Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth - PubMed (original) (raw)

. 2011 Jun 24;42(6):719-30.

doi: 10.1016/j.molcel.2011.04.025.

Dong Li, Di Zhao, Ruiting Lin, Yajing Chu, Heng Zhang, Zhengyu Zha, Ying Liu, Zi Li, Yanping Xu, Gang Wang, Yiran Huang, Yue Xiong, Kun-Liang Guan, Qun-Ying Lei

Affiliations

Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth

Lei Lv et al. Mol Cell. 2011.

Abstract

Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Acetylation at K305 Decreases PKM2 Enzyme Activity

(A) PKM2 is acetylated. Flag-PKM2 was transfected into 293T cells followed by treatment with TSA and NAM for the indicated time, and PKM2 acetylation and protein levels were analyzed by western blot with indicated antibody, respectively. (B) Mutation of K305 decreases PKM2 acetylation. The indicated plasmids were cotransfected into 293T cells, and protein was immunoprecipitated (IP) for acetylation analysis. Acetylation levels were normalized against β-actin. (C) Characterization of acetyl-PKM2 (K305) antibody. The indicated plasmids were transfected into 293T cells, and acetylation level of IPed Flag-PKM2 was measured by the site-specific K305 acetylation antibody. (D) Endogenous PKM2 is acetylated at K305. 293T cells were treated with TSA and NAM. Endogenous PKM2 was immunoprecipitated, and protein levels and acetylation of K305 were determined by western blot with indicated antibodies (left panel). Relative PKM2 K305 acetylation over protein level was quantified (right panel). Error bars represent ± SD for triplicate experiments. (E) K305Q mutant decreases PKM2 enzyme activity. Flag-tagged wild-type and mutant PKM2 protein were expressed in 293T cells and purified by IP. The enzyme activity was measured and normalized against protein level. Mean values of relative enzyme activity of triplicate experiments with standard deviation (±SD) are presented. (F) NAM and TSA treatment decreases PKM2 wild-type but not mutant enzyme activity. Flag-tagged wild-type and mutant PKM2 protein were expressed in 293T cells and treated with or without NAM and TSA, then purified by IP. The PKM2 enzyme activity was measured and normalized against protein level. Mean values of relative enzyme activity of triplicate experiments with standard deviation (±SD) are presented. (G) K305Q mutation decreases the binding affinity toward PEP. The activities of wild-type and mutant PKM2 were assayed with increasing concentrations of ADP or PEP as indicated. Error bars represent ±SD for triplicate experiments.

Figure 2

Figure 2. Glucose and PCAF Regulate PKM2 Acetylation and Protein Levels

(A) High glucose increases PKM2 acetylation at K305 and decreases PKM2 protein level. HeLa cells were cultured with 25 mM glucose medium. The steady-state level and K305 acetylation of endogenous PKM2 were analyzed by western blot. PKM2 protein and acetylation levels were normalized against β-actin. (B) PCAF binds with PKM2. 293T cells were transfected with indicated plasmids, and PCAF-PKM2 association was examined by IP and western blot. (C) PCAF increases PKM2 acetylation at K305. 293T cells were transfected with indicated plasmids, and PKM2 acetylation at K305 was determined by western blot. (D) High glucose promotes the interaction between PCAF and PKM2. The indicated plasmids were cotransfected into 293T cells, followed by indicated glucose concentration treatment, and the interaction between PKM2 and PCAF was examined by immunoprecipitation. (E) Knocking down PCAF decreases endogenous K305 acetylation of PKM2. siRNA oligo targeting PCAF was transfected into 293T cells, and the levels of endogenous PCAF, PKM2 protein, and K305 acetylation were determined by western blot. (F) PCAF negatively regulates PKM2 activity. PCAF expression plasmid or siRNA oligo targeting PCAF was cotransfected into 293T cells with a vector expressing Flag-PKM2. PKM2 was immunoprecipitated and activity was assayed. The mean value of triplicate experiments and standard deviation are presented. (G) PCAF decreases endogenous PKM2 protein level. PCAF plasmid was transfected into 293T cells and endogenous PKM2 protein level was measured by western blot. (H) TSA and NAM treatment decreases the level of wild-type PKM2 but not the PKM2K305Q and PKM2K305R mutants. wild-type, K305Q, and K305R mutants expressing stable cells were treated with or without TSA and NAM, followed by western blot to determine PKM2 protein levels. PKM2 protein levels were normalized against β-actin.

Figure 3

Figure 3. Glucose-Induced PKM2 Degradation Requires CMA

(A) High glucose induces moderate increase of PKM2 mRNA. HeLa cells were cultured with the increasing concentrations of glucose. The level of PKM2 mRNA was determined by qPCR and normalized against GAPDH. Error bars represent ±SD for triplicate experiments. (B) Inhibition of deacetylases does not significantly affect PKM2 mRNA level. HeLa cells cultured in 25 mM glucose were treated with or without NAM and TSA. The levels of PKM2 mRNA were determined by qPCR using GAPDH as an internal control. The level of PKM2 mRNA in the cells treated with or without NAT and TSA are shown. Error bars represent ±SD for triplicate experiments. (C) PKM2 is not degraded by the 26S proteasome pathway. 293T cells were treated with proteasome inhibitor MG132, and the PKM2 protein level was analyzed by western blotting. PEPCK, a known proteasome substrate, was included as a control. (D) Leupeptin accumulates K305 acetylation and PKM2 protein. 293T cells were treated with or without Leupeptin. The levels of total and acetylated PKM2 were determined by western blot. PKM2 level was normalized against β-actin. (E) CMA activator 6-aminonicotinamide decreases PKM2 protein levels. 293T cells were treated with or without 6-aminonicotinamide (6-AN), and PKM2 level was determined by western blot. (F) Serum withdrawal decreases PKM2 protein. PKM2 level was determined by western blot after serum withdrawal as indicated in 293T cells. (G) HSC70 knockdown accumulates PKM2. Plasmid expressing HSC70 antisense was transfected into 293T cells. HSC70 knockdown efficiency and PKM2 level were determined by western blot. (H) LAMP2A knockdown accumulates PKM2. LAMP2A was stably knocked down in HeLa cells by shRNA. LAMP2A knockdown efficiency was determined by Q-PCR while PKM2 level was determined by western blot. b-actin protein (second bottom panel) and mRNA (bottom panel, determined by qPCR) are shown as controls. (I) LAMP2A knockdown blocks the glucose effect on PKM2 protein levels. HeLa cell pools stably expressing LAMP2A shRNA were cultured in the presence of increasing concentrations of glucose. PKM2 expression level was determined by western blot.

