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Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression

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Science Advances  11 Jul 2018:
Vol. 4, no. 7, eaas9184
DOI: 10.1126/sciadv.aas9184
  • Fig. 1 Phosphorylation of the TRM9L disordered domain.

    (A) Schematic of human TRM9L with conserved N- and C-terminal methyltransferase domains labeled in blue. The nonhomologous internal domain predicted to be an intrinsically disordered is in green. (B) Immunoblot analysis of total lysate of HCT116 cells expressing FLAG-TRM9L harvested either by trypsin or mechanical dislodgement (scraping). Lysates were treated without (−) or with (+) CIP. Red and black arrows denote phosphorylated and unphosphorylated forms of TRM9L, respectively. (C) Two-dimensional immunoblot analysis of HCT116 cell lysates showing multiple phosphorylation states. Lysates from cells harvested through trypsin were subject to IEF, followed by SDS-PAGE and finally probed by immunoblotting against FLAG-TRM9L. Lysates were treated without (−) or with (+) CIP as in (B). Red and black arrows denote phosphorylated and unphosphorylated forms of TRM9L, respectively. (D) Representative MS/MS spectrum of the tryptic TRM9L peptide spanning phosphorylated Ser214 (SHpSVGYEPAMAR). b and y ions detected in the MS/MS spectrum were labeled in red. Phosphorylation of Ser214 is established by the presence of y10, y10-H3PO4, b3, and b3-H3PO4 ions. (E) Primary sequence of the intrinsically disordered domain of TRM9L with phosphorylated residues labeled in red. Sequences consistent with the canonical 14-3-3 binding motif and the RSK phosphorylation motif were boxed. amu, atomic mass unit; m/z, mass/charge ratio.

  • Fig. 2 Oxidative stress induces hyperphosphorylation of TRM9L.

    (A) HCT116 + FLAG-TRM9L cells treated at the indicated concentration of H2O2 were harvested by mechanical dislodgement and analyzed by immunoblot for FLAG-TRM9L. (B) TRM9L phosphorylation status in HCT116 cells after 10 min of exposure with the indicated dose of H2O2. (C) Kinetics of TRM9L hyperphosphorylation in HCT116 cells after exposure to 880 μM H2O2. For (B) and (C), relative phosphorylation represents the ratio of phosphorylated TRM9L to unphosphorylated TRM9L signal at each time point or dose; data represent means ± SD (n = 3). (D) Two-dimensional gel analysis reveals multiple sites of H2O2-induced TRM9L phosphorylation. HCT116 + FLAG-TRM9L cells were mock-treated or treated with 880 μM H2O2, harvested by mechanical dislodgement, and analyzed by 2D gel. Lysates prepared from cells treated with H2O2 were treated without (−) or with (+) CIP. (E) Menadione (Men) and H2O2, but not γ-radiation, induce TRM9L phosphorylation. HCT116 + FLAG-TRM9L cells were mock-treated or treated with the indicated dose of menadione, H2O2, or γ-radiation followed by mechanical harvesting and immunoblot analysis of cell lysates for FLAG-TRM9L. (F) Quantitative phosphoproteomic reveals a H2O2-induced increase in TRM9L phosphorylation at Ser255 (white) and Ser291 (hatched) but not Ser214 (black) in HCT116 + FLAG-TRM9L cells; data represent means ± SD (n = 3 at each dose of H2O2). (G) H2O2-induced Ser380 phosphorylation, but not other sites, determines the 1D gel mobility shift. HCT116 cells expressing the indicated TRM9L variants were mock-treated or exposed to 880 μM H2O2 followed by immunoblot analysis of cell lysates for FLAG-TRM9L. Gy, gray.

  • Fig. 3 H2O2-induced hyperphosphorylation of TRM9L is dependent on the ERK- and RSK-signaling pathways.

    (A) Simplified schematic of the MEK/ERK signaling pathways transduced through RSK. Kinase inhibitors used in this study are denoted. (B) H2O2-induced Thr202/Tyr204 dual phosphorylation of ERK1/2 and hyperphosphorylation of TRM9L are dependent on MEK1/2 activation. HCT116 + FLAG-TRM9L cells that were untreated or exposed to 880 μM H2O2 with or without the pretreatment of MEK1/2 inhibitor U0126 were harvested by mechanical dislodgement and analyzed by immunoblot for the indicated proteins. (C and D) H2O2-induced hyperphosphorylation of TRM9L is inhibited by pretreatment of the RSK inhibitors BI-D1870 (C) and AZD7762 (D). Top: HCT116 + FLAG-TRM9L cells were left untreated or treated with the indicated concentration of H2O2 in the presence of vehicle [dimethyl sulfoxide (DMSO)] or the indicated RSK inhibitor followed by immunoblot analysis. Bottom: Quantification of H2O2-induced hyperphosphorylation of Ser380 in the presence of DMSO or RSK inhibitor. Data represent means ± SD (n = 3).

  • Fig. 4 Phosphorylation-dependent interaction of TRM9L with 14-3-3 proteins.

