The role of receptor internalization in CD95 signaling
2006; Springer Nature; Volume: 25; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7601016
ISSN1460-2075
AutoresKyeong‐Hee Lee, Christine Feig, Vladimir Tchikov, Robert Schickel, Cora Hallas, Stefan Schütze, Marcus E. Peter, Andrew C. Chan,
Tópico(s)NF-κB Signaling Pathways
ResumoArticle23 February 2006free access The role of receptor internalization in CD95 signaling Kyeong-Hee Lee Kyeong-Hee Lee Department of Immunology, Genentech Inc., South San Francisco, CA, USA Search for more papers by this author Christine Feig Christine Feig The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Vladimir Tchikov Vladimir Tchikov Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Robert Schickel Robert Schickel The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Cora Hallas Cora Hallas Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Stefan Schütze Stefan Schütze Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Marcus E Peter Marcus E Peter The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Andrew C Chan Corresponding Author Andrew C Chan Department of Immunology, Genentech Inc., South San Francisco, CA, USA Search for more papers by this author Kyeong-Hee Lee Kyeong-Hee Lee Department of Immunology, Genentech Inc., South San Francisco, CA, USA Search for more papers by this author Christine Feig Christine Feig The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Vladimir Tchikov Vladimir Tchikov Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Robert Schickel Robert Schickel The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Cora Hallas Cora Hallas Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Stefan Schütze Stefan Schütze Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany Search for more papers by this author Marcus E Peter Marcus E Peter The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA Search for more papers by this author Andrew C Chan Corresponding Author Andrew C Chan Department of Immunology, Genentech Inc., South San Francisco, CA, USA Search for more papers by this author Author Information Kyeong-Hee Lee1,‡, Christine Feig2,‡, Vladimir Tchikov3,‡, Robert Schickel2, Cora Hallas3, Stefan Schütze3, Marcus E Peter2 and Andrew C Chan 1 1Department of Immunology, Genentech Inc., South San Francisco, CA, USA 2The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, USA 3Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany ‡These authors contributed equally to this work *Corresponding author. Department of Immunology, Genentech Inc., MS-34, Bldg. 12-281, 1 DNA Way, South San Francisco, CA 94080, USA. Tel.: +1 650 225 8104; Fax: +1 650 225 8136; E-mail: [email protected] or [email protected] The EMBO Journal (2006)25:1009-1023https://doi.org/10.1038/sj.emboj.7601016 These authors shared senior authorship PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-κB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates. Introduction Surface receptors transduce signals derived from the extracellular milieu to evoke a diverse range of cellular responses. This process is initiated upon ligand binding and transduced through the spatial and temporal regulation of physical interactions of receptors with intracellular signaling molecules. Gain- or loss-of function mutants alter the normal balance of cellular homeostasis that, in turn, can induce oncogenesis and/or developmental arrest. For many receptors, triggering by ligand results in receptor clustering that is followed by downregulation of activated surface receptors through endocytosis and subsequent lysosomal degradation (Ceresa and Schmid, 2000). These latter steps typically attenuate signaling via removal of activated receptor complexes. Recent studies, however, indicate that receptor internalization can target activated receptors to the endocytic compartment, and contributes to both the intensity of signaling and assembly of signaling complexes (Miaczynska et al, 2004b). CD95 (CD95/APO-1/TNFRSF6) is a prototypic death receptor belonging to the tumor necrosis factor (TNF) receptor superfamily (Li-Weber and Krammer, 2003). Interactions of CD95 with its ligand, CD95L (CD178/FasL/TNSF6), play a pivotal role in the regulation of peripheral tolerance and lymphoid homeostasis. Natural mutations within CD95 and CD95L in humans and mice are associated with the development of autoimmune lymphoproliferative syndromes (Nagata, 1999). CD95 is expressed on the surface of cells as preassociated