Research ArticleCELL BIOLOGY

The protein kinase activity of fructokinase A specifies the antioxidant responses of tumor cells by phosphorylating p62

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Science Advances  24 Apr 2019:
Vol. 5, no. 4, eaav4570
DOI: 10.1126/sciadv.aav4570
  • Fig. 1 KHK-A is required for oxidative stress–enhanced p62 oligomerization and Nrf2 activation in HCC cells.

    (A to D and G and H) Immunoblot analyses were performed with the indicated antibodies. (A) Huh7 and Hep3B cells expressing Flag-p62 were treated with or without hypoxia for 6 hours in the presence of 10 μM chloroquine (CQ; a lysosome inhibitor). Immunoprecipitation analyses with an anti-Flag antibody were performed. Hif 1α, hypoxia-inducible factor 1α; WB, Western blot; IP, immunoprecipitation; IgG, immunoglobulin G. (B) Huh7 and Hep3B cells with or without expressing KHK short hairpin RNA (shRNA) and with or without reconstituted expression of the indicated proteins were transfected with vectors expressing Flag-p62 and HA-p62 and treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). After incubation with the reversible cross-linking agent DSP (0.4 mg/ml) for 2 hours, the cells were lysed in a buffer containing 1% SDS to solubilize all proteins. The lysates were subjected to immunoprecipitation analyses with an anti-Flag antibody after diluting SDS to 0.1%. (C) Huh7 cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated proteins were treated with or without hypoxia for 6 hours and lysed and analyzed by reducing (containing 2.5% β-mercaptoethanol) and nonreducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE) to detect p62 aggregation. (D) Huh7 and Hep3B cells with or without expression of KHK shRNA were reconstituted with or without expression of the indicated KHK proteins. After stimulation with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM), the cells were lysed in a lysis buffer with 1% Triton X-100. The insoluble fraction was lysed in a lysis buffer with 1% SDS. WCL, whole-cell lysate. (E and F) Huh7 cells with or without KHK shRNA expression and with or without reconstituted expression of Flag-tagged rKHK-A or rKHK-C were stimulated with or without hypoxia for 6 hours. Immunofluorescent analyses were performed with the indicated antibodies (E). The numbers of puncta in 100 cells were counted and quantified. Data are shown as means ± SD of 100 cells per group. A two-tailed Student’s t test was used. **P < 0.01 (F). (G) Huh7 and Hep3B cells expressing KHK shRNA with or without reconstituted expression of the indicated proteins were treated with or without hypoxia and lysosome inhibitor CQ (10 μM) for the indicated periods of time. (H) The indicated cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated proteins were treated with or without hypoxia for 12 hours. The nuclear fractions were prepared. PCNA, proliferating cell nuclear antigen. (I) The indicated cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated proteins were transfected with quinone oxidoreductase 1 (NQO1)–ARE-luc and pRL-TK (Renilla luciferase control reporter vector) plasmids. Starting at 18 hours after transfection, cells were treated with or without hypoxia for 12 hours and harvested for luciferase activity analyses. The data are presented as means ± SD from triplicate samples. **P < 0.01.

  • Fig. 2 AMPK phosphorylates KHK-A and promotes the association between KHK-A and p62.

    Immunoprecipitation or immunoblot analyses were performed with the indicated antibodies. (A) Huh7 cells transfected with Flag–KHK-A were pretreated with SB203580 (10 μM), SP600125 (25 μM), U0126 (20 μM), or compound C (5 μM) before hypoxia stimulation for 6 hours in the presence of CQ (10 μM). (B) Huh7 cells expressing Flag–KHK-C or Flag–KHK-A were cotransfected with HA-AMPKα and treated with or without hypoxia for 6 hours or H2O2 (0.5 mM) for 1 hour in the presence of the lysosome inhibitor CQ (10 μM). (C) Huh7 cells transfected with Flag–KHK-A were pretreated with or without compound C (5 μM) for 30 min before hypoxia stimulation for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). ACC1, acetyl-CoA carboxylase 1. (D) WT and AMPKα1/2 double-knockout (DKO) mouse embryonic fibroblasts (MEFs) infected with lentivirus expressing Flag–KHK-A were treated with or without A769662 (0.5 mM) for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (E and F) Bacterially purified GST–KHK-A, GST–KHK-C (E), or GST–KHK-A S80A (F) was incubated with or without bacterially purified active AMPK in the presence of [γ-32P]ATP. Autoradiography and immunoblot analyses were performed. (G) Huh7 cells expressing WT Flag–KHK-A, Flag–KHK-C, or Flag–KHK-A S80A were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (H) WT and AMPKα1/2 DKO MEFs were cultured with or without hypoxia for 6 hours. (I) An in vitro AMPK kinase assay was performed by mixing bacterially purified WT GST–KHK-A, GST–KHK-A S80A, or GST–KHK-C on glutathione agarose beads with or without purified active AMPK in the presence of AMP and ATP for 1 hour. The glutathione agarose beads were then washed and incubated with purified His-p62 for a GST pulldown analysis. (J) Huh7 cells expressing Flag–KHK-A, Flag–KHK-C, or Flag–KHK-A S80A were treated with or without hypoxia for 4 hours in the presence of the lysosome inhibitor CQ (10 μM). (K) Huh7 cells expressing Flag–KHK-A and HA-AMPKα were pretreated with N-acetyl-l-cysteine (NAC) (5 mM) or Trolox (100 μM) for 30 min before hypoxia stimulation for 6 hours. (L) Huh7 cells expressing Flag–KHK-A were pretreated with NAC (5 mM) or Trolox (100 μM) for 30 min before hypoxia stimulation for 6 hours in the presence of the lysosome inhibitor CQ (10 μM).

