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TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

Morioka S, Broglie P, Omori E, Ikeda Y, Takaesu G, Matsumoto K, Ninomiya-Tsuji J - J. Cell Biol. (2014)

Bottom Line: We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation.Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro.Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695.

ABSTRACT
TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

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Hyperactivation of TAK1 promotes RIPK3-dependent cell death. (A) HeLa cells were transfected with 1 µg of Flag-TAB1 (TAB1), DsRedMT7-TAK1 (TAK1), HA-RIPK3 (RIPK3), or their control vectors for the total amount of 3 µg. At 48 h after transfection, cells were stimulated with 200 ng/ml TNF, 1 µM Smac mimetic, and 20 µM Z-VAD (T/S/Z) for 6 h. DsRed-positive transfected cells were gated, and cell death was analyzed by annexin V and fixable viability dye eFlour 780 staining. Percentages of fixable viability dye–positive cells in the transfected cells are shown (three independent experiments; mean ± SD; **, P < 0.01; P = 0.0043 and P = 0.0011 from the left). (B) HeLa cells were transfected as shown in A and treated with 200 ng/ml TNF alone or together with 1 µM Smac mimetic and 20 µM Z-VAD (S/Z). Activity of TAK1 was monitored by immunoblotting with anti-phospho-TAK1. Expression levels of TAB1, TAK1, RIPK1, and RIPK3 were also analyzed. β-Actin is shown as a control. (C) HeLa cells were transfected with expression vectors for RIPK3 with TAB1 and TAK1. Cell lysates were incubated with lambda protein phosphatase. Mobility shift of RIPK3 was observed by immunoblotting. β-Actin is shown as a control. (D) The Flag-tagged TAK1 wild-type (TAK1) or kinase-dead (TAK1 KD) TAK1 together with T7-tagged TAB1 (TAB1) and GFP-tagged wild-type (RIPK3) or kinase-dead (D161N; RIPK3 KD) RIPK3 were separately expressed in HEK293 cells. TAK1–TAB1 complex and RIPK3 were immunoprecipitated with anti-Flag or anti-RIPK3 antibody, respectively. TAK1–TAB1 complex was released from antibody beads, activated by incubation with ATP, and then mixed with RIPK3 or RIPK3 KD. RIPK3 kinase activity was assessed by in vitro kinase assay. Left panel, autoradiograph; right panels, immunoblot analysis of the immunoprecipitates with anti-TAK1 and anti-RIPK3 antibodies. Co-precipitated endogenous TAK1 and RIPK1 in the protein preparations were also determined by immunoblotting (right panels). Because HEK293 cells do not express RIPK3, endogenous RIPK3 was not examined.
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fig5: Hyperactivation of TAK1 promotes RIPK3-dependent cell death. (A) HeLa cells were transfected with 1 µg of Flag-TAB1 (TAB1), DsRedMT7-TAK1 (TAK1), HA-RIPK3 (RIPK3), or their control vectors for the total amount of 3 µg. At 48 h after transfection, cells were stimulated with 200 ng/ml TNF, 1 µM Smac mimetic, and 20 µM Z-VAD (T/S/Z) for 6 h. DsRed-positive transfected cells were gated, and cell death was analyzed by annexin V and fixable viability dye eFlour 780 staining. Percentages of fixable viability dye–positive cells in the transfected cells are shown (three independent experiments; mean ± SD; **, P < 0.01; P = 0.0043 and P = 0.0011 from the left). (B) HeLa cells were transfected as shown in A and treated with 200 ng/ml TNF alone or together with 1 µM Smac mimetic and 20 µM Z-VAD (S/Z). Activity of TAK1 was monitored by immunoblotting with anti-phospho-TAK1. Expression levels of TAB1, TAK1, RIPK1, and RIPK3 were also analyzed. β-Actin is shown as a control. (C) HeLa cells were transfected with expression vectors for RIPK3 with TAB1 and TAK1. Cell lysates were incubated with lambda protein phosphatase. Mobility shift of RIPK3 was observed by immunoblotting. β-Actin is shown as a control. (D) The Flag-tagged TAK1 wild-type (TAK1) or kinase-dead (TAK1 KD) TAK1 together with T7-tagged TAB1 (TAB1) and GFP-tagged wild-type (RIPK3) or kinase-dead (D161N; RIPK3 KD) RIPK3 were separately expressed in HEK293 cells. TAK1–TAB1 complex and RIPK3 were immunoprecipitated with anti-Flag or anti-RIPK3 antibody, respectively. TAK1–TAB1 complex was released from antibody beads, activated by incubation with ATP, and then mixed with RIPK3 or RIPK3 KD. RIPK3 kinase activity was assessed by in vitro kinase assay. Left panel, autoradiograph; right panels, immunoblot analysis of the immunoprecipitates with anti-TAK1 and anti-RIPK3 antibodies. Co-precipitated endogenous TAK1 and RIPK1 in the protein preparations were also determined by immunoblotting (right panels). Because HEK293 cells do not express RIPK3, endogenous RIPK3 was not examined.

