Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation

Abstract

Despite the fact that most cancer cells display high glycolytic activity, cancer cells selectively express the less active M2 isoform of pyruvate kinase (PKM2). Here we demonstrate that PKM2 expression makes a critical regulatory contribution to the serine synthetic pathway. In the absence of serine, an allosteric activator of PKM2, glycolytic efflux to lactate is significantly reduced in PKM2-expressing cells. This inhibition of PKM2 results in the accumulation of glycolytic intermediates that feed into serine synthesis. As a consequence, PKM2-expressing cells can maintain mammalian target of rapamycin complex 1 activity and proliferate in serine-depleted medium, but PKM1-expressing cells cannot. Cellular detection of serine depletion depends on general control nonderepressible 2 kinase-activating transcription factor 4 (GCN2-ATF4) pathway activation and results in increased expression of enzymes required for serine synthesis from the accumulating glycolytic precursors. These findings suggest that tumor cells use serine-dependent regulation of PKM2 and GCN2 to modulate the flux of glycolytic intermediates in support of cell proliferation.

Fig. 1.

Serine increases PKM2 activity and glycolytic rate. (A) In vitro PKM2 activity increases in the presence of serine. Assay performed with a series of serine concentrations as indicated with and without 10 μM FBP (data represent mean ± SEM, n = 3). (B) Equal numbers of H1299 cells were incubated with various concentrations of serine for 16 h, cells were trypsinized, stained with Trypan blue, and counted (data represent mean ± SEM, n = 3). (C) Glucose consumption and lactate production in B were measured as described in Materials and Methods and normalized to cell number (data represent mean ± SEM, n = 3). (D) The lactate production/glucose consumption ratio increases with the serine concentration (data represent mean ± SEM, n = 3).

Fig. 2.

PKM2 expressing cells are more resistant to serine starvation than PKM1-expressing cells. (A) To generate cell lines expressing PKM1 or PKM2, H1299 cells expressing Flag-tagged (Left) or HA-tagged (Right) mouse PKM1 or PKM2 were transiently transfected with 20 nM nontargeting siRNA (NT) or PKM siRNA. After 48 h, PKM1/2 expression was measured using immunoblot. (B) The levels of [U-¹³C] PEP, [U-¹³C] 3-PG, and [U-¹³C] serine in Flag-M1/M2 cells were measured using LC-MS. Flag-M1/M2 cells were incubated in serine-free medium for 24 h and then labeled with [U-¹³C₆] glucose for 3 h. This process results in essentially complete labeling of PEP and 3-PG; thus, the data for these metabolites reflect total cellular concentrations. For serine, labeling is incomplete and the enhanced labeling reflects increased flux (data represent mean ± SEM, n = 3). *P < 0.05, two-tailed Student t test. (C) PKM2 confers resistance to serine starvation. Flag-M1/M2 (Left) or HA-M1/M2 (Right) cells were incubated with or without serine for 24 or 48 h, and cell proliferation was measured using an MTT assay. The ratios of proliferation between (−) serine and (+) serine media were calculated and plotted (data represent mean ± SEM, n = 3).

Fig. 3.

PKM2 maintains mTORC1 activity upon serine depletion. (A) M2 cells, but not M1 cells, maintain mTORC1 activity upon serine deprivation. Flag-M1/M2 (Left) or HA-M1/M2 (Right) cells were incubated with/without serine for 16 h, and mTOR activities were measured by immunoblotting for p-S6K and p-S6. Data are representative of three independent experiments. (B) Leucine withdrawal reduces mTORC1 activity in both M1 and M2 cells. Flag-M1/M2 cells were incubated with/without leucine for 16 h.

Fig. 4.

Blocking endogenous serine synthetic pathway by knocking down PSAT1. (A) Knocking down PSAT1 in tumor cells. DLD1 and H1299 cells were transfected with 50 nM nontargeting siRNA or PSAT1 siRNA. After 48 h, PSAT1 expression was measured using immunoblot. (B) Knocking down PSAT1 reduces tumor cell proliferation under serine starvation. Cells were transfected with PSAT1 siRNA as described earlier. After 48 h, transfected cells were incubated in media with or without serine for 24 or 48 h. Cell proliferation was measured using an MTT assay. The ratios of proliferation between (−) serine and (+) serine media were calculated and plotted (data represent mean ± SEM, n = 3). (C) Knocking down PSAT1 inhibits S6 phosphorylation upon serine depletion. H1299 cells were transfected with PSAT1 siRNA as described earlier. After 48 h, transfected cells were incubated with/without serine for 16 h. (D) Knocking down PSAT1 reduces proliferation of M2 cells significantly, but not M1 cells. HA-M1/M2 H1299 cells were transfected with PSAT1 siRNA as described before. After 48 h, transfected cells were incubated with or without serine for 24 or 48 h and cell proliferation was measured using an MTT assay. The ratios of proliferation between (−) serine and (+) serine media were calculated and plotted (data represent mean ± SEM, n = 3).

Fig. 5.

The induction of enzymes in the serine synthetic pathway under amino acid starvation depends on GCN2-ATF4 pathway. (A) DLD1 cells expressing nontargeting shRNA or ATF4 shRNA were starved in media without serine or glutamine for 6 h, and mRNA levels were measured using real-time PCR (data represent mean ± SD, n = 3). *P_PHGDH = 0.0011, P_PSAT1 = 0.0106, P_PSPH = 0.0038. (B) Wild-type, GCN2^−/− and ATF4^−/− MEFs were starved in the media without serine or glutamine for 6 h, and mRNA levels were measured using real-time PCR (data represent mean ± SD, n = 3). *P_PHGDH = 0.0124, P_PSAT1 = 0.0001, P_PSPH = 0.0004. (C) ATF4 is necessary for cell proliferation under serine starvation. (Upper) DLD1 cells expressing ATF4 shRNA (shATF4) or nontargeting shRNA (shNT) were incubated in media with or without serine for 24, 48, and 72 h. Cell proliferation was measured using an MTT assay. The ratios of proliferation between (−) serine and (+) serine media were calculated and plotted (data represent mean ± SEM, n = 3). (Lower) The protein levels of ATF4 and PSAT1 after 24-h serine withdrawal were measured using immunoblot. (D) Flag-M1/M2 cells were incubated with or without serine for 24 h. ATF4 and PSAT1 expression were measured using immunoblot. Data are representative of three independent experiments.