  • Fig. 3 KHK-A acts as a protein kinase and phosphorylates p62 at S28.

    Immunoblot analyses were performed with the indicated antibodies. (A) An in vitro AMPK kinase assay was performed by mixing bacterially purified WT GST–KHK-A, GST–KHK-A S80A, or GST on glutathione agarose beads with or without purified active AMPK in the presence of AMP and ATP for 1 hour. The glutathione agarose beads were then washed and incubated with purified His-p62 in the presence of [γ-32P]ATP. Autoradiography was performed. (B) Huh7 cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated proteins were treated with or without A769662 (0.5 mM) for 4 hours in the presence of the lysosome inhibitor CQ (10 μM). (C) WT and AMPKα1/2 DKO MEFs were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (D) Huh7 and Hep3B cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated KHK proteins were pretreated with or without compound C (5 μM) for 30 min before hypoxia stimulation for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (E) Huh7 and Hep3B cells with or without expressing KHK shRNA and with or without reconstituted expression of the indicated proteins were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (F and G) Parental Huh7 and Hep3B cells and the indicated clones of cells with knock-in of KHK-A S80A (F) or p62 S28A (G) expression were stimulated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM).

  • Fig. 4 KHK-A–mediated p62 S28 phosphorylation is required for oxidative stress–enhanced p62 oligomerization and Nrf2 activation.

    (A to G and I) Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. (A) Huh7 and Hep3B cells with or without knock-in of KHK-A S80A expression were transfected with the vectors expressing Flag-p62 and HA-p62 and treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). After incubation with DSP (0.4 mg/ml) for 2 hours, the cells were lysed in a buffer containing 1% SDS to solubilize all proteins. The lysates were subjected to immunoprecipitation analyses with an anti-Flag antibody after diluting SDS to 0.1%. (B) Huh7 cells expressing the indicated p62 proteins were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). After incubation with DSP (0.4 mg/ml) for 2 hours, the cells were lysed in a buffer containing 1% SDS to solubilize all proteins. The lysates were subjected to immunoprecipitation analyses with an anti-Flag antibody after diluting SDS to 0.1%. (C) Huh7 and Hep3B cells with or without knock-in expression of p62 S28A were treated with or without hypoxia for 6 hours. The whole-cell lysates were analyzed by reducing and nonreducing SDS-PAGE to detect p62 aggregation. (D and E) Huh7 and Hep3B cells with or without knock-in expression of KHK-A S80A (D) or p62 S28A (E) were stimulated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (F) Huh7 cells with or without knock-in of KHK-A S80A expression were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (G) Endogenous p62-depleted Huh7 cells with or without reconstituted expression of WT Flag-rp62, rp62 S28A, or rp62 K7R were treated with or without hypoxia for 6 hours in the presence of the lysosome inhibitor CQ (10 μM). (H) Huh7 cells with or without knock-in of KHK-A S80A (top) or p62 S28A (bottom) expression were stimulated with or without hypoxia for 6 hours. Immunofluorescence analyses were performed with the indicated antibodies. Numbers of puncta in 100 cells were counted and quantified. Data are shown as means ± SD of 100 cells per group. A two-tailed Student’s t test was used. **P < 0.001. (I) Huh7 and Hep3B cells with or without knock-in of KHK-A S80A (top) or p62 S28A (bottom) expression were treated with or without hypoxia for 12 hours. The nuclear fractions were prepared. (J) Huh7 and Hep3B cells with or without knock-in of KHK-A S80A (top) or p62 S28A (bottom) expression were transfected with NQO1-ARE-luc and pRL-TK plasmids. After treatment with or without hypoxia for 12 hours, the cells were harvested for luciferase activity analyses. The data are presented as means ± SD from triplicate samples. **P < 0.001.

  • Fig. 5 p62 S28 phosphorylation reduces ROS production and promotes cancer cell survival.

    The data are presented as means ± SD from three independent experiments. **P < 0.001. A two-tailed Student’s t test was used. (A, B and E) LO2, Huh7, and Hep3B cells with or without expression of KHK shRNA were reconstituted with or without expression of the indicated KHK proteins. After treatment of the cells with or without hypoxia for 36 hours, ROS levels (A), intracellular NADP+ and NADPH levels (B), and the percentages of apoptotic cells (E) were measured. (C, D, and F) Huh7 cells with or without knock-in of KHK-A S80A (left) or p62 S28A (right) expression were treated with or without hypoxia for 36 hours. ROS levels (C), intracellular NADP+ and NADPH levels (D), and the percentages of apoptotic cells (F) were measured. (G to I) Huh7 cells with reconstituted expression of the indicated KHK-A proteins (top) or p62 proteins (bottom) were stably transfected with or without HA-tagged constitutively active Nrf2 and treated with or without hypoxia for 36 hours. ROS levels (G), intracellular NADP+ and NADPH levels (H), and the percentages of apoptotic cells (I) were measured.

