PMC

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 (). 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.

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-FLIP_L, caspase-8, and actin was determined by immunoblotting. Pro-c8, caspase-8 precursor; c8 p43, caspase-8 cleavage product; FlipL p43/p41, c-FLIP_L cleavage product; *, unspecific band. (B) Caspase-8, c-FLIP_L, 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.

Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO;lpr/+ embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO;lpr/+ liver. Apoptotic (TUNEL⁺) cells are absent in Raf-1 KO;lpr/+. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO;lpr/+ pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO;lpr/+ pup (right). The experiments were performed exclusively with F2 animals.

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 ezrin^pT567 (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.

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 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 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.

Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P < 0.02; **, P < 0.01, according to a t test comparing KO with WT cells. (B) The surface expression of Fas but not of TNFRI is altered in Raf-1 KO MEFs. WT and KO cells were stained with FITC-conjugated αFas (left) or with hamster α-mouse TNFRI antibody followed by FITC-conjugated goat α-hamster antibody (right) and were analyzed by flow cytometry. Dashed lines, isotype control (iso). (C and D) Fas is slightly overexpressed in Raf-1–deficient cells. (C) Different amounts of whole cell lysates from WT and KO cells were analyzed by αFas immunoblotting. Ponceau staining of the membrane is shown as a loading control. (D) Fas mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker. Molecular mass markers (in kilodaltons, C; or bp, D) are shown on the left.

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 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 ezrin^pT567. 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-ezrin^1-310 (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 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.

Heterozygosity at the lpr locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1 ^–/– ;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1 ^–/– ;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.