homotrimers and, upon CD95L binding, undergoes a conformational change to reveal its cytoplasmic death domain (DD) to favor homotypic interactions with other DD-containing proteins (Itoh and Nagata, 1993; Boldin et al, 1995; Chinnaiyan et al, 1995; Siegel et al, 2000). Additional interactions mediated through the N-terminal ‘death effector domain’ (DED) of FADD with DED domains encoded within procaspase-8 and -10 assemble the death-inducing signaling complex (DISC) (Peter and Krammer, 2003). Efficient DISC assembly provides a molecular scaffold concentrating cysteine proteases to induce autoproteolytic cleavage of caspase-8 and, in turn, subsequent activation of the apoptotic pathway. CD95-mediated apoptosis is transduced through two general modes (Algeciras-Schimnich et al, 2003; Barnhart et al, 2003). Type I cells exhibit rapid receptor internalization and form large amounts of DISC, while type II cells are more dependent upon the mitochondrial amplification pathway and exhibit quantitatively less and slower DISC assembly. We demonstrate here that CD95 internalization in type I cells plays a previously unrecognized requisite role in CD95L-induced activation of apoptotic pathways. In contrast, engagement of CD95 without receptor internalization results in activation of nonapoptotic signaling pathways. Hence, the subcellular compartment of CD95 signaling activates divergent biochemical pathways to promote distinct survival or apoptotic cellular fates. Results Expression of a plasma membrane localized PIP2-specific 5-phosphatase modulates PIP2 levels and inhibits CD95L-induced CD95 internalization and apoptosis We have previously demonstrated that disruption of filamentous actin inhibits CD95 internalization, a process that normally proceeds through a clathrin-mediated endocytic compartment, and renders cells more resistant to CD95-mediated apoptosis (Algeciras-Schimnich et al, 2002; Algeciras-Schimnich and Peter, 2003). As cellular levels of PIP2 (PtdIns(4,5)P2) have been shown to regulate clathrin-mediated endocytosis (Martin, 2001), we employed an enzymatic approach using the Saccharomyces cerevisiae Inp54 5-phosphatase (INP54p) that hydrolyzes PIP2 to PI(4)P (Stolz et al, 1998). Targeting of a green fluorescent protein (GFP)–INP54p fusion protein to the plasma membrane (PM) was achieved by attaching a myristoylation/palmitoylation sequence from the Fyn protein tyrosine kinase (Shenoy-Scaria et al, 1993; Raucher et al, 2000) (Supplementary Figure 1A, middle panels). Expression of FynC-GFP-INP54p reduced PIP2 levels in >98% of transfected GFP+, but not of GFP−, cells (Supplementary Figure 1B, middle panel). In contrast, expression of a mutant in which Cys 3 and 6, important for palmitoylation and PM localization, were mutated to Ser (designated as FynS-GFP-INP54p) resulted in a cytoplasmic distribution (Supplementary Figure 1A, right panels) and lesser effects on PIP2 levels Supplementary Figure 1B, bottom panel). PIP2 levels were unaffected in cells expressing a control FynC-GFP cDNA (top panel). We next investigated the functional consequences of reduced PIP2 in CD95 function. BJAB cells, transiently transfected with FynC-GFP-INP54p, FynS-GFP-INP54p or FynC-GFP, were stimulated with Flag-tagged (Flag-)CD95L (SuperFasL, Apotech) and the degree of apoptosis was assessed by TUNEL staining. In FynC-GFP+ or FynS-GFP-INP54p+ cells, ∼65% of cells were TUNEL+ following CD95L stimulation Supplementary Figure 1C, left and right panels). In contrast, 90% using a dual-transfection protocol. Data shown are representative of two experiments. (B) Defective DISC assembly in PM-targeted INP54p-expressing cells. BJAB cells were treated as described in (A). CD95L–CD95 complexes were immunoprecipitated by use of anti-Flag Ab-coupled beads and analyzed for associated DISC proteins (lanes 1–8). Cell lysates were also analyzed for DISC proteins and CD95 expression (lanes 9 and 10). Data shown are representative of three experiments. (C) BJAB cells transiently transfected with FynC-GFP-INP54p were stimulated with Flag-CD95L for the indicated times. Permeabilized cells were visualized by deconvolution microscopy for GFP (green) in the left panels and stained for CD95 (red) and F-actin (blue) in the middle panels. Quantitative image analysis with RPVs recorded for CD95 fluorescence signals is shown in the right panels. Red indicates the highest and blue represents the lowest fluorescence intensity. RPV=relative pixel value. Data shown are representative of >150 cells analyzed. (D) Inhibition of CD95 internalization by FynC-GFP-INP54p. BJAB cells, transfected with FynC-GFP (top) or FynC-GFP-INP54p (bottom), were incubated with Flag-CD95L at 37°C for 15 (green) or 30 mins (red). As a control, cells were incubated with Flag-CD95L on ice (0 min, black). The remaining surface CD95 was detected by staining with an anti-CD95 mAb (DX2) and analyzed by flow cytometry. Data shown are representative of four experiments. Download figure Download PowerPoint As CD95L binding to CD95 was not altered by FynC-GFP-INP54p Supplementary Figure 3A), we assessed the effects of FynC-GFP-INP54p on CD95 clustering. Cells were incubated with Flag-CD95L at 4°C, activation was induced by incubation at 37°C, and localization of CD95 analyzed by deconvolution microscopy. In wt BJAB cells (GFP− cells), CD95L induced small ‘patch-like’ receptor clusters at the PM within 5 min after stimulation (Figure 1C, panels 4–6, left cell). Expression of FynC-GFP-INP54p did not affect the ability of CD95L to induce CD95 clustering at the PM 5 min following activation (Figure 1C, panels 4–6, GFP+ cells). By 15 and 30 min, the clustered CD95 in GFP− cells had internalized to intracellular compartments (Figure 1C, panels 7–12, left cell). In contrast, CD95 remained clustered at the PM for at least 30 min in FynC-GFP-INP54p+ cells without any significant internalization following CD95L stimulation (Figure 1C, panels 7–12, right cell). These results were further supported by flow-cytometric studies. In BJAB cells transfected with control FynC-GFP, CD95 downregulation was detected within 15 min following CD95L activation (Figure 1D, top panel). In contrast, FynC-GFP-INP54p+ cells were unable to downregulate surface CD95 even 30 min following CD95L activation (bottom panel). Similar results were found for SKW6.4 cells Supplementary Figure 2D). Membrane-bound CD95L (mCD95L)-induced apoptosis requires CD95 internalization Our data so far suggested that CD95-mediated apoptosis in response to a soluble form of CD95L (sCD95L) stimulation requires receptor internalization. However, it is widely assumed that the physiologic stimulus for CD95 is more likely to be mCD95L. We therefore incubated murine CT26 cells expressing human mCD95L (CT26mCD95L) with SKW6.4 cells. No soluble CD95L could be detected in CT26mCD95L cells or concentrated supernatant derived from cultures of these cells Supplementary Figure 4B and data not shown). In SKW6.4 cells, CD95 was efficiently internalized when co-incubated with CT26mCD95L, but not in untreated SKW6.4 cells (Figure 2A and (Supplementary Figure 4A and B). Correspondingly, CT26mCD95L cells induced a time-dependent processing of procaspase-8 Supplementary Figure 4C). When SKW6.4 cells were overlaid on adherent CT26 cells, CD95 internalized in SKW6.4 cells contacting CT26mCD95L, but not control CT26, cells (Figure 2B, bottom panels). These data suggest that mCD95L induces internalization of CD95 as much as sCD95L. Figure 2.Membrane-bound CD95L induces internalization of CD95. (A) Internalization of CD95 on SKW6.4 cells following activation by mCD95L. SKW6.4 cells were incubated with FITC-DX2 on ice. Cells were then left untreated or incubated with detached CT26 cells expressing human mCD95L for 1 h. Nuclei were stained with DAPI and CD95 was visualized by confocal microscopy. The left bottom panel represents an early stage with intact nucleus and internalized CD95-containing vesicles (arrowheads). The right bottom panel shows a more advanced stage of apoptosis with nuclear fragmentation. (B) CD95 on SKW6.4 cells internalizes at the contact side with mCD95L-expressing CT26 cells. SKW6.4 cells (labeled S) with a bound biotin-labeled anti-CD95 mAb were plated on top of adherent CT26 or CT26mCD95L cells (labeled C) and incubated as indicated. CD95 was visualized by staining with streptavidin Alexa Flour 488. Nuclei were visualized by DAPI staining. (C) Internalization of CD95 on BJAB cells following activation by cleavage-resistant mCD95L(DA4). BJAB cells (labeled B) were incubated with chicken DT40 cells (labeled D) (left panel) or DT40 cells expressing human mCD95L(DA4) (right panel) for 1 h and then analyzed by deconvolution microscopy for CD95 (red), surface hIgM (green) and DAPI staining (blue). (D) Inhibition of mCD95L-induced apoptosis in FynC-GFP-INP54p-expressing cells. BJAB cells were transfected with FynC-GFP-INP54p or control FynC-GFP and incubated with DT40 cells expressing a noncleavable mutant of