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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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Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1−/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P < 0.01 according to a t test comparing WT, vector (V), KC, or KD cells with KO cells.
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fig4: Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1−/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P < 0.01 according to a t test comparing WT, vector (V), KC, or KD cells with KO cells.

Mentions: We have previously shown that in unstimulated and migrating fibroblasts, the formation of a Raf-1–Rok-α complex restrains the activation of Rok-α independently of Raf-1 kinase activity (Ehrenreiter et al., 2005). Among the consequences of Rok-α hyperactivation in Raf-1 KO cells are the disorganization of the vimentin cytoskeleton, which results in the relocalization of Rok-α to the plasma membrane, and the constitutive phosphorylation of ezrin on T567 (Ehrenreiter et al., 2005). The rapid collapse of vimentin structures and the hyperphosphorylation of ezrin in Fas-stimulated KO cells, which are hallmarks of Rok-α activation, suggested that Raf-1 might serve a similar function during Fas stimulation. Indeed, Rok-α was quickly translocated to the membrane of KO cells upon Fas stimulation, where it often resided in structures similar to small blebs (Fig. 4 A, arrowheads). In contrast, in WT cells, Fas stimulation resulted in an even more defined localization of Rok-α to the vimentin cytoskeleton (Fig. 4 A). Consistent with a role of Raf-1 in the regulation of Rok-α activity and localization, increasing amounts of Rok-α were detectable in endogenous Raf-1 immunoprecipitates (IPs) from Fas-induced WT cells (Fig. 4 B). As described for migration, the stable expression of either kinase-competent (KC) or kinase-dead (KD) Raf-1 in KO cells rescued the increase in Fas expression (Fig. 4, C and D) and ezrin phosphorylation/localization (Fig. 4 D) as well as the cytoskeletal defects (Ehrenreiter et al., 2005). Consistently, both KC and KD clones showed normal sensitivity to Fas-mediated apoptosis. TNFα-induced apoptosis was not affected by the reintroduction of KC or KD Raf-1 (Fig. 4 E). Vector-transfected cells and KO MEFs behaved indistinguishably.


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)

Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1−/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P < 0.01 according to a t test comparing WT, vector (V), KC, or KD cells with KO cells.
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fig4: Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1−/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P < 0.01 according to a t test comparing WT, vector (V), KC, or KD cells with KO cells.
Mentions: We have previously shown that in unstimulated and migrating fibroblasts, the formation of a Raf-1–Rok-α complex restrains the activation of Rok-α independently of Raf-1 kinase activity (Ehrenreiter et al., 2005). Among the consequences of Rok-α hyperactivation in Raf-1 KO cells are the disorganization of the vimentin cytoskeleton, which results in the relocalization of Rok-α to the plasma membrane, and the constitutive phosphorylation of ezrin on T567 (Ehrenreiter et al., 2005). The rapid collapse of vimentin structures and the hyperphosphorylation of ezrin in Fas-stimulated KO cells, which are hallmarks of Rok-α activation, suggested that Raf-1 might serve a similar function during Fas stimulation. Indeed, Rok-α was quickly translocated to the membrane of KO cells upon Fas stimulation, where it often resided in structures similar to small blebs (Fig. 4 A, arrowheads). In contrast, in WT cells, Fas stimulation resulted in an even more defined localization of Rok-α to the vimentin cytoskeleton (Fig. 4 A). Consistent with a role of Raf-1 in the regulation of Rok-α activity and localization, increasing amounts of Rok-α were detectable in endogenous Raf-1 immunoprecipitates (IPs) from Fas-induced WT cells (Fig. 4 B). As described for migration, the stable expression of either kinase-competent (KC) or kinase-dead (KD) Raf-1 in KO cells rescued the increase in Fas expression (Fig. 4, C and D) and ezrin phosphorylation/localization (Fig. 4 D) as well as the cytoskeletal defects (Ehrenreiter et al., 2005). Consistently, both KC and KD clones showed normal sensitivity to Fas-mediated apoptosis. TNFα-induced apoptosis was not affected by the reintroduction of KC or KD Raf-1 (Fig. 4 E). Vector-transfected cells and KO MEFs behaved indistinguishably.

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