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. 2016 Feb 15;6:21524.
doi: 10.1038/srep21524.

Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

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Free PMC article

Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

Chang-Liang He et al. Sci Rep. .
Free PMC article

Abstract

In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells.

Figures

Figure 1
Figure 1. Quantitative Phosphoproteomic Approach to Survey PKM2 Substrates.
(A) Schematic diagram of the PKM2 substrate survey strategy. Proteins in RCC lysate were treated as indicated in the blue brackets, the resulted peptides were labeled, mixed and enriched for phosphopeptides, followed by LC-MS/MS analysis to identify PKM2 substrates. (B) Proteins in a lysate of RCC were de-phosphorylated with alkaline phosphatase and precipitated with acetone. The redissolved proteins were treated with recombinant PKM2 or PKM2K270M (K270M), employing PEP as the phosphor-donor. The levels of P-Ser, P-Thr and P-Tyr in proteins were determined after treatments.
Figure 2
Figure 2. Bioinformatics analysis of the substrates of PKM2.
(A) The PKM2 phosphorylated serine (upper) and threonine (lower) sites identified were aligned and their spanning amino acid residues were visualized with WebLogo. (B–D) The solvent accessibility (B), secondary structures distributions (C) and sub-cellular localizations (D) of PKM2 substrates were predicted with NetSurfP 1.1, ESpritz and WoLF PSORT, respectively. (E,F) The enriched biological processes (E) and the enriched KEGG pathways (F) of identified substrates are demonstrated with heatmaps.
Figure 3
Figure 3. Serine202 and serine203 are phosphorylated by PKM2.
(A) MS/MS spectra of tryptic peptides from the PKM2-treated HEK293T proteome that led to the identification of phosphorylation of serine202 (left) or serine 203 (right) of AKT1S1. (B) Flag-tagged AKT1S1 and Myc-tagged PKM2 were co-expressed in HEK293T cells. The PKM2 co-purified with the AKT1S1 was detected by Myc antibody. (C) Flag-tagged AKT1S1 and HA-tagged PKM2 were co-expressed in HEK293T cells. The AKT1S1 co-purified with the PKM2 was detected by Flag antibody. (D)Purified AKT1S1 was treated with recombinant PKM2 in the presence and absence of PEP, and the levels of P-Ser of AKT1S1 in the reaction mixture after treatment were determined. Numerical values below the gels indicate quantification of the bands relative to untreated AKT1S1 (hereinafter). (E) Flag-tagged AKT1S1 was co-expressed with PKM2. P-ser levels of AKT1S1 from cells cultured with and without TEPP-46 (100 nM) supplementation were determined. (F) Flag-tagged AKT1S1 was co-expressed with PKM2 or PKM2Y105F (Y105F), P-ser levels of AKT1S1 purified from different cells were determined. (G) Flag-tagged AKT1S1 was co-expressed with either HA-tagged PKM2 or HA-tagged PKM2K270 mutant (K270M) in HeLa cells. The P-Ser levels of Flag bead-purified AKT1S1 from each culture were determined and quantified. (I) Purified AKT1S1 was treated with either purified PKM2 or purified K270M. The P-S202, P-S203 and P-S202/203 levels of each treated AKT1S1 were determined by site-specific antibodies and quantified. The relative intensities of phosphorylation signals were normalized to those of untreated AKT1S1. (J) Flag-tagged PKM2 or Flag-tagged K270M was overexpressed in HEK293T cells. The endogenous P-S202/203 levels of AKT1S1 of each culture were determined and the relative intensities of P-S202/203 signals were normalized to that of HEK293T cells. (K) The endogenous P-S202/203 levels of HEK293T cells before and after PKM2 knockdown by independent shRNAs were compared. The PKM2 knockdown efficiency was confirmed by western blot.
Figure 4
Figure 4. PKM2 releases AKT1S1 from raptor and promotes AKT1S1-14-3-3 interaction by phosphorylating S202/203 of AKT1S1.
(A) Purified raptor was incubated with AKT1S1, AKT1S1 + PEP or AKT1S1 + PEP + PKM2. After incubation, AKT1S1 was purified by Flag beads. The amount of raptor co-purified with AKT1S1 and the PS202/203 level of AKT1S1 were determined and normalized to that of AKT1S1 incubated with raptor without other components. (B) Raptor and AKT1S1 were co-expressed with either PKM2 or K270M in HEK293T cells. The amount of raptor co-precipitated with AKT1S1 from different cells was determined. (C) Raptor was co-expressed with either AKT1S1 or 2SA in HEK293T cells. The amount of raptor co-immunoprecipitated with AKT1S1 or 2SA in the presence and absence of PKM2 was compared. (D) Raptor was co-expressed with either AKT1S1 or 2SE in HEK293T cells. The amount of raptor co-immunoprecipitated with AKT1S1 or 2SE in the presence and absence of PKM2 was determined and raptor signals were normalized to that co-expressed with AKT1S1 alone. (E) Raptor was co-expressed with either S202A or S203A in HEK293T cells. The amount of raptor co-immunoprecipitated with S202A or S203A in the presence and absence of PKM2 was determined and compared. (F) 14-3-3 and AKT1S1 were co-expressed with either PKM2 or K270M in HEK293T cells. The amount of 14-3-3 co-immunoprecipitated with AKT1S1 was compared under different co-expression conditions.
