Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes

Abstract

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.

Fig. 1.

Proliferation and generation of lactate in human T lymphocytes. (A) The proportion of cells in S phase [bromodeoxyuridine (BrdU) incorporation and DNA content analysis by flow cytometry; details provided in SI Materials and Methods] at different times after initiation of proliferation. Result representative of three independent experiments. (B) Percentage of cells in S phase and production of lactate per 24 h at different times after initiation of proliferation; n = 3, mean ± SEM. (C) The appearance of PFKFB3 and geminin, and the disappearance of Cdh1, at different times after initiation of proliferation. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. AU, arbitrary units.

Fig. 2.

Effect of overexpression of Cdh1 on PFKFB3, lactate generation, and T-lymphocyte proliferation. All data shown in this figure were obtained 72 h after initiation of proliferation. (A) Overexpression of Cdh1 promoted the degradation of PFKFB3 and geminin, but did not affect the KEN box-mutant form of PFKFB3. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (B) The proportion of cells in S phase was greatly reduced in cells overexpressing Cdh1 compared with those expressing EV. Coexpression of PFKFB3 mut did not prevent the Cdh1-induced reduction in proliferation. Result representative of three experiments. (C) In Cdh1-overexpressing cells, both proliferation and lactate generation were significantly reduced. In cells cotransfected with Pfkfb3 mut, the generation of lactate was similar to EV values but proliferation was still reduced; n = 3, mean ± SEM; *P < 0.05 versus cells containing EV. AU, arbitrary units.

Fig. 3.

Effect of silencing Pfkfb3 on lactate generation and proliferation. (A) Pfkfb3 was silenced in T lymphocytes using shRNA (Pfkfb3 shRNA). Cell lysates were analyzed by Western blotting 72 h after initiation of proliferation. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (B) The proportion of T lymphocytes in S phase 72 h after transfection with Pfkfb3 shRNA was far less than that of T lymphocytes transfected with scrambled shRNA. Result representative of three experiments. (C) Both proliferation and lactate generation were significantly reduced in T lymphocytes 72 h after transfection with Pfkfb3 shRNA compared with those transfected with scrambled shRNA; n = 3, mean ± SEM; *P < 0.05 versus cells containing scrambled shRNA. AU, arbitrary units.

Fig. 4.

Pfkfb3 gene expression and the activity of APC/C-Cdh1. (A) The expression of Pfkfb3 mRNA in T lymphocytes increased with time after activation. The relative expression of Pfkfb3 was determined by RT-qPCR as described in SI Materials and Methods. Results are the mean ± SEM of three independent experiments; *P < 0.05 versus nonactivated (N.A.) T lymphocytes. (B) Incubation of proliferating T lymphocytes (48 h) with the proteasome inhibitor MG132 significantly enhanced the amounts of PFKFB3 and geminin detected. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (C) PFKFB3 protein was immunoprecipitated with anti-ubiquitin antibodies from lysates of T lymphocytes activated to proliferate for 48 h and treated with DMSO (−) or MG132 (10 μM) for 2 h. The immunoprecipitated proteins were subjected to Western blotting and detected with anti-PFKFB3 as detailed in SI Materials and Methods. Figure representative of three experiments. (D) Overexpression of Cdh1 markedly reduced the appearance of PFKFB3 72 h after initiation of proliferation. However incubation with MG132 prevented degradation of the protein. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (E) The expression of Pfkfb3 mRNA at 72 h after initiation of proliferation was similar in cells transfected with EV, with Cdh1, and with Cdh1 in the presence of MG132. Pfkfb3 mRNA was quantified by RT-qPCR as described in SI Materials and Methods. Values are mean ± SEM of three independent experiments.

Fig. 5.

GLS1 activity in T-lymphocyte proliferation. (A) Percentage of cells in S phase and generation of ammonia at different times after initiation of proliferation; n = 3, mean ± SEM. (B) Time course of GLS1, geminin, and Cdh1 expression after initiation of proliferation. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (C) Incubation of proliferating T lymphocytes (48 h) with MG132 significantly enhanced the amounts of GLS1 and PFKFB3 detected. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (D) GLS1 protein was immunoprecipitated with anti-ubiquitin antibodies from lysates of T lymphocytes activated to proliferate for 48 h and treated with DMSO (−) or MG132 (10 μM) for 2 h. The immunoprecipitated proteins were subjected to Western blotting and detected with anti-GLS1. Figure representative of three experiments. (E) Overexpression of Cdh1 in HEK 293 cells promoted degradation of overexpressed GLS1 but this effect was markedly reduced in cells cotransfected with ΔGls1. Proteins from a Western blot representative of thre independent experiments were quantified by densitometry. (F) The C-terminal region of human GLS1 and of a truncated form in which the KEN box has been deleted (ΔGLS1).

Fig. 6.

Effect of silencing Gls1 on ammonia generation and proliferation. (A) Gls1 was silenced in T lymphocytes using shRNA (Gls1 shRNA). Cell lysates were analyzed by Western blotting 72 h after initiation of proliferation. Proteins from a Western blot representative of three independent experiments were quantified by densitometry. (B) The proportion of T lymphocytes in S phase 72 h after transfection with Gls1 shRNA was significantly lower than that of T lymphocytes transfected with scrambled shRNA. Result representative of three experiments. (C) Both proliferation and rate of ammonia generation were significantly reduced in T lymphocytes 72 h after transfection with Gls1 shRNA compared with those transfected with scrambled shRNA; n = 3, mean ± SEM; *P < 0.05 versus cells containing scrambled shRNA. AU, arbitrary units.