C, Right) Mean of three independent experiments.

Fig. 6.

Proliferation is restored in PFKFB3-silenced cells by overexpression of PFK1. (A) qRT-PCR demonstrated that there was ∼90% reduction in Pfkfb3 mRNA levels in PFKFB3-silenced cells 48 h after transfection. (B) Immunoblots from asynchronously proliferating cells 48 h after transfection with nontargeting control siRNA plus empty vector (−/− lane), with Pfkfb3-siRNA plus empty vector or with Pfkfb3-siRNA plus PFK1. (C) Flow cytometry profiles of DNA content (Left) and derived cell cycle phase distribution (Right) 48 h after transfection with nontargeting control siRNA, with Pfkfb3-siRNA, or with Pfkfb3-siRNA plus PFK1. (A) Mean ± SEM of three independent experiments. (B; and C, Left) Representative of three independent experiments. (C, Right) Mean of three independent experiments.