Research ArticleMetabolism

The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose

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Science Signaling  03 Sep 2019:
Vol. 12, Issue 597, eaaw8000
DOI: 10.1126/scisignal.aaw8000
  • Fig. 1 Proteomics analysis of the response to 2DG in yeast reveals transcriptional induction of the 2DG6P phosphatases Dog1 and Dog2.

    (A) Volcano plot representing changes in protein abundance in total protein extracts of wild-type (WT) yeast in response to 2DG (0.2%), obtained by MS-based proteomics and analyzed with MaxQuant software. The x axis corresponds to the log2 value of the abundance ratio [label-free quantification (LFQ)] between 2DG treatment and the negative control. The y axis represents the ─log10 of the P value of the statistical t test for each quantified protein (n = 3 independent biological replicates). Lines: threshold with an FDR of 0.01. (B) Gene ontology (GO) analysis of the proteins identified as up-regulated in response to 2DG treatment along with their P value and the proteins included in each category. (C) Western blot on total protein extracts of yeast cells expressing endogenously tagged Dog1-GFP or Dog2-GFP, before and after 2DG addition for the indicated times, using an anti-GFP antibody. A longer exposure is displayed for Dog1-GFP cells to highlight the higher abundance of Dog1 after 2DG addition. Rsp5, whose levels did not change upon 2DG addition in all of our experiments, is used as a loading control (n = 2 independent experiments). (D) β-Galactosidase assays of WT yeast cells expressing LacZ under the control of the pDOG1 or pDOG2 promoters, before and after 2DG treatments for 3 hours (±SEM, n = 3 independent experiments, t test). A.U., arbitrary units. (E) Serial dilutions of cultures from the indicated strains were spotted onto SD plates containing no DG or 0.05% 2DG and grown for 3 days at 30°C (n = 2 independent experiments). (F) β-Galactosidase assays of WT and hog1∆ strains expressing LacZ under the control of the pDOG2 promoter, before and after 2DG treatments for 3 hours (±SEM, n = 3 independent experiments, t test). (G) Western blot on total protein extracts of WT and hog1∆ cells endogenously expressing a Dog2-GFP fusion, before and after 2DG addition for 3 hours, using an anti-GFP antibody. Total protein was visualized in gels using a trihalo compound. Glc, glucose. (H) Relative expression of Dog2-GFP under the same conditions as (G) after normalization to total protein and using WT/untreated as a reference (±SEM, n = 3 independent experiments, t test).

  • Fig. 2 2DG treatment induces Dog2 expression through glycosylation defects that trigger ER stress and the UPR.

    (A) WT cells were grown overnight to mid-log phase in synthetic complete (SC) medium, centrifuged, and resuspended in SC medium containing mannose (2%) or not, and treated with 0.2% 2DG or tunicamycin (Tm; 1 μg/ml) for 4 hours. Total protein extracts were Western-blotted for carboxypeptidase Y (CPY) (n = 3 independent experiments). Glc, glucose. Glycos., glycosylation. (B) Schematic of the UPR signaling pathway in yeast showing how ER stress triggers Ire1-mediated splicing of the pre-mRNA encoding the transcription factor Hac1 and the subsequent induction of UPR target genes. (C) β-Galactosidase assays on WT and hac1∆ cells expressing LacZ under the control of a UPR-inducible promoter (pUPRE1) and treated with 0.2% 2DG or tunicamycin (1 μg/ml) for 3 hours (±SEM, n = 3 independent experiments, t test). (D) β-Galactosidase assays on WT cells expressing LacZ under the control of the DOG2 promoter and treated as in (C) (±SEM, n = 3 independent experiments, t test). (E) β-Galactosidase assays on WT and hac1∆ cells expressing LacZ under the control the DOG2 promoter, before and after 3-hour 2DG treatments (±SEM, n = 3 independent experiments, t test). (F) Western blot for GFP on total protein extracts of WT and hac1∆ cells endogenously expressing a Dog2-GFP fusion, before and after 3-hour treatment with 2DG or tunicamycin. (G) Relative expression of Dog2-GFP under the same conditions as (F) after normalization to total protein and using WT/untreated as a reference (±SEM, n = 3 independent experiments, t test). (H) Serial dilutions of cultures from the indicated strains were spotted onto SC plates (supplemented with 2% mannose when indicated) containing no DG or 0.05% 2DG, and were grown for 3 days at 30°C (n = 2 independent experiments). (I) Serial dilutions of cultures from the indicated strains overexpressing DOG2 (pGPD-DOG2) or not (Ø) were spotted onto SC-Ura plates containing 0, 0.05, or 0.2% 2DG. The plates were scanned after 3 days of incubation at 30°C (n = 2 independent experiments).