  • Fig. 6 KHK-A–mediated p62 S28 phosphorylation promotes hepatocellular tumorigenesis and is associated with clinical aggressiveness of human HCC.

    (A) Huh7 (1 × 106) cells with or without knock-in of KHK-A S80A (top) or p62 S28A (bottom) expression were intrahepatically injected into athymic nude mice (n = 7 per group). The mice were euthanized and examined for tumor growth 28 days after injection. The arrows point to the tumors. Tumor volumes were calculated (right). Data represent the means ± SD of seven mice. **P < 0.01 by a two-tailed Student’s t test. (B) IHC analyses of tumor tissues were performed with an anti-Ki67 antibody. Ki67-positive cells were quantified in 10 microscope fields. **P < 0.01 by a two-tailed Student’s t test. (C and D) IHC analyses of xenograft tumors from nude mice were performed with the indicated antibodies. (E) IHC staining of 30 human HCC and matched nontumor tissue samples was performed with the indicated antibodies. Representative photos of stains of two cases are shown. (F) IHC staining of 90 human HCC specimens was performed with the indicated antibodies. Representative images from the staining of four different specimens are shown. (G) Kaplan-Meier plots of the overall survival rates in human HCC specimens (n = 90) in groups with high (staining score, 4 to 8) and low (staining score, 0 to 3) expression of KHK-A pS80, p62 pS28, and KHK-C. The P values were calculated using the log-rank test. (H) The mechanism underlying differentiated responses of normal hepatocytes and HCC cells to oxidative stress. Oxidative stress results in AMPK-mediated phosphorylation of KHK-A S80, primarily expressed in HCC cells, but not of KHK-C, primarily expressed in normal hepatocytes. S80-phosphorylated KHK-A acts as a protein kinase and phosphorylates p62 S28, resulting in p62 oligomerization, p62 and Keap1 aggregation, and nuclear translocation and activation of Nrf2, which lead to counteracting ROS production, maintaining tumor cell survival, and promoting HCC development. In contrast, normal hepatocytes, lacking KHK-A–mediated antioxidative response, have high levels of ROS production and increased apoptosis. Ub, ubiquitin.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaav4570/DC1

    Fig. S1. KHK-A is required for oxidative stress–enhanced p62 oligomerization.

    Fig. S2. KHK-A is required for Nrf2 activation upon oxidative stress.

    Fig. S3. AMPK phosphorylates KHK-A and promotes the association between KHK-A and p62.

    Fig. S4. AMPK-mediated KHK-A S80 phosphorylation inhibits the interaction between KHK-A and PRPS1 and PRPS1 T225 phosphorylation.

    Fig. S5. KHK-A acts as a protein kinase and phosphorylates p62 at S28.

    Fig. S6. KHK-A is required for oxidative stress–induced but not proteasomal stress−induced p62 aggregation.

    Fig. S7. KHK-A–mediated p62 S28 phosphorylation is required for oxidative stress–enhanced p62 oligomerization and Nrf2 activation.

    Fig. S8. KHK-A mediated p62 S28 phosphorylation reduces ROS production and promotes cancer cell survival without altering autophagy initiation.

    Fig. S9. The phosphorylation-mimicking KHK-A S80E and p62 S28E mutations promote p62 oligomerization and Nrf2 activation.

    Fig. S10. KHK-A–mediated p62 S28 phosphorylation promotes hepatocellular tumorigenesis and is associated with the clinical aggressiveness of human HCC.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. KHK-A is required for oxidative stress–enhanced p62 oligomerization.
    • Fig. S2. KHK-A is required for Nrf2 activation upon oxidative stress.
    • Fig. S3. AMPK phosphorylates KHK-A and promotes the association between KHK-A and p62.
    • Fig. S4. AMPK-mediated KHK-A S80 phosphorylation inhibits the interaction between KHK-A and PRPS1 and PRPS1 T225 phosphorylation.
    • Fig. S5. KHK-A acts as a protein kinase and phosphorylates p62 at S28.
    • Fig. S6. KHK-A is required for oxidative stress–induced but not proteasomal stress−induced p62 aggregation.
    • Fig. S7. KHK-A–mediated p62 S28 phosphorylation is required for oxidative stress–enhanced p62 oligomerization and Nrf2 activation.
    • Fig. S8. KHK-A mediated p62 S28 phosphorylation reduces ROS production and promotes cancer cell survival without altering autophagy initiation.
    • Fig. S9. The phosphorylation-mimicking KHK-A S80E and p62 S28E mutations promote p62 oligomerization and Nrf2 activation.
    • Fig. S10. KHK-A–mediated p62 S28 phosphorylation promotes hepatocellular tumorigenesis and is associated with the clinical aggressiveness of human HCC.

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