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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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Raf-1 modulates cytoskeletal changes during Fas activation and is required for Fas internalization. (A) Fas is concentrated in patches and clusters at the surface of Raf-1 KO MEFs. MEFs were stained with FITC-conjugated αFas for 45 min on ice (0). Cells were then washed, incubated for 30 min at 37°C, and analyzed using a confocal microscope. Fas clustering was observed in 72 ± 4% of KO cells. (B) Defective internalization in Raf-1–deficient MEFs. Cells were treated with 1 μg/ml αFas at 4°C to allow binding, washed, and either kept at 4°C or transferred to 37°C to allow internalization. Internalization was determined by comparing the amount of αFas left on the surface of cells incubated at 37°C with that present on the surface of the cells kept at 4°C. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025; **, P < 0.015, according to a t test comparing KO with WT cells. (C–E) Dramatic cytoskeletal remodeling in Raf-1 KO MEFs. Cells were treated with FITC-conjugated αFas as described in A and were stained with rhodamin-conjugated phalloidin (C), with antibodies against vimentin (D), or with ezrinpT567 (E) followed by the appropriate fluorochrome-conjugated antibodies. As the amount of Fas on the surface of WT cells is too low to be detectable, the merge is shown only for the KO cells. 74 ± 7% untreated KO cells showed the cytoskeletal phenotypes. Fas-induced shrinkage was observed in 70 ± 4% of the KO cells, and uropod formation was observed in 35 ± 3% of WT cells.
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fig2: Raf-1 modulates cytoskeletal changes during Fas activation and is required for Fas internalization. (A) Fas is concentrated in patches and clusters at the surface of Raf-1 KO MEFs. MEFs were stained with FITC-conjugated αFas for 45 min on ice (0). Cells were then washed, incubated for 30 min at 37°C, and analyzed using a confocal microscope. Fas clustering was observed in 72 ± 4% of KO cells. (B) Defective internalization in Raf-1–deficient MEFs. Cells were treated with 1 μg/ml αFas at 4°C to allow binding, washed, and either kept at 4°C or transferred to 37°C to allow internalization. Internalization was determined by comparing the amount of αFas left on the surface of cells incubated at 37°C with that present on the surface of the cells kept at 4°C. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025; **, P < 0.015, according to a t test comparing KO with WT cells. (C–E) Dramatic cytoskeletal remodeling in Raf-1 KO MEFs. Cells were treated with FITC-conjugated αFas as described in A and were stained with rhodamin-conjugated phalloidin (C), with antibodies against vimentin (D), or with ezrinpT567 (E) followed by the appropriate fluorochrome-conjugated antibodies. As the amount of Fas on the surface of WT cells is too low to be detectable, the merge is shown only for the KO cells. 74 ± 7% untreated KO cells showed the cytoskeletal phenotypes. Fas-induced shrinkage was observed in 70 ± 4% of the KO cells, and uropod formation was observed in 35 ± 3% of WT cells.

Mentions: Consistent with the results in Fig. 1, Fas staining was faint, distributed evenly on the cell surface of WT MEFs, and did not appreciably change upon Fas stimulation. In KO cells, the staining was brighter, and Fas was visualized as a rim around the cells and occasionally in patches and clusters that became more prominent upon Fas stimulation (Fig. 2 A). In addition, Fas internalization was significantly reduced in KO fibroblasts (Fig. 2 B). These phenotypes suggested a possible defect in Fas-stimulated cytoskeletal rearrangement, particularly in view of the cytoskeletal anomalies reported in migrating Raf-1 KO cells (Ehrenreiter et al., 2005). Upon Fas stimulation, WT cells produced long protrusions that were brightly stained with an antibody against phosphorylated ezrin (pT567), which was hardly detectable in unstimulated WT cells (Fig. 2 E). These structures are reminiscent of the uropods observed in T lymphocytes—long (at least one third of the whole cell body) and large bulbs transiently protruding from the cell surface—whose formation depends on the phosphorylation of ezrin on T567 (Lee et al., 2004). In T lymphocytes, functionally active Fas colocalizes with ezrin in the uropodes (Parlato et al., 2000); in adherent Raf-1 WT fibroblasts, however, the amount of Fas was too low to be detectable. As previously described, in unstimulated KO fibroblasts, the actin was detected in a rim around the cells, and the vimentin cytoskeleton was disorganized (Ehrenreiter et al., 2005). Upon Fas stimulation, bright patches of actin appeared, which partially colocalized with Fas. In addition, the vimentin cytoskeleton collapsed and was visualized as a dense perinuclear structure and at the tips of the short protrusions induced by Fas in these cells (Fig. 2, C and D). Although full-fledged uropods could not be observed in KO fibroblasts, these small, Fas-induced protrusions may be interpreted as an attempt to form such structures. In contrast to the WT, unstimulated KO fibroblasts contained significant amounts of ezrinpT567 (Figs. 2 E, top; and 4 D, bottom) localized to microvilli, which is in line with previous data (Takeuchi et al., 1994). In the KO, however, ezrinpT567 staining increased and concentrated in large spots, which partially colocalized with Fas (Fig. 2 E). The presence of hyperphosphorylated ezrin in KO cells could be confirmed by immunoblotting (Fig. 3 B).