  • Fig. 3 2DG activates the MAPK-based CWI pathway, which is required for 2DG tolerance and additionally contributes to the regulation of Dog2 expression.

    (A) Schematic of the CWI pathway showing the various components and their requirement for growth on 2DG (see color code in the inset) based on drop tests shown in (B). (B) Serial dilutions of cultures from the indicated deletion strains were spotted onto SD plates containing no DG or 0.05% 2DG and grown for 3 days at 30°C (n = 2 independent experiments). (C) β-Galactosidase assays on WT and slt2∆ cells expressing LacZ under the control of the DOG2 promoter, before and after 3-hour 2DG treatments (±SEM, n = 3 independent experiments, t test). (D) Western blot on total protein extracts of WT and slt2∆ cells expressing an endogenously tagged Dog2-GFP fusion, before and after 3-hour treatment with 2DG, using an anti-GFP antibody. (E) Relative expression of Dog2-GFP under the same conditions as (D) after normalization to total protein and using WT/untreated as a reference (±SEM, n = 3 independent experiments, t test). (F) Serial dilutions of cultures from the indicated strains were spotted onto SC plates (supplemented with 2% mannose when indicated) containing no DG or 0.05% 2DG and were grown for 3 days at 30°C (n = 2 independent experiments).

  • Fig. 4 DOG2, but not DOG1, is negatively controlled by glucose availability through transcriptional repression by Mig1-Mig2 and the kinase Snf1.

    (A) Schematic of the glucose repression pathway showing how Snf1 (the yeast homolog of AMPK), PP1 (composed of Glc7 and Reg1 subunits), and their downstream transcriptional repressors Mig1/Mig2 regulate glucose-repressed genes in response to glucose availability or absence (such in the presence of lactate). (B) WT and snf1∆ strains, both expressing endogenously tagged Dog1-TAP and Dog2-GFP fusions, were grown overnight in SC medium and then either treated with 0.2% 2DG or switched to an SC lactate medium for 4 hours. Dog1-TAP was detected with the peroxidase–anti-peroxidase (PAP) complex and Dog2-GFP with anti-GFP antibodies, phosphorylated (p) Snf1 with anti-phospho-AMPK and total Snf1 with an anti-polyHis tag (because Snf1 contains a stretch of 13 histidine residues that can be used for its detection) (n = 2 independent experiments). (C) β-Galactosidase activity on WT and snf1∆ cells expressing LacZ under the control of the DOG1 or the DOG2 promoter, before and after 3-hour growth in lactate. The fold induction after transfer to lactate is indicated for each promoter in each strain (±SEM, n = 4 independent experiments). (D) WT and snf1∆ strains, transformed either with a genomic clone containing both DOG1 and DOG2 under the control of its own promoter (pend:DOG) or with a vector containing DOG2 under the control of the strong GPD promoter (pGPD:DOG2), were grown, serially diluted, and spotted onto SC plates (SC-Leu or SC-Ura) with or without 0.2% DG, and grown for 3 days at 30°C (n = 2 independent experiments). (E) β-Galactosidase assays on WT and the indicated deletion mutants expressing LacZ under the control the DOG2 promoter after overnight growth in SC medium (exponential phase) (±SEM, n = 6 independent experiments). (F) The indicated strains, all expressing an endogenously tagged Dog2-GFP fusion, were grown overnight in SC medium (to exponential phase). Total protein extracts were immunoblotted with anti-GFP antibodies. Rsp5 was used as a loading control (n = 2 independent experiments). (G) Serial dilutions of cultures of the indicated mutants were spotted on yeast extract-peptone-dextrose (YPD) plates containing 0, 0.2, or 0.5% of 2DG. Plates were scanned after 3 days of growth at 30°C (n = 3 independent experiments).

