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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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Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 (O'Neill et al., 2004). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.
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fig8: Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 (O'Neill et al., 2004). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.

Mentions: In agreement with this hypothesis, if we restore Fas internalization, we reduce the sensitivity of KO MEFs to Fas-induced apoptosis. But how does this work? Our experiments clearly indicate a role for Rok-α downstream signaling (more specifically for ezrin) in agonist-induced Fas internalization. In WT fibroblasts, ezrinpT567 can be detected only upon Fas stimulation and is concentrated in uropod-like protrusions (one to two per cell), the site of Fas accumulation in T cells (Parlato et al., 2000). In addition, interfering with ezrin function in WT MEFs results in decreased Fas internalization. The opposite happens in KO cells (Fig. 5), in which ezrin is constitutively phosphorylated and is hyperphosphorylated upon Fas activation. These results indicate that a tight control of ezrin activation is necessary for correct Fas signaling. Together with the evidence in a previous study (Parlato et al., 2000), they suggest a model in which ezrin phosphorylation is necessary for uropod development and Fas clustering and, in turn, for DISC formation. However, if too much ezrin is phosphorylated or if ezrin phosphorylation persists for too long, large Fas clusters that cannot be internalized, ineffective DISC formation, and inefficient substrate cleavage result (Fig. 8). Fas internalization requires both actin and caspase-8 activity (Algeciras-Schimnich et al., 2002), and it has been suggested that DISC internalization may depend on the degradation of substrates involved in cortical actin reorganization by DISC-associated caspase-8. In this situation, an excess of ezrinp567, which acts as an actin filament/plasma membrane cross-linker and may, therefore, prevent cortical actin reorganization, is probably counterproductive (Bretscher et al., 2002). Indeed, Fas-stimulated, caspase-dependent dephosphorylation of ezrin has been shown previously (Kondo et al., 1997). In WT fibroblasts, Fas activation increases the interaction between Raf-1 and Rok-α and, thereby, prevents the hyperactivation of the latter kinase and its substrates, favoring cortical actin rearrangements and Fas internalization. Thus, Rok-α downstream signaling has an impact on both the strength and duration of the apoptotic signal, and restraining Rok-α is the common molecular basis of the essential function of Raf-1 in apoptosis and migration.


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)

Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 (O'Neill et al., 2004). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.
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Related In: Results  -  Collection

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fig8: Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 (O'Neill et al., 2004). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.
Mentions: In agreement with this hypothesis, if we restore Fas internalization, we reduce the sensitivity of KO MEFs to Fas-induced apoptosis. But how does this work? Our experiments clearly indicate a role for Rok-α downstream signaling (more specifically for ezrin) in agonist-induced Fas internalization. In WT fibroblasts, ezrinpT567 can be detected only upon Fas stimulation and is concentrated in uropod-like protrusions (one to two per cell), the site of Fas accumulation in T cells (Parlato et al., 2000). In addition, interfering with ezrin function in WT MEFs results in decreased Fas internalization. The opposite happens in KO cells (Fig. 5), in which ezrin is constitutively phosphorylated and is hyperphosphorylated upon Fas activation. These results indicate that a tight control of ezrin activation is necessary for correct Fas signaling. Together with the evidence in a previous study (Parlato et al., 2000), they suggest a model in which ezrin phosphorylation is necessary for uropod development and Fas clustering and, in turn, for DISC formation. However, if too much ezrin is phosphorylated or if ezrin phosphorylation persists for too long, large Fas clusters that cannot be internalized, ineffective DISC formation, and inefficient substrate cleavage result (Fig. 8). Fas internalization requires both actin and caspase-8 activity (Algeciras-Schimnich et al., 2002), and it has been suggested that DISC internalization may depend on the degradation of substrates involved in cortical actin reorganization by DISC-associated caspase-8. In this situation, an excess of ezrinp567, which acts as an actin filament/plasma membrane cross-linker and may, therefore, prevent cortical actin reorganization, is probably counterproductive (Bretscher et al., 2002). Indeed, Fas-stimulated, caspase-dependent dephosphorylation of ezrin has been shown previously (Kondo et al., 1997). In WT fibroblasts, Fas activation increases the interaction between Raf-1 and Rok-α and, thereby, prevents the hyperactivation of the latter kinase and its substrates, favoring cortical actin rearrangements and Fas internalization. Thus, Rok-α downstream signaling has an impact on both the strength and duration of the apoptotic signal, and restraining Rok-α is the common molecular basis of the essential function of Raf-1 in apoptosis and migration.

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