    (A) Silver stain of FLAG-SBP–tagged ALKBH8 or TRM9L affinity-purified from transiently transfected HEK 293T cells. Arrows point to bait proteins, while p28 indicates copurifying proteins processed for LC-MS peptide identification. “*” denotes antibody heavy/light chain from acid elution. (B) LC-MS identification of proteins in the p28 gel band. The 14-3-3 proteins that were identified, number of unique peptides, and total number of peptides from the p28 gel slice are noted. (C) Silver stain of FLAG affinity purifications from extracts prepared from HEK 293T cells stably expressing either empty vector or FLAG-SBP-TRM9L. Arrow points to bait protein, while p28 represents copurifying proteins processed for LC-MS protein identification. “*” is antibody heavy/light chain from acid elution. (D) LC-MS identification of proteins present in total elutions from purifications in (C). All 14-3-3 proteins that were identified are noted with the number of unique peptides and total number of peptides. (E) Immunoblot verification of 14-3-3 proteins copurifying with Strep-tagged TRM9L variants transiently expressed in HEK 293T cells. Strep-TRM9L-WT copurifies biotin elution with 14-3-3 gamma, epsilon, and eta, while TRM9L S214A and S255A lacked copurification of 14-3-3 proteins. “Input” represents 2% of total protein extract, while biotin elution represents 15% of total purification. (F) Immunoblot showing that all, while “biotin elution” represents 30% of total purification. TRM9L variants except S214A and S255A copurify with 14-3-3 epsilon when transiently expressed in HEK 293T cells. “Input” represents 4% of total protein extract , while “biotin elution” represents 30% of total purification. (G) Immunoblot verification of 14-3-3 copurification with TRM9L from SW620 cells. One hundred percent of elutions from FLAG affinity purifications from SW620 cells stably expressing FLAG-SBP-TRM9L were loaded. For “input,” 4% of starting input extracts were loaded.

  • Fig. 5 Multiple TRM9L phosphorylation sites link ROS signaling and tumor growth suppression.

    (A to D) TRM9L re-expression sensitizes colon cancer cell lines to oxidative stress but not γ-radiation. Survival of the indicated human cell lines 24 hours after treatment with H2O2 or ionizing radiation was measured by trypan blue staining. Data represent means ± SD, and asterisks denote significance (P ≤ 0.05) in a Student’s t test. (E and F) Sensitization of colon cancer cell lines by TRM9L re-expression to H2O2 is dependent on MEK-ERK signaling. Survival of the indicated human cell lines 24 hours after treatment with H2O2 with or without the pretreatment of the RSK inhibitor was measured as in (A) to (D). (G) Tumor growth suppression is dependent on TRM9L hyperphosphorylation. Xenografts of SW620 cell lines expressing the indicated TRM9L variants or LacZ (as a negative control) were implanted onto chick CAMs followed by analysis of tumor cell growth. Relative to WT TRM9L–expressing cells, tumor growth (CAM xenografts) is suppressed by phosphorylation at Ser214 (P < 0.0007), Ser255 (P < 0.0001), and Ser380 (P < 0.01). Bars represent means ± SD for the data representing individual tumor samples, with a Mann-Whitney test for significance.

  • Fig. 6 Phosphorylation of human TRM9L is a critical regulator of oxidative stress survival and tumor growth suppression.

    Oxidative stress conditions induce TRM9L hyperphosphorylation through the MEK-RSK signaling pathway. Hyperphosphorylation of TRM9L inhibits cell proliferation and suppresses tumor cell growth.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/7/eaas9184/DC1

    Supplementary Text

    Fig. S1. TRM9L transcript level predicts clinical prognosis.

    Fig. S2. TRM9L has a unique domain predicted to be intrinsically disordered and highly phosphorylated.

    Fig. S3. MS/MS spectra of phosphorylated tryptic peptides from TRM9L.

    Fig. S4. MS/MS spectra of phosphorylated tryptic peptides from TRM9L S380A mutant treated with H2O2.

    Fig. S5. Copurification of 14-3-3 proteins with TRM9L is dependent on serine residues S214 and S255.

    Fig. S6. Sensitivity of TRM9L expressing SW620 cells to the 14-3-3 antagonist BV02.

    Fig. S7. Validation of TRM9L expression levels in SW620 stable cell lines.

    Table S1. Kinase inhibitors screened for activity to block phosphorylation-dependent gel mobility shift of TRM9L.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • Fig. S1. TRM9L transcript level predicts clinical prognosis.
    • Fig. S2. TRM9L has a unique domain predicted to be intrinsically disordered and highly phosphorylated.
    • Fig. S3. MS/MS spectra of phosphorylated tryptic peptides from TRM9L.
    • Fig. S4. MS/MS spectra of phosphorylated tryptic peptides from TRM9L S380A mutant treated with H2O2.
    • Fig. S5. Copurification of 14-3-3 proteins with TRM9L is dependent on serine residues S214 and S255.
    • Fig. S6. Sensitivity of TRM9L expressing SW620 cells to the 14-3-3 antagonist BV02.
    • Fig. S7. Validation of TRM9L expression levels in SW620 stable cell lines.
    • Table S1. Kinase inhibitors screened for activity to block phosphorylation-dependent gel mobility shift of TRM9L.

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