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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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Inefficient DISC formation but increased caspase activation in Raf-1 KO cells. (A) DISC formation is inefficient in Raf-1–deficient MEFs. Cells were either left untreated (0) or were treated with 2 μg/ml αFas plus 5 μg/ml Chx. At the indicated times, the DISC was collected using protein A–Sepharose beads. The presence of Fas, FADD, c-FLIPL, caspase-8, and actin was determined by immunoblotting. Pro-c8, caspase-8 precursor; c8 p43, caspase-8 cleavage product; FlipL p43/p41, c-FLIPL cleavage product; *, unspecific band. (B) Caspase-8, c-FLIPL, and caspase-3 are rapidly cleaved, and ezrin is hyperphosphorylated in KO MEFs treated with αFas. MEFs were treated with αFas/Chx as described in A. Whole cell lysates collected at the indicated times were analyzed by immunoblotting. Molecular mass markers are shown in kilodaltons on the left.
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fig3: Inefficient DISC formation but increased caspase activation in Raf-1 KO cells. (A) DISC formation is inefficient in Raf-1–deficient MEFs. Cells were either left untreated (0) or were treated with 2 μg/ml αFas plus 5 μg/ml Chx. At the indicated times, the DISC was collected using protein A–Sepharose beads. The presence of Fas, FADD, c-FLIPL, caspase-8, and actin was determined by immunoblotting. Pro-c8, caspase-8 precursor; c8 p43, caspase-8 cleavage product; FlipL p43/p41, c-FLIPL cleavage product; *, unspecific band. (B) Caspase-8, c-FLIPL, and caspase-3 are rapidly cleaved, and ezrin is hyperphosphorylated in KO MEFs treated with αFas. MEFs were treated with αFas/Chx as described in A. Whole cell lysates collected at the indicated times were analyzed by immunoblotting. Molecular mass markers are shown in kilodaltons on the left.

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)

Inefficient DISC formation but increased caspase activation in Raf-1 KO cells. (A) DISC formation is inefficient in Raf-1–deficient MEFs. Cells were either left untreated (0) or were treated with 2 μg/ml αFas plus 5 μg/ml Chx. At the indicated times, the DISC was collected using protein A–Sepharose beads. The presence of Fas, FADD, c-FLIPL, caspase-8, and actin was determined by immunoblotting. Pro-c8, caspase-8 precursor; c8 p43, caspase-8 cleavage product; FlipL p43/p41, c-FLIPL cleavage product; *, unspecific band. (B) Caspase-8, c-FLIPL, and caspase-3 are rapidly cleaved, and ezrin is hyperphosphorylated in KO MEFs treated with αFas. MEFs were treated with αFas/Chx as described in A. Whole cell lysates collected at the indicated times were analyzed by immunoblotting. Molecular mass markers are shown in kilodaltons on the left.
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fig3: Inefficient DISC formation but increased caspase activation in Raf-1 KO cells. (A) DISC formation is inefficient in Raf-1–deficient MEFs. Cells were either left untreated (0) or were treated with 2 μg/ml αFas plus 5 μg/ml Chx. At the indicated times, the DISC was collected using protein A–Sepharose beads. The presence of Fas, FADD, c-FLIPL, caspase-8, and actin was determined by immunoblotting. Pro-c8, caspase-8 precursor; c8 p43, caspase-8 cleavage product; FlipL p43/p41, c-FLIPL cleavage product; *, unspecific band. (B) Caspase-8, c-FLIPL, and caspase-3 are rapidly cleaved, and ezrin is hyperphosphorylated in KO MEFs treated with αFas. MEFs were treated with αFas/Chx as described in A. Whole cell lysates collected at the indicated times were analyzed by immunoblotting. Molecular mass markers are shown in kilodaltons on the left.
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