Fig 1: Sensitivity to TNF-induced septic shock in Dapk1-/- mice and DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a, b Control and Dapk1-/- mice (n = 4 in each group) were injected with mouse TNF (1.0 µg/g body weight), and survival (a) and rectal body temperature (b) were determined at the indicated time points. P values (a) for Long-rank (Mantel-–Cox) test. Mean ± SD (b) are shown. *P < 0.05, **P < 0.01 (b) for two-way ANOVA followed by a Tukey’s multiple comparison test. (c) Normal TNF-triggered activation or NF-?B, ERK, JNK, and AKT in Dapk1-/- BMDMs. WT and Dapk1-/- BMDMs were treated with TNF (20 ng/ml), and cell lysates were prepared and levels of pI?Ba, I?Ba, pERK, ERK, pJNK, JNK, pAKT, and AKT were determined for the indicated time points. (d, f) Diminished TNF-induced phosphorylation of RIPK1(S321), MK2 and p38 MAPK in Dapk1-/- BMDMs. WT and Dapk1-/- BMDMs were treated with TNF (20 ng/ml), and levels of pIKK, IKK, pRIPK1(S321), RIPK1, p-p38, p38 (d), pMK2 and MK2 (f) were determined in cell lysates for the indicated time points. Data are representative of three independent experiments. (e) The extents of RIPK1(S321) and p38 MAPK activation from three independent experiments in (d) were quantitated using normalized intensity of pRIPK1(S321) and p-p38 in WT BMDMs at 15 min as 1. Mean ± SD are shown. *P < 0.05, ***P < 0.001 for two-way ANOVA followed by a Sidak’s multiple comparison test. (g) Inhibition of p38 MAPK or MK2 confers susceptibility to necroptosis in WT macrophages. WT BMDMs were treated with zVAD, AT-406, SB203589 (1 µM) and PF3644022 (2 µM), as indicated, and the extent of necroptosis determined. Values are mean ± SD of triplicates in a single experiment. **p < 0.01, ***p < 0.001 for unpaired t-test. Results have been repeated in two independent experiments.
Fig 2: DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1-/- BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis in DAPK1-deficient cells. DAPK1-/- HT-29 cells were transfected with empty vector (EV), or transfected with WT DAPK1 or DAPK1[K42A]. Cells were treated with zVAD (Ctrl) or zVAD + BV6, and viability determined by ATP assay. c Either WT DAPK or DAPK1[K42A] increases p38 MAPK activation in DAPK1-null cells. DAPK1-/- HT-29 cells, mock transduced, transduced with WT DAPK1 or DAPK1[K42A] were treated with TNF (20 ng/ml) for the indicated time points, and the levels of DAPK1, p38 MAPK and phospho-p38 MAPK were determined. d Overexpression of p38 MAPK inhibits necroptosis. DAPK1-/- HT-29 cells were transfected with empty vector (EV), p38 MAPK or MKK3. The extent of necroptosis induced by zVAD+BV6 was determined by ATP release. e Interaction between DAPK1 and p38 MAPK. HEK293T cells were transfected with DAPK1-FLAG and/or p38-HA as indicated. Cell lysates were prepared 24 later, and precipitated with anti-FLAG or anti-HA. f p38 MAPK binds DAPK in vitro. Recombinant human p38 MAPK (200 ng) was incubated with purified DAPK (100 ng), as indicated. p38 MAPK was pulled down by anti-p38 MAPK (ab170099, Abcam), and the presence of DAPK and p38 MAPK in immunoprecipitants determined by anti-FLAG and anti-His, respectively. Results have been independently confirmed using another anti-p38 MAPK (7218, Cell Signaling). g DAPK1 promotes MKK3-directed p38 MAPK phosphorylation. Recombinant p38 MAPK (50 ng), MKK3 (100 ng), and DAPK1-FLAG (25 ng or 75 ng) was incubated as indicated in ATP-containing kinase buffer at 30 °C for 1 h. The amounts of p38 MAPK, MKK3, DAPK1 and phospho-p38 MAPK was determined by immunoblots. h Binding of different DAPK1 mutants to p38 MAPK. HEK293T cells were transfected with different mutants of DAPK1-FLAG and p38-HA as indicated. The presence of p38-HA in anti-FLAG precipitates was determined. i Failure of DAPK1(?Cyt) to increase p38 MAPK activation. DAPK1, DAPK1(?Cyt) or DAPK1(?CAM) was evaluated for its ability to increase MKK3-directed p38 MAPK activation described in (g). Values are mean ± SD of triplicates in a single experiment. *P < 0.05, ***P < 0.001 (b, d) for two-way ANOVA followed by a Tukey’s multiple comparison test. Results have been repeated in three (a–c, e, f) or two (d, g–i) independent experiments.
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