Research ArticleCELL BIOLOGY

Stress-responsive MTK1 SAPKKK serves as a redox sensor that mediates delayed and sustained activation of SAPKs by oxidative stress

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Science Advances  24 Jun 2020:
Vol. 6, no. 26, eaay9778
DOI: 10.1126/sciadv.aay9778
  • Fig. 1 Oxidative stress activates MTK1 in a GADD45-independent manner.

    (A to C) HEK293 cells stably expressing Myc-MTK1 (M57 cells) or kinase-defective Myc-MTK1(K/R) were treated with MMS, CPT-11, CDDP, etoposide, vinblastine, nocodazole, thapsigargin, tunicamycin, heat shock, or H2O2 (A), with TPA (B), or with UV, sorbitol, anisomycin, or ionomycin (C) for the indicated times. (D) M57 cells were pretreated with cycloheximide (CHX) (15 μg/ml for 30 min) or emetine (25 μg/ml for 15 min), followed by treatment with etoposide, TPA, or H2O2. (A to D) Myc-MTK1 or Myc-MTK1(K/R) was immunoprecipitated from cell extracts, and phosphorylation of its activation site was detected by immunoblotting using an anti–phospho-MTK1(T1493) antibody (top rows). The same filters were reprobed with an anti-Myc antibody (second rows). (E and F) HEK293 cells were treated with TPA (for 120 min) or H2O2 (for 60 min). In (E), total RNA was extracted and analyzed for GADD45α, β, or γ mRNA expression (fold change) using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Data are means ± SEM (n = 3). **P < 0.02; ns, not significant. In (F), cell extracts were probed for GADD45β or β-actin (loading control). Where indicated, the cells were pretreated for 30 min with CHX. (G) HEK293 cells were stimulated with H2O2 (for 60 min). Immunoprecipitated endogenous MTK1 was probed with anti–P-MTK1 or anti-MTK1 antibodies.

  • Fig. 2 Oxidative stress–induced MTK1 activation requires both oxidation and reduction of MTK1.

    (A and B) HEK293 (A) and M57 cells (B) were stimulated with various concentrations of H2O2 for 10 min. Cell extracts were electrophoresed under nonreducing (−2ME) or reducing (+2ME) conditions, and probed for endogenous MTK1 (A) and Myc-MTK1 (B) by immunoblotting. Ox-MTK1, oxidized MTK1. (C) M57 cells were treated with H2O2 (1 mM). Cell extracts were electrophoresed under nonreducing conditions and immunoblotted with anti-Myc antibody (top). Immunoprecipitated Myc-MTK1 was electrophoresed under reducing conditions and probed with anti–P-MTK1 or anti-Myc antibodies (middle and bottom). (D) M57 cells were pretreated with the catalase inhibitor aminotriazol and stimulated with H2O2 (0.5 mM). Oxidation (top), phosphorylation (middle), and the expression level (bottom) of Myc-MTK1 were assessed as in (C). (E) Schematic diagram of MTK1 structure. GBD, GADD45-binding domain; AID, autoinhibitory domain; CCD, coiled-coil domain; KD, kinase domain. (F to H and J) HEK293 cells expressing Myc-MTK1 or its mutant derivatives (ΔGBD, 3C/S, C163S, C193S, or C218S) were treated with H2O2. Oxidation (F and G) and phosphorylation (H and J) of Myc-MTK1 were monitored as in (C). (I) Cos7 cells were cotransfected with Myc-MTK1 (WT, ΔGBD, or 3C/S) and GADD45β. Immunoprecipitated Myc-MTK1 was probed with anti–P-MTK1 or anti-Myc antibodies.

  • Fig. 3 Reduction of oxidized MTK1 induces its conformational change.

    (A and B) Reduction of MTK1 disrupts the inhibitory N-C domain interaction. Cos7 cells were transfected with Myc-MTK1-C, Flag-MTK1-N (WT or its 3C/S mutant), or Flag-GADD45β as indicated and were stimulated with H2O2 for the indicated times. Myc-MTK1-C was then immunoprecipitated (IP), and coprecipitating Flag-MTK1-N was detected by immunoblotting (top). In (A), coprecipitating Flag-GADD45β is also shown (second). In (B), where indicated, the transfected cells were pretreated with the Trx-reductase inhibitor 2,4-dinitro-1-chlorobenzene (DNCB) (100 μM) before H2O2 stimulation. (C) HEK293 cells stably expressing Myc-MTK1 or its 3P (L997P/I1001P/V1008P) mutant were stimulated with H2O2. Immunoprecipitated Myc-MTK1 was probed with anti–P-MTK1 (top). (D and E) HEK293 cells were transfected with Flag-MTK1 together with Myc-MTK1 or its 3C/S mutant as indicated and were treated with or without H2O2. Immunoprecipitated Flag-MTK1 was then probed for coprecipitating Myc-MTK1 by Western blotting (top rows) under reducing (D) or nonreducing (E) conditions. In (E), asterisks indicate the immunoglobulin G (IgG) heavy chain. (A to E) The levels of protein expression are shown in the lower rows.

  • Fig. 4 Trx-mediated reduction of oxidized MTK1 elicits its kinase activity.