human mCD95L(DA4) at a ratio of 1:5 (BJAB:DT40) for the indicated times. Apoptosis of GFP+ cells was assessed by staining with Annexin V. Data shown are representative of two independent experiments. (E) Inhibition of mCD95L-mediated CD95 downregulation in FynC-GFP-INP54p-expressing cells. BJAB cells, transfected as described in D, were incubated with DT40 cells expressing mCD95L(DA4) for 30 or 60 min. Surface CD95 expression on GFP+ cells was assessed by flow cytometry. Data shown are representative of two independent experiments. Download figure Download PowerPoint We have previously shown that unmodified sCD95L does not induce CD95 apoptosis in type I cells (Algeciras-Schimnich et al, 2003). However, to exclude that the internalization and apoptosis observed in cells exposed to mCD95L were not due to very small amounts of secreted sCD95L, we expressed a mutant form of human mCD95L (designated as DA4) that cannot be cleaved from the membrane surface (Tanaka et al, 1998) on the surface of chicken DT40 B cells Supplementary Figure 3B). Deconvolution microscopy confirmed that CD95 internalized into the cytoplasmic compartment when incubated with mCD95L(DA4)-expressing DT40 cells (Figure 2C, right panel), but not when incubated with control DT40 cells (left panel). We next tested whether apoptosis induced by mCD95L was affected by FynC-GFP-INP54p expression. While DT40 cells expressing the CD95L(DA4) mutant induced apoptosis in FynC-GFP+ BJAB cells (Figure 2D, left panels), apoptosis was significantly attenuated at 16 and 24 h following engagement of CD95L(DA4) in FynC-GFP-INP54p+ BJAB cells (right panels). Concurrently, surface CD95 was downregulated in FynC-GFP+ BJAB cells at 30 and 60 min, but this was significantly compromised in FynC-GFP-INP54p+ BJAB cells (Figure 2E). Expression of FynC-GFP-INP54p in BJAB cells inhibited CD95L-induced apoptosis irrespective of the degree of CD95 oligomerization. Expression of FynC-GFP-INP54p inhibited apoptosis and CD95 downregulation induced via soluble and plate-bound crosslinked Flag-CD95L or anti CD95 mAb (CH-11) Supplementary Figure 5A). Finally, CD95 activation of BJAB cells using beads covalently coupled with an anti-CD95 mAb or Flag-CD95L was also inhibited by expression of FynC-GFP-INP54p Supplementary Figure 5B and data not shown). In contrast to the agonistic anti-CD95 mAbs, treatment of H9 cells with an antagonistic anti-CD95 mAb (ZB4) failed to induce CD95 internalization Supplementary Figure 5C). Hence, independent of the methodology of stimulation, expression of FynC-GFP-INP54p inhibits CD95 internalization and apoptosis. Moreover, apoptosis is dependent on internalization of CD95 in cells treated with sCD95L or mCD95L. Clathrin-mediated endocytosis is required for CD95-induced apoptosis Our data suggested that modulation of PIP2 levels through INP54p rendered cells resistant to CD95-mediated apoptosis by blocking internalization of CD95. However, modulation of PIP2 could result in global cellular changes that could cause cells to become resistant to CD95-mediated apoptosis by mechanisms other than receptor internalization. CD95 has been suggested to internalize through a clathrin-mediated endocytic pathway (Algeciras-Schimnich et al, 2002). To specifically interfere with this form of receptor internalization, we targeted expression of the AP-2 adaptor complex and clathrin heavy chain (CHC) proteins using RNA interference. Transfection of siRNAs specific for CHC or AP-2(α±μ2) adaptor subunits resulted in significant reduction in their levels of protein expression (Figure 3A, lanes 2–6) (Motley et al, 2003). Figure 3.Clathrin-mediated endocytosis is required for CD95 downregulation and apoptosis. (A) Knockdown of CHC, AP-2 μ2 and AP-2 α using siRNAs in BJAB cells. Efficiency of knockdown was monitored by blotting for the indicated proteins. Blotting for actin was used as a control for protein loading (bottom). (B) BJAB cells transfected with the indicated siRNAs, as described in (A), were incubated with Flag-CD95L for 30 min and surface CD95 expression assessed by flow cytometry. Red histograms indicate cells stimulated for 30 min, while the gray shadowed areas indicate basal levels of CD95 without CD95L stimulation. Data shown are representative of three experiments. (C) BJAB cells, as described in (A) and (B), were incubated in the presence (grey) or absence (white) of Flag-CD95L for 16 h and apoptotic cells quantified by staining with Annexin V and 7-AAD. Data shown are representative