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Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Piazzolla D, Meissl K, Kucerova L, Rubiolo C, Baccarini M - J. Cell Biol. (2005)

Bottom Line: Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha.Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts.Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, Campus Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

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Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P < 0.01 according to a t test comparing Rok-α with SCR siRNA–transfected cells of either genotype. (E) KO cells transfected with Rok-α or SCR siRNA were stained with antibodies against Fas and ezrinpT567. 81 ± 3% of the cells displayed the phenotypes shown. (F and G) DN ezrin restores normal Fas internalization and Fas sensitivity in KO cells. (F) Morphology and Fas staining of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG). Reduced surface Fas clustering was displayed by 89 ± 4% of the DN-Ez-eG–transfected cells. (G, top) The effect of eG and Ez-eG on Fas-induced apoptosis (200 ng/ml αFas and 5 μg/ml Chx for 16 h) was determined by FACS analysis of Annexin V–positive cells. (bottom) The effect of eG and DN-Ez-eG on Fas internalization was determined as described in Fig. 2 B. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.01 (top) and *, P < 0.04 (bottom) according to a t test comparing eG with DN-Ez-eG–expressing cells of either genotype. (A–G) Transfection with the empty vector (eG) or with SCR siRNA did not alter the phenotype of KO cells.
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fig5: Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P < 0.01 according to a t test comparing Rok-α with SCR siRNA–transfected cells of either genotype. (E) KO cells transfected with Rok-α or SCR siRNA were stained with antibodies against Fas and ezrinpT567. 81 ± 3% of the cells displayed the phenotypes shown. (F and G) DN ezrin restores normal Fas internalization and Fas sensitivity in KO cells. (F) Morphology and Fas staining of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG). Reduced surface Fas clustering was displayed by 89 ± 4% of the DN-Ez-eG–transfected cells. (G, top) The effect of eG and Ez-eG on Fas-induced apoptosis (200 ng/ml αFas and 5 μg/ml Chx for 16 h) was determined by FACS analysis of Annexin V–positive cells. (bottom) The effect of eG and DN-Ez-eG on Fas internalization was determined as described in Fig. 2 B. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.01 (top) and *, P < 0.04 (bottom) according to a t test comparing eG with DN-Ez-eG–expressing cells of either genotype. (A–G) Transfection with the empty vector (eG) or with SCR siRNA did not alter the phenotype of KO cells.

Mentions: Rok-α–dependent ezrin deregulation is responsible for the defects in Fas clustering and internalization in Raf-1–deficient cells. To determine whether hyperactivation of Rok-α was responsible for the increased clustering of Fas and for Fas hypersensitivity in KO cells, we transfected Raf-1–deficient cells with plasmids expressing a dominant-negative (DN) form of this protein. KD Rok-α (eG–Rok-α KD) abrogated both ezrin phosphorylation and Fas clustering (Fig. 5, A and B); however, as a result of the toxicity of the construct, it was not possible to investigate its effects on Fas-induced apoptosis. Therefore, we used small interfering RNA (siRNA) to silence Rok-α in KO and WT fibroblasts. 72 h after transfection with Rok-α siRNA, the expression of Rok-α, but not of the related kinase Rok-β, was radically reduced in cells of either genotype (Fig. 5 C). Silencing Rok-α abrogated the hypersensitivity of KO cells to Fas-induced apoptosis (Fig. 5 D) and dramatically reduced Fas clustering and ezrin phosphorylation (Fig. 5 E). Scrambled (SCR) siRNA had no effect on Rok-α expression, Fas clustering, Fas-induced apoptosis, or ezrin phosphorylation (Fig. 5, C–E). To determine whether ezrin hyperphosphorylation was causally linked to increased Fas clustering and to hypersensitivity to Fas-induced apoptosis, we transfected WT and KO cells with DN ezrin (DN-Ez-eG or ezrin1-310), which interferes with the function of endogenous ERM and microvilli formation (Crepaldi et al., 1997). DN ezrin abrogated Fas clustering (Fig. 5 F) and significantly decreased Fas-induced apoptosis in KO cells while causing a reproducible, albeit not significant, increase in Fas-induced apoptosis in WT cells (Fig. 5 G, top). In addition, DN ezrin had opposite effects on the internalization in WT and KO cells: it decreased internalization in the former and increased it in the latter (Fig. 5 G, bottom). Together with the data in Figs. 2 E, 3 B, and 4 D, these results show that in WT cells, ezrin is a positive regulator of Fas internalization and that it may modulate apoptosis by regulating the amount of Fas available for further stimulation at the cell surface. In contrast, in KO fibroblasts, hyperphosphorylation of ezrin prevents internalization and prolongs the death signal, thereby determining the difference in Fas sensitivity between WT and KO cells.


Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Piazzolla D, Meissl K, Kucerova L, Rubiolo C, Baccarini M - J. Cell Biol. (2005)

Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P < 0.01 according to a t test comparing Rok-α with SCR siRNA–transfected cells of either genotype. (E) KO cells transfected with Rok-α or SCR siRNA were stained with antibodies against Fas and ezrinpT567. 81 ± 3% of the cells displayed the phenotypes shown. (F and G) DN ezrin restores normal Fas internalization and Fas sensitivity in KO cells. (F) Morphology and Fas staining of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG). Reduced surface Fas clustering was displayed by 89 ± 4% of the DN-Ez-eG–transfected cells. (G, top) The effect of eG and Ez-eG on Fas-induced apoptosis (200 ng/ml αFas and 5 μg/ml Chx for 16 h) was determined by FACS analysis of Annexin V–positive cells. (bottom) The effect of eG and DN-Ez-eG on Fas internalization was determined as described in Fig. 2 B. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.01 (top) and *, P < 0.04 (bottom) according to a t test comparing eG with DN-Ez-eG–expressing cells of either genotype. (A–G) Transfection with the empty vector (eG) or with SCR siRNA did not alter the phenotype of KO cells.
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fig5: Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P < 0.01 according to a t test comparing Rok-α with SCR siRNA–transfected cells of either genotype. (E) KO cells transfected with Rok-α or SCR siRNA were stained with antibodies against Fas and ezrinpT567. 81 ± 3% of the cells displayed the phenotypes shown. (F and G) DN ezrin restores normal Fas internalization and Fas sensitivity in KO cells. (F) Morphology and Fas staining of KO cells expressing eGFP (eG) or eGFP-ezrin1-310 (DN-Ez-eG). Reduced surface Fas clustering was displayed by 89 ± 4% of the DN-Ez-eG–transfected cells. (G, top) The effect of eG and Ez-eG on Fas-induced apoptosis (200 ng/ml αFas and 5 μg/ml Chx for 16 h) was determined by FACS analysis of Annexin V–positive cells. (bottom) The effect of eG and DN-Ez-eG on Fas internalization was determined as described in Fig. 2 B. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.01 (top) and *, P < 0.04 (bottom) according to a t test comparing eG with DN-Ez-eG–expressing cells of either genotype. (A–G) Transfection with the empty vector (eG) or with SCR siRNA did not alter the phenotype of KO cells.
Mentions: Rok-α–dependent ezrin deregulation is responsible for the defects in Fas clustering and internalization in Raf-1–deficient cells. To determine whether hyperactivation of Rok-α was responsible for the increased clustering of Fas and for Fas hypersensitivity in KO cells, we transfected Raf-1–deficient cells with plasmids expressing a dominant-negative (DN) form of this protein. KD Rok-α (eG–Rok-α KD) abrogated both ezrin phosphorylation and Fas clustering (Fig. 5, A and B); however, as a result of the toxicity of the construct, it was not possible to investigate its effects on Fas-induced apoptosis. Therefore, we used small interfering RNA (siRNA) to silence Rok-α in KO and WT fibroblasts. 72 h after transfection with Rok-α siRNA, the expression of Rok-α, but not of the related kinase Rok-β, was radically reduced in cells of either genotype (Fig. 5 C). Silencing Rok-α abrogated the hypersensitivity of KO cells to Fas-induced apoptosis (Fig. 5 D) and dramatically reduced Fas clustering and ezrin phosphorylation (Fig. 5 E). Scrambled (SCR) siRNA had no effect on Rok-α expression, Fas clustering, Fas-induced apoptosis, or ezrin phosphorylation (Fig. 5, C–E). To determine whether ezrin hyperphosphorylation was causally linked to increased Fas clustering and to hypersensitivity to Fas-induced apoptosis, we transfected WT and KO cells with DN ezrin (DN-Ez-eG or ezrin1-310), which interferes with the function of endogenous ERM and microvilli formation (Crepaldi et al., 1997). DN ezrin abrogated Fas clustering (Fig. 5 F) and significantly decreased Fas-induced apoptosis in KO cells while causing a reproducible, albeit not significant, increase in Fas-induced apoptosis in WT cells (Fig. 5 G, top). In addition, DN ezrin had opposite effects on the internalization in WT and KO cells: it decreased internalization in the former and increased it in the latter (Fig. 5 G, bottom). Together with the data in Figs. 2 E, 3 B, and 4 D, these results show that in WT cells, ezrin is a positive regulator of Fas internalization and that it may modulate apoptosis by regulating the amount of Fas available for further stimulation at the cell surface. In contrast, in KO fibroblasts, hyperphosphorylation of ezrin prevents internalization and prolongs the death signal, thereby determining the difference in Fas sensitivity between WT and KO cells.

Bottom Line: Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha.Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts.Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, Campus Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

Show MeSH
Related in: MedlinePlus