Mentions: In an effort to determine the mechanism by which Tab2 deficiency activates TNF-induced RIPK3-dependent cell death, we focused on hyperactivation and sustained activation of TAK1 and asked whether it is the cause of RIPK3-dependent cell death. We used HeLa cells because they are highly transfectable and known to be deficient in RIPK3 expression (He et al., 2009). We activated TAK1 using TAK1-binding protein 1 (TAB1), which is a constitutively associated TAK1 binding partner, and co-overexpression of TAK1 and TAB1 highly activates TAK1 (Kishimoto et al., 2000). TAK1 and TAB1 were overexpressed in the presence and absence of RIPK3 in HeLa cells, which were analyzed for cell death (Fig. 5). Activation of TAK1 or expression of RIPK3 alone did not profoundly induce cell death but RIPK3 moderately increased annexin V– and cell permeability dye double-positive necrotic cell death in the presence of treatment with TNF together with an inhibitor of cIAP, Smac mimetic (Wang et al., 2008), and Z-VAD (T/S/Z) treatment (Fig. 5 A), which is consistent with the earlier study (He et al., 2009). However, when TAK1 and TAB1 were coexpressed with RIPK3, cell death was dramatically enhanced (Fig. 5 A). We also reproduced this result in mouse dermal fibroblasts. Overexpression of TAK1 and TAB1 or RIPK3 alone only marginally increased necrotic cell death in dermal fibroblasts, but overexpression of TAK1, TAB1, and RIPK3 together highly enhanced cell death (Fig. S4). Overexpression of TAK1 and TAB1 produces an active form of TAK1 and we found that it induced a slower migrating additional RIPK3 band (Fig. 5 B). The retardation of RIPK3 migration was abolished by lambda phosphatase treatment (Fig. 5 C), suggesting that RIPK3 is phosphorylated by TAK1. We then examined whether TAK1 activates RIPK3 by the in vitro kinase assay (Fig. 5 D). An active and kinase-dead TAK1, RIPK3, and kinase-dead RIPK3 were prepared from HEK293 cells separately overexpressing these proteins, and proteins were mixed as indicated and subjected to in vitro kinase assay. Kinase activity was assessed by its autophosphorylation of RIPK3. RIPK3 was basally active (lane 3), which is consistent with an earlier study (Cho et al., 2009), and the activity was further increased by TAK1 (lane 4). The kinase-dead RIPK3 was not detectably phosphorylated by TAK1 (lane 7). Thus, phosphorylation of wild-type RIPK3 (lanes 3–5) is likely to be predominantly mediated by RIPK3 autophosphorylation. We note that endogenous RIPK1 was co-precipitated with RIPK3 (Fig. 5 D, far right panels). This raises the possibility that co-precipitated RIPK1 in the RIPK3 preparation may be involved in activity of RIPK3 in these in vitro assays. However, endogenous RIPK1 was not coprecipitated with TAK1 (Fig. 5 D, second from right panels), indicating that adding TAK1 preparation to the kinase assay mixture did not increase total amount of RIPK1. Thus, the increase of RIPK3 activity (lane 4) over basal RIPK3 activity (lane 3) is mediated by TAK1 but not due to increase amount of RIPK1. These data demonstrate that an active TAK1 activates RIPK3, leading to necrosis.


TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

Morioka S, Broglie P, Omori E, Ikeda Y, Takaesu G, Matsumoto K, Ninomiya-Tsuji J - J. Cell Biol. (2014)

Hyperactivation of TAK1 promotes RIPK3-dependent cell death. (A) HeLa cells were transfected with 1 µg of Flag-TAB1 (TAB1), DsRedMT7-TAK1 (TAK1), HA-RIPK3 (RIPK3), or their control vectors for the total amount of 3 µg. At 48 h after transfection, cells were stimulated with 200 ng/ml TNF, 1 µM Smac mimetic, and 20 µM Z-VAD (T/S/Z) for 6 h. DsRed-positive transfected cells were gated, and cell death was analyzed by annexin V and fixable viability dye eFlour 780 staining. Percentages of fixable viability dye–positive cells in the transfected cells are shown (three independent experiments; mean ± SD; **, P < 0.01; P = 0.0043 and P = 0.0011 from the left). (B) HeLa cells were transfected as shown in A and treated with 200 ng/ml TNF alone or together with 1 µM Smac mimetic and 20 µM Z-VAD (S/Z). Activity of TAK1 was monitored by immunoblotting with anti-phospho-TAK1. Expression levels of TAB1, TAK1, RIPK1, and RIPK3 were also analyzed. β-Actin is shown as a control. (C) HeLa cells were transfected with expression vectors for RIPK3 with TAB1 and TAK1. Cell lysates were incubated with lambda protein phosphatase. Mobility shift of RIPK3 was observed by immunoblotting. β-Actin is shown as a control. (D) The Flag-tagged TAK1 wild-type (TAK1) or kinase-dead (TAK1 KD) TAK1 together with T7-tagged TAB1 (TAB1) and GFP-tagged wild-type (RIPK3) or kinase-dead (D161N; RIPK3 KD) RIPK3 were separately expressed in HEK293 cells. TAK1–TAB1 complex and RIPK3 were immunoprecipitated with anti-Flag or anti-RIPK3 antibody, respectively. TAK1–TAB1 complex was released from antibody beads, activated by incubation with ATP, and then mixed with RIPK3 or RIPK3 KD. RIPK3 kinase activity was assessed by in vitro kinase assay. Left panel, autoradiograph; right panels, immunoblot analysis of the immunoprecipitates with anti-TAK1 and anti-RIPK3 antibodies. Co-precipitated endogenous TAK1 and RIPK1 in the protein preparations were also determined by immunoblotting (right panels). Because HEK293 cells do not express RIPK3, endogenous RIPK3 was not examined.
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fig5: Hyperactivation of TAK1 promotes RIPK3-dependent cell death. (A) HeLa cells were transfected with 1 µg of Flag-TAB1 (TAB1), DsRedMT7-TAK1 (TAK1), HA-RIPK3 (RIPK3), or their control vectors for the total amount of 3 µg. At 48 h after transfection, cells were stimulated with 200 ng/ml TNF, 1 µM Smac mimetic, and 20 µM Z-VAD (T/S/Z) for 6 h. DsRed-positive transfected cells were gated, and cell death was analyzed by annexin V and fixable viability dye eFlour 780 staining. Percentages of fixable viability dye–positive cells in the transfected cells are shown (three independent experiments; mean ± SD; **, P < 0.01; P = 0.0043 and P = 0.0011 from the left). (B) HeLa cells were transfected as shown in A and treated with 200 ng/ml TNF alone or together with 1 µM Smac mimetic and 20 µM Z-VAD (S/Z). Activity of TAK1 was monitored by immunoblotting with anti-phospho-TAK1. Expression levels of TAB1, TAK1, RIPK1, and RIPK3 were also analyzed. β-Actin is shown as a control. (C) HeLa cells were transfected with expression vectors for RIPK3 with TAB1 and TAK1. Cell lysates were incubated with lambda protein phosphatase. Mobility shift of RIPK3 was observed by immunoblotting. β-Actin is shown as a control. (D) The Flag-tagged TAK1 wild-type (TAK1) or kinase-dead (TAK1 KD) TAK1 together with T7-tagged TAB1 (TAB1) and GFP-tagged wild-type (RIPK3) or kinase-dead (D161N; RIPK3 KD) RIPK3 were separately expressed in HEK293 cells. TAK1–TAB1 complex and RIPK3 were immunoprecipitated with anti-Flag or anti-RIPK3 antibody, respectively. TAK1–TAB1 complex was released from antibody beads, activated by incubation with ATP, and then mixed with RIPK3 or RIPK3 KD. RIPK3 kinase activity was assessed by in vitro kinase assay. Left panel, autoradiograph; right panels, immunoblot analysis of the immunoprecipitates with anti-TAK1 and anti-RIPK3 antibodies. Co-precipitated endogenous TAK1 and RIPK1 in the protein preparations were also determined by immunoblotting (right panels). Because HEK293 cells do not express RIPK3, endogenous RIPK3 was not examined.