    (A to D) HEK293 cells stably expressing Myc-MTK1 (A to C) or its 3C/S mutant (D) were transfected with Flag-GADD45β, Flag-Trx (WT, C35S, or C32S/C35S) (A), Flag-NRX (WT or C208S) (B), or Flag-TRP32 (WT or C37S) (C) as indicated, and treated with H2O2 for 20 min. Immunoprecipitated Flag-tagged proteins were probed for coprecipitating Myc-MTK1 (top). The levels of protein expression are shown in the lower rows. Flag-GADD45β served as a positive control for MTK1 binding. (E) M57 cells were treated with H2O2. Immunoprecipitated Myc-MTK1 was probed for coprecipitating endogenous Trx (top). C, control IgG. (F) M57 cells were pretreated with or without the Trx-reductase inhibitor 2,4-dinitro-1-chlorobenzene (DNCB) (100 μM) and stimulated with H2O2. Oxidation (top), phosphorylation (middle), and the expression level (bottom) of Myc-MTK1 were assessed as in Fig. 2C. (G) Oxidized Myc-MTK1 was immunopurified from H2O2-treated M57 cells, and incubated with recombinant Trx or Trx(C32S/C35S) for 30 min in vitro. Adenosine triphosphate was then added to initiate the kinase reaction and incubated for the indicated times. The reaction mixture was probed with anti–P-MTK1 (top) and anti-Myc (middle) antibodies. The total amounts of Trx and Trx(C32S/C35S) were also probed (bottom). (H) The intensity of the P-MTK1 bands in (G) was quantified. Error bars, SEM (n = 3). *P < 0.05; **P < 0.02.

  • Fig. 5 MTK1 mediates delayed and sustained activation of SAPKs by oxidative stress.

    (A) Parental HEK293 cells (WT), MTK1 knock-out cells (MTK1KO), and MTK1KO cells reexpressing Myc-MTK (MTK1KO + Myc-MTK1) were treated with H2O2 (1 mM) for the indicated times. Phosphorylation levels of p38, JNK, MKK3/6, and MKK4 were analyzed by immunoblotting using appropriate phospho-specific antibodies (second, fourth, sixth, and eighth rows). The expression levels of MTK1, p38, JNK, MKK3/6, and MKK4 in cell lysates are also shown (top, third, fifth, seventh, and bottom). (B and D) ASK1 mediates early activation of SAPKs by oxidative stress. HEK293 cells and ASK1-KO cells (clones #1 and #2) were stimulated with H2O2 [1 mM for (B) and 0.1 mM for (D)]. Cell lysates were analyzed by immunoblotting for the expression levels and phosphorylation states of the indicated proteins as in (A). (C) MTK1 and ASK1 are major mediators of SAPK activation by oxidative stress. HEK293 and MTK1/ASK1 double-KO cells (clones #1 and #2) were stimulated with H2O2 (1 mM). The expression levels and phosphorylation states of the indicated proteins were analyzed by immunoblotting. (E) MTK1 promotes H2O2-induced cell death. The indicated cells were treated with H2O2 (0.5 mM) for 6 hours. Cell viability was assessed using the CCK8 assay. Data are means ± SEM (n = 3). *P < 0.05; ***P < 0.01.

  • Fig. 6 MTK1 is critical for respiratory burst-induced IL-6 production in MFs.

    (A and E) MFs were stimulated with H2O2 (1 mM) (A) or zymosan (0.2 mg/ml) (E). Cell lysates were electrophoresed under nonreducing conditions and probed for endogenous MTK1. (B) Immunoblot analysis of MTK1 expression in THP-1 cells transduced with two different shRNAs against MTK1 (#1 or #2). (C) Immunoblot analyses of phosphorylated or total p38 and JNK in H2O2-stimulated MFs. (D) ROS production in MFs treated with (+) or without (−) zymosan was visualized using the fluorescent ROS probe CellROX Green. DIC, differential interference contrast. Scale bar, 30 μm. (F) Control MFs and MFs silenced for MTK1 (shMTK1#2) or/and ASK1 (siASK1) were stimulated with zymosan. Phosphorylation states of p38 and JNK were analyzed by immunoblotting. (G) Expression levels of TNFα (left) and IL-6 (right) mRNAs in zymosan-treated MFs were analyzed using qRT-PCR. (H) MFs were pretreated with N-acetyl cysteine (NAC) (left) or p38 and JNK inhibitors (SB/SP) (right), followed by stimulation with zymosan. IL-6 mRNA expression was then quantified. (I) qRT-PCR analyses of GADD45α, β, and γ expression in zymosan-treated MFs. TPA-treated HEK293 cells served as a positive control. All data were normalized by β-actin mRNA expression. (G to I) Error bars, SEM (n = 3). *P < 0.05; ***P < 0.01. (J) A schematic model of oxidative stress–induced SAPK activation. MTK1 and ASK1 are major mediators of oxidative stress–induced SAPK signaling. However, they have different time and dose-response characteristics.

Supplementary Materials

  • Supplementary Materials

    Stress-responsive MTK1 SAPKKK serves as a redox sensor that mediates delayed and sustained activation of SAPKs by oxidative stress

    Moe Matsushita, Takanori Nakamura, Hisashi Moriizumi, Hiroaki Miki, Mutsuhiro Takekawa

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