Figure 4

Figure 4. PKM2 Acetylation Promotes Its Binding with HSC70 and Uptake by Lysosome

(A) Acetyl mimetic K305Q mutation increases the binding between PKM2 and HSC70. The plasmids were cotransfected into 293T cells as indicated, the binding between PKM2 and HSC70 were examined by IP-western analysis. (B) Inhibition of deacetylases increases PKM2-HSC70 binding. The plasmids were cotransfected into 293T cells as indicated, followed by NAM+TSA treatment. PKM2-HSC70 binding was determined by IP-western analysis, and the levels of total and acetylated PKM2 were determined by western blot (lower panels). (C) HSC70 reduces acetylated PKM2. The plasmids were cotransfected into 293T cells as indicated and followed by NAM+TSA treatment, and PKM2 acetylation at K305 was determined by western blot. (D) The HCS70 preferentially interacts with K305-acetylated PKM2. HA-PKM2-transfected cells were immunoprecipitated with HSC70 antibody (indicated as IP). Relative acetylation of total cellular PKM2 (Input) and the HSC70-associated pKM2 (IP) were determined by western blot with indicated antibodies. (E) Endogenous PKM2 associates with HSC70. Cell lysate from H1299 cells was immunoprecipated with PKM2 antibody and followed by western blot analysis as indicated. (F) Glucose promotes the PKM2-HSC70 association. Indicated plasmids were cotransfected into 293T cells followed by treatment with media with different glucose concentrations. The binding between PKM2 and HSC70 was analyzed by IP-western. (G) Identification of a HSC70 binding motif in PKM2. The binding between PKM2 mutants (generated on K305Q plasmid) and HSC70 was analyzed by IP-western. (H) PCAF increases PKM2 acetylation and shifts it to higher molecular weight fractions. The indicated plasmids were cotransfected into 293T cells, and cell lysates were separated by gel filtration, followed by western analysis. Fraction numbers and elution of molecular weight markers (MW) are indicated. The acetylated PKM2 is exclusively present in the high molecular weight fractions. (I) Inhibition of deacetylases promotes lysosomal uptake of PKM2. Flag-tagged wild-type or HSC70-binding deficient mutant PKM2 was immunopurified from 293T cells treated with or without TSA and NAM, and incubated with the lysosomes isolated from rat liver. Lysosomes were reisolated and the associated PKM2 (either inside or binding to the surface) was determined by western blot. The presence of protease inhibitors (cocktail) should block lysosomal degradation, thus such conditions should measure both PKM2 binding to lysosomes and uptake, whereas in the absence of cocktail this assay only measures PKM2 binding to lysosomes. (J) The HSC70 binding-defective PKM2-393L/394A mutant has weak lysosomal binding. Experiments are similar to those in (I), except PKM2 mutant was used.

Figure 5

Figure 5. Accumulation of Glycolytic Intermediates and NADPH in Cells Expressing Acetylation Mimetic K305Q Mutant

(A) Cells expressing acetylation mimetic K305Q mutant of PKM2 accumulate higher levels of glycolytic intermediates. Metabolites in H1299 cells wild-type (H1299/PKM2) or K305Q mutant (H1299/PKM2K305Q) PKM2 were analyzed by LC-MSMS. The ratios of individual metabolites in H1299/PKM2 expressing over H1299/PKM2K305Q-expressing cells are shown from three independent samples. PKM2 knockdown efficiency and re-expression were determined by western blot. Error bars represent ±SD for triplicate experiments. (B) H1299/PKM2K305Q cells accumulate NADPH. Cells used in the assay were the same as in (A). NADPH was measured using a NADPH kit according to the manufacturer’s protocol. Error bars represent ±SD for triplicate experiments.

Figure 6

Figure 6. PKM2 K305Q Promotes Cell Proliferation and Tumor Growth

(A) The expression of PKM2 and PKM2K305Q in xenograft. Whole cell tracts were prepared from either original stable H1299/PKM2 and H1299/PKM2K305Q pools or xenograft tumors, followed by western blot. (B) PKM2K305Q promotes cell proliferation. 3 × 104 H1299/PKM2 or H1299/PKM2K305Q cells were seeded in each well. Cell numbers were counted every 24 hr. Error bars represent ±SD for triplicate experiments. (C and D) PKM2K305Q mutant promotes xenograft tumor growth. Nude mice injected on the left side with H1299/PKM2K305Q cells and the right side with H1299/PKM2 cells. The xenograft tumors were measured over time and dissected at the endpoint and shown as H1299/PKM2 (upper row) and H1299/PKM2K305Q (lower row) in (C). Quantification of average width of tumors over time is shown in (D). Error bars represent ±SD for ten tumors.

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