Figure 5
Figure 5. PKM2 activates mTORC1 signaling and promotes cell proliferation by phosphorylating S202/203 of AKT1S1.
(A) Raptor was co-expressed with 4E-BP1 in HeLa cells and in PKM2 overexpressing HeLa cells. The amount of raptor co-immunoprecipitated with 4E-BP1 in different cells was determined by western blot and normalized to that of without PKM2 expression. (B) Raptor was co-expressed with 4E-BP1 in HeLa cells and in PKM2 knockdown HeLa cells. The amount of 4E-BP1 co-immunoprecipitated with raptor in different cells and was compared. (C) The levels of P-T389-S6K and P-T37/46-4EBP in HEK293T cells and in PKM2 overexpressing HEK293T cells were detected and quantified under with or without rapamycin supplementation in the culture media, respectively. All intensities of P-T389-S6K and P-T37/46-4EBP signals were normalized to those of untreated cells. (D) The levels of P-T37/46-4EBP were determined by immunofluorescence in HeLa cells and PKM2 overexpressing HeLa cells with and without rapamycin supplementation in the culture media, respectively. Bar scales are 25 μm. (E) EGF effects on the levels of endogenous P-T389-S6K and P-T37/46-4EBP of HEK293T cells and PKM2 knockdown HEK293T cells were determined and quantified relative to that of non-treated cells. (F) AKT1S1 was knocked down by shRNA in HEK293T cells (bottom). The levels of P-T389-S6K and P-T37/46-4EBP in response to PKM2 overexpression were determined in AKT1S1 knockdown cells after shRNA resistant AKT1S1, 2SA and 2SE were each re-introduced into cells. P-T389-S6K and P-T37/46-4EBP signals were normalized relative to untreated cells. (G) Growth curves of HEK293T cells, the PKM2 overexpressing HEK293T cells, the AKT1S1 knockdown HEK293T cells and PKM2 overexpressing AKT1S1 knockdown HEK293T cells were determined. Shown are the average values (n = 3) with SD. Knockdown efficiency of AKT1S1 is demonstrated in (F). (H–J) The levels of endogenous P-T389-S6K and P-T37/46-4EBP of HEK293T cells were detected under serum starvation (SS, H), amino acids starvation (AAs Starv., I) and both (J) were detected. Amino acids starvation was achieved by culturing cells in basal DMEM with all other ingredients except amino acids.
Figure 6
Figure 6. PKM2 inhibits autophagy by activating mTORC1.
(A) Cells were cultured under either normal DMEM or serum starvation (SS) conditions. The levels of LC3B II and p62 in HeLa cells and HeLa cells expressing PKM2 were determined and normalized to those of untreated cells, respectively. (B) Cells were cultured under either normal DMEM or SS conditions. The LC3B levels in response to PKM2 overexpression were detected by immunofluorescence. Shown are representative immunofluorescence results (left) and average value of quantization of triplicate experiments with S.D.(right). Bar scales are 100 μm. (C) Under SS, the levels of LC3B and p62 in HEK293T cells expressing or not expressing PKM2 were compared in both the absence and presence of rapamycin. Signals of LC3B and p62 were quantified relative to those of neither PKM2 nor rapamycin was treated cells. (D) PKM2 was overexpressed in HEK293T cells cultured in SS and chloroquine (100 nM)-supplemented media. The PKM2 effects on the levels of LC3B and levels of p62 were determined and compared in each cells. Signals of LC3B and p62 were quantified relative to those of untreated cells. (E) Autophagy was induced by serum starvation in HEK293T cells, the effects of TEPP-46 on levels of LC3B and p62 were detected in HEK293T cells and in HEK293T cells overexpressing PKM2. TEPP-46 effects on autophagy markers were also examed in HEK293T cells without serum starvation as control. (F) The levels of LC3B and p62 of HEK293T cells were determined in the overexpressing of AKT1S1 or 2SA and co-expression of AKT1S1 and PKM2 or 2SA and PKM2. Signals of LC3B and p62 were quantified relative to those of untreated cells.
Figure 7
Figure 7. Correlation among PKM2 overexpression, S202/203 phosphorylation and mTORC1 activation in RCC and breast cancers.
(A,B) PKM2, P-S202/203, P- S2448-mTOR, p-T37/46-4EBP and LC3B levels of the same patient were detected in RCC (A) and breast cancer (B) tissues and adjacent normal tissues. Representative IHC (left) and statistic (right, n = 10) results are shown. For RCC samples, normal and tumor tissues are marked by N and T, respectively. For breast cancer, tumor and normal tissues are marked by red and yellow arrows, respectively. Pathologic results were confirmed by experienced pathologists. Bar scales were 100 μm, heights of breast cancer samples were compressed to 1/2. (C) Schematic diagram of the PKM2-mTORC1 regulatory loop. Overexpression of PKM2 leads accumulation of anabolic intermediates and activation of mTOR signaling that promotes utilization of anabolic intermediates and inhibits autophagy. Meanwhile, mTOR activates PKM2 to form a positive loop to enhance the anabolic processes. Signals that activate either PKM2 or mTORC1 can result in both anabolic functions.

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