  • Fig. 5 The characterization of spontaneous 2DG-resistant strains identifies mutants showing increased Dog2 expression, including a new mutant allele of CYC8.

    (A) Twenty-four clones showing a spontaneous resistance to 0.2% 2DG were isolated. The β-galactosidase activity of these mutants, due to the expression of the LacZ reporter driven by the DOG2 promoter, was measured after overnight growth in SC medium (to exponential phase) (±SEM, n = 4 independent experiments, t test). Colors represent the identity of the mutants as determined in fig. S6 (A and B) for reg1 and fig. S6 (C to E) for hxk2 and (B) to (G) for cyc8. (B) Schematic of the domain organization of the Cyc8 protein, showing the Poly(Q) and Poly(QA) repeats and the N-terminal tetratricopeptide (TPR) repeats. Red: mutation identified by whole-genome resequencing of the spontaneous 2DG-resistant mutants #9 and #10. (C) A WT strain, the mutant strains #9 and #10, and the reg1∆ mutant (used as a positive control) were grown in SC medium (to exponential phase). Total protein extracts were immunoblotted for invertase [invertase is heavily glycosylated and migrates as a smear (116)] (n = 2 independent experiments). (D) WT and mutants #9 and #10 were transformed with low-copy (centromeric) plasmid either empty or containing WT CYC8 or mutant cyc8 (Gln320*), spotted on SC-Leu or SC-Leu + 0.2% 2DG medium, and grown for 3 days at 30°C. Middle: the control plate was scanned and then washed for 1 min under a constant flow of water, and then scanned again (n = 2 independent experiments). (E) β-Galactosidase activity on WT and mutants #9 and #10 expressing LacZ under the control the DOG2 promoter and transformed with an empty vector or a low-copy vector containing WT CYC8, after growth in SC medium (normalized to the value of the WT, ±SEM, n = 3 independent experiments, t test). (F) Western blot on total protein extracts of WT and mutants #9 and #10 cells expressing an endogenously tagged Dog2-GFP fusion and transformed with either an empty plasmid or a low-copy (centromeric) plasmid containing WT CYC8 after growth in SC medium, using an anti-GFP antibody. (G) Relative expression of Dog2-GFP under the same conditions as (F) after normalization by total proteins and using the WT control as a reference (±SEM, n = 3 independent experiments, t test). (H) Serial dilutions of cultures of the indicated mutants were spotted on SC medium or SC + 0.2% 2DG medium and grown for 3 days at 30°C (n = 2 independent experiments). mut, mutant.

  • Fig. 6 The human phosphatase HDHD1 has a 2DG6P phosphatase activity and its overexpression leads to 2DG resistance in HeLa cells.

    (A) Multiple protein sequence alignment of yeast Dog1, Dog2, E. coli yniC, and the human proteins HDHD1, HDHD4, and PSPH aligned with ClustalX 2.0. The highly conserved catalytic aspartates are displayed in yellow. The first six amino acids of PSPH were truncated to optimize the N-terminal alignment of its catalytic aspartates with the other phosphatases. (B) Serial dilutions of WT and dog1dog2∆ strains transformed with the indicated plasmids were spotted on SC-Ura medium with or without 0.05% 2DG and were scanned after 3 days of growth at 30°C (n = 2 independent experiments). (C) Serial dilutions of dog1dog2∆ strains transformed with an empty vector or vectors allowing the expression of an HDHD1 or its predicted catalytic mutant, HDHD1-DD>AA (in which the N-terminal catalytic aspartates were mutated to alanines), were spotted on SC-Ura medium with or without 0.05% 2DG and were scanned after 3 days of growth at 30°C (n = 2 independent experiments). (D) Recombinant, His-tagged HDHD1 and HDHD1-DD>AA were expressed in bacteria and purified for in vitro enzymatic tests; 0.7 μg was loaded on a gel to show homogeneity of the protein purification. (E) In vitro 2DG6P phosphatase activity of HDHD1 and HDHD1>DDAA as measured by assaying glucose release from 2DG6P (n = 3 independent experiments). (F) Growth of HeLa cells transfected with an empty vector (□) or with a construct allowing the overexpression of HDHD1 (○) over time in the absence (open symbols) or presence (filled symbol) of 5 mM 2DG. The number of cells is normalized to that of the untransformed/untreated cells after 3 days (±SEM, n = 3 independent experiments, t test).