Mentions: In an effort to determine the mechanism by which Tab2 deficiency activates TNF-induced RIPK3-dependent cell death, we focused on hyperactivation and sustained activation of TAK1 and asked whether it is the cause of RIPK3-dependent cell death. We used HeLa cells because they are highly transfectable and known to be deficient in RIPK3 expression (He et al., 2009). We activated TAK1 using TAK1-binding protein 1 (TAB1), which is a constitutively associated TAK1 binding partner, and co-overexpression of TAK1 and TAB1 highly activates TAK1 (Kishimoto et al., 2000). TAK1 and TAB1 were overexpressed in the presence and absence of RIPK3 in HeLa cells, which were analyzed for cell death (Fig. 5). Activation of TAK1 or expression of RIPK3 alone did not profoundly induce cell death but RIPK3 moderately increased annexin V– and cell permeability dye double-positive necrotic cell death in the presence of treatment with TNF together with an inhibitor of cIAP, Smac mimetic (Wang et al., 2008), and Z-VAD (T/S/Z) treatment (Fig. 5 A), which is consistent with the earlier study (He et al., 2009). However, when TAK1 and TAB1 were coexpressed with RIPK3, cell death was dramatically enhanced (Fig. 5 A). We also reproduced this result in mouse dermal fibroblasts. Overexpression of TAK1 and TAB1 or RIPK3 alone only marginally increased necrotic cell death in dermal fibroblasts, but overexpression of TAK1, TAB1, and RIPK3 together highly enhanced cell death (Fig. S4). Overexpression of TAK1 and TAB1 produces an active form of TAK1 and we found that it induced a slower migrating additional RIPK3 band (Fig. 5 B). The retardation of RIPK3 migration was abolished by lambda phosphatase treatment (Fig. 5 C), suggesting that RIPK3 is phosphorylated by TAK1. We then examined whether TAK1 activates RIPK3 by the in vitro kinase assay (Fig. 5 D). An active and kinase-dead TAK1, RIPK3, and kinase-dead RIPK3 were prepared from HEK293 cells separately overexpressing these proteins, and proteins were mixed as indicated and subjected to in vitro kinase assay. Kinase activity was assessed by its autophosphorylation of RIPK3. RIPK3 was basally active (lane 3), which is consistent with an earlier study (Cho et al., 2009), and the activity was further increased by TAK1 (lane 4). The kinase-dead RIPK3 was not detectably phosphorylated by TAK1 (lane 7). Thus, phosphorylation of wild-type RIPK3 (lanes 3–5) is likely to be predominantly mediated by RIPK3 autophosphorylation. We note that endogenous RIPK1 was co-precipitated with RIPK3 (Fig. 5 D, far right panels). This raises the possibility that co-precipitated RIPK1 in the RIPK3 preparation may be involved in activity of RIPK3 in these in vitro assays. However, endogenous RIPK1 was not coprecipitated with TAK1 (Fig. 5 D, second from right panels), indicating that adding TAK1 preparation to the kinase assay mixture did not increase total amount of RIPK1. Thus, the increase of RIPK3 activity (lane 4) over basal RIPK3 activity (lane 3) is mediated by TAK1 but not due to increase amount of RIPK1. These data demonstrate that an active TAK1 activates RIPK3, leading to necrosis.

Bottom Line: We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation.Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro.Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695.

ABSTRACT
TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Show MeSH
Related in: MedlinePlus