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)

Raf-1 modulates cytoskeletal changes during Fas activation and is required for Fas internalization. (A) Fas is concentrated in patches and clusters at the surface of Raf-1 KO MEFs. MEFs were stained with FITC-conjugated αFas for 45 min on ice (0). Cells were then washed, incubated for 30 min at 37°C, and analyzed using a confocal microscope. Fas clustering was observed in 72 ± 4% of KO cells. (B) Defective internalization in Raf-1–deficient MEFs. Cells were treated with 1 μg/ml αFas at 4°C to allow binding, washed, and either kept at 4°C or transferred to 37°C to allow internalization. Internalization was determined by comparing the amount of αFas left on the surface of cells incubated at 37°C with that present on the surface of the cells kept at 4°C. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025; **, P < 0.015, according to a t test comparing KO with WT cells. (C–E) Dramatic cytoskeletal remodeling in Raf-1 KO MEFs. Cells were treated with FITC-conjugated αFas as described in A and were stained with rhodamin-conjugated phalloidin (C), with antibodies against vimentin (D), or with ezrinpT567 (E) followed by the appropriate fluorochrome-conjugated antibodies. As the amount of Fas on the surface of WT cells is too low to be detectable, the merge is shown only for the KO cells. 74 ± 7% untreated KO cells showed the cytoskeletal phenotypes. Fas-induced shrinkage was observed in 70 ± 4% of the KO cells, and uropod formation was observed in 35 ± 3% of WT cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171328&req=5

fig2: Raf-1 modulates cytoskeletal changes during Fas activation and is required for Fas internalization. (A) Fas is concentrated in patches and clusters at the surface of Raf-1 KO MEFs. MEFs were stained with FITC-conjugated αFas for 45 min on ice (0). Cells were then washed, incubated for 30 min at 37°C, and analyzed using a confocal microscope. Fas clustering was observed in 72 ± 4% of KO cells. (B) Defective internalization in Raf-1–deficient MEFs. Cells were treated with 1 μg/ml αFas at 4°C to allow binding, washed, and either kept at 4°C or transferred to 37°C to allow internalization. Internalization was determined by comparing the amount of αFas left on the surface of cells incubated at 37°C with that present on the surface of the cells kept at 4°C. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025; **, P < 0.015, according to a t test comparing KO with WT cells. (C–E) Dramatic cytoskeletal remodeling in Raf-1 KO MEFs. Cells were treated with FITC-conjugated αFas as described in A and were stained with rhodamin-conjugated phalloidin (C), with antibodies against vimentin (D), or with ezrinpT567 (E) followed by the appropriate fluorochrome-conjugated antibodies. As the amount of Fas on the surface of WT cells is too low to be detectable, the merge is shown only for the KO cells. 74 ± 7% untreated KO cells showed the cytoskeletal phenotypes. Fas-induced shrinkage was observed in 70 ± 4% of the KO cells, and uropod formation was observed in 35 ± 3% of WT cells.
Mentions: Consistent with the results in Fig. 1, Fas staining was faint, distributed evenly on the cell surface of WT MEFs, and did not appreciably change upon Fas stimulation. In KO cells, the staining was brighter, and Fas was visualized as a rim around the cells and occasionally in patches and clusters that became more prominent upon Fas stimulation (Fig. 2 A). In addition, Fas internalization was significantly reduced in KO fibroblasts (Fig. 2 B). These phenotypes suggested a possible defect in Fas-stimulated cytoskeletal rearrangement, particularly in view of the cytoskeletal anomalies reported in migrating Raf-1 KO cells (Ehrenreiter et al., 2005). Upon Fas stimulation, WT cells produced long protrusions that were brightly stained with an antibody against phosphorylated ezrin (pT567), which was hardly detectable in unstimulated WT cells (Fig. 2 E). These structures are reminiscent of the uropods observed in T lymphocytes—long (at least one third of the whole cell body) and large bulbs transiently protruding from the cell surface—whose formation depends on the phosphorylation of ezrin on T567 (Lee et al., 2004). In T lymphocytes, functionally active Fas colocalizes with ezrin in the uropodes (Parlato et al., 2000); in adherent Raf-1 WT fibroblasts, however, the amount of Fas was too low to be detectable. As previously described, in unstimulated KO fibroblasts, the actin was detected in a rim around the cells, and the vimentin cytoskeleton was disorganized (Ehrenreiter et al., 2005). Upon Fas stimulation, bright patches of actin appeared, which partially colocalized with Fas. In addition, the vimentin cytoskeleton collapsed and was visualized as a dense perinuclear structure and at the tips of the short protrusions induced by Fas in these cells (Fig. 2, C and D). Although full-fledged uropods could not be observed in KO fibroblasts, these small, Fas-induced protrusions may be interpreted as an attempt to form such structures. In contrast to the WT, unstimulated KO fibroblasts contained significant amounts of ezrinpT567 (Figs. 2 E, top; and 4 D, bottom) localized to microvilli, which is in line with previous data (Takeuchi et al., 1994). In the KO, however, ezrinpT567 staining increased and concentrated in large spots, which partially colocalized with Fas (Fig. 2 E). The presence of hyperphosphorylated ezrin in KO cells could be confirmed by immunoblotting (Fig. 3 B).

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