  • Fig. 7 Working model.

    Glucose phosphorylation triggers the onset of the glucose-repression pathway in which PP1 inactivates Snf1. This leads to the lack of phosphorylation of Mig1 and Mig2, which remain in the nucleus to mediate the glucose repression of genes such as DOG2. The deletion of REG1, HXK2, or MIG1 and MIG2 or a mutation in CYC8 leads to 2DG resistance, which is at least partially mediated through increased expression of DOG2, leading to the dephosphorylation of 2DG6P. In contrast, the deletion of SNF1 causes an increased sensitivity to 2DG, which can be rescued by the deletion of MIG1 and MIG2 or by Dog2 overexpression. In parallel, 2DG6P causes (i) ER stress and triggers the UPR pathway, which stimulates DOG2 expression through the transcription factor Hac1, and (ii) the CWI pathway, likely through interference with polysaccharide and cell wall synthesis, which also induces DOG2 through the transcription factor Rlm1.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/597/eaaw8000/DC1

    Fig. S1. Characterization of other candidates showing increased abundance after 2DG treatment.

    Fig. S2. Hog1 signaling responds to 2DG, but DOG1 expression is not regulated by Hog1.

    Fig. S3. DOG1 expression is regulated by the UPR pathway.

    Fig. S4. Slt2 participates in the regulation of DOG1 expression.

    Fig. S5. Cis regions involved in the regulation of the DOG2 promoter by glucose.

    Fig. S6. Identification of reg1 and hxk2 mutants within the isolated spontaneous 2DG-resistant mutants.

    Fig. S7. Multiple protein sequence alignment of Hxk2 orthologs and positions of the mutations identified.

    Fig. S8. HDHD1 but not its close homologs HDHD4 or PSPH allow resistance to 2DG.

    Table S1. Single-nucleotide variants in clones #9 and #10 as compared to the WT strain, as identified by whole-genome resequencing.

    Table S2. Yeast strains used in this study.

    Table S3. Plasmids used in this study.

    Table S4. Antibodies used in this study.

    Data file S1. Proteomic response to 2DG treatment.

    References (117121)

  • The PDF file includes:

    • Fig. S1. Characterization of other candidates showing increased abundance after 2DG treatment.
    • Fig. S2. Hog1 signaling responds to 2DG, but DOG1 expression is not regulated by Hog1.
    • Fig. S3. DOG1 expression is regulated by the UPR pathway.
    • Fig. S4. Slt2 participates in the regulation of DOG1 expression.
    • Fig. S5. Cis regions involved in the regulation of the DOG2 promoter by glucose.
    • Fig. S6. Identification of reg1 and hxk2 mutants within the isolated spontaneous 2DG-resistant mutants.
    • Fig. S7. Multiple protein sequence alignment of Hxk2 orthologs and positions of the mutations identified.
    • Fig. S8. HDHD1 but not its close homologs HDHD4 or PSPH allow resistance to 2DG.
    • Table S1. Single-nucleotide variants in clones #9 and #10 as compared to the WT strain, as identified by whole-genome resequencing.
    • Table S2. Yeast strains used in this study.
    • Table S3. Plasmids used in this study.
    • Table S4. Antibodies used in this study.
    • References (117121)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Proteomic response to 2DG treatment.

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