of three experiments. (D) CD95L-induced association of FADD with CD95 is inhibited by AP-2 and CHC siRNAs. Cells were transfected with control (lanes 1 and 2), AP-2 (lane 3) or CHC (lane 4) siRNAs, activated with Flag-CD95L for 30 min and FADD association with activated CD95 assessed by immunoprecipitating CD95L–CD95 complexes. Lanes 5–7 demonstrate comparable levels of FADD and CD95 in all cells. (E) FADD and caspase-8 association with CD95 is inhibited by CHC siRNAs. BJAB cells were transfected with control (lanes 1–4) and CHC (lanes 5–8) siRNAs and activated with Flag-CD95L for the indicated times. Association of FADD and caspase-8 with activated CD95 was assessed by immunoprecipitating for CD95L and immunoblotting for FADD, caspase-8 and CD95. (F) Clathrin-mediated endocytosis is required for CD95-mediated apoptosis in PBTs. Activated human CD4+ PBTs were transfected with siRNAs for AP-2(α+μ2) and a GFP cDNA to monitor expression efficiency. Sorted GFPhi cells were analyzed for CD95 downregulation (left) and apoptosis (right) following Flag-CD95L activation (30 min and 6 h, respectively). Red histograms indicate cells stimulated with CD95L while grey shadowed areas represent untreated cells. % apoptotic cells are quantified on the right. Data shown are representative of two experiments. Download figure Download PowerPoint Correspondingly, knockdown of AP-2 (α or μ2) alone resulted in a moderate decrease in CD95L-induced downregulation of surface CD95 (Figure 3B, panels 2 and 3). Knockdown of both AP-2 (α and μ2) subunits or CHC resulted in a greater compromise in CD95 downregulation (panels 4 and 5). Finally, knockdown of both AP-2(α+μ2) and CHC resulted in the greatest inhibition of CD95 downregulation, though the basal level of surface CD95 expression was also decreased (panel 6). The degree of compromise observed in CD95 downregulation directly correlated with the degree of apoptosis induced by CD95L. Gene knockdown of either AP-2(α+μ2) or CHC resulted in ∼50–80% decrease in CD95L-induced apoptosis, respectively, and the combination of AP-2(α+μ2) and CHC siRNAs, which demonstrated the greatest inhibition in CD95L-induced CD95 downregulation, resulted in total protection from CD95L-induced apoptosis (Figure 3C). Interestingly, the inducible association of FADD with CD95 was compromised in cells transfected with AP-2(α+μ2) or CHC siRNAs (Figure 3D, lanes 3 and 4). The lack of FADD association severely reduced the formation of the DISC as neither FADD nor caspase-8 co-immunoprecipitated with CD95 at 5, 15 or 30 min following CD95 activation in cells transfected with CHC siRNAs (Figure 3E). These results suggest a role of receptor internalization in assembly of the DISC. Finally, we analyzed the role of CD95 internalization in CD95L-induced apoptosis with peripheral human T lymphocytes (PBTs). PBTs were activated through CD3/CD28 and then transfected with siRNAs for AP-2(α+μ2) and GFP, the latter of which was utilized to monitor expression. GFPhi cells were purified by cell sorting and analyzed for CD95 internalization and apoptosis. Transfection of AP-2(α+μ2) siRNAs in PBTs resulted in inhibition of CD95 downregulation following CD95L activation (Figure 3F, left bottom panel). Correspondingly, these cells demonstrated compromised CD95L-induced apoptosis (Figure 3F, right bottom panel). In summary, our data indicated that inhibition of CD95 internalization in type I cells as well as in primary T lymphocytes attenuated recruitment of DISC components to CD95 receptors and apoptosis. Recruitment of DISC components following CD95 internalization To directly follow the recruitment of DISC components to activated CD95 and to compare receptor signaling between type I and type II cells, we made use of a novel method to isolate receptor-containing internalized vesicles that has been used to detect internalizing TNF receptor and its signaling components (Schneider-Brachert et al, 2004). In this method, cells were incubated with biotinylated anti-CD95 (anti-APO-1) mAb, followed by addition of streptavidin coupled magnetic nanoparticles. Following internalization, cells were homogenized and magnetic vesicles isolated in a free flow apparatus employing a high-gradient magnetic field. Western blotting of receptor-containing vesicles for endosomal and lysosomal markers was performed to assess the different endocytic maturation stages of receptor-containing vesicles. Consistent with the ability of CD95 to internalize in type I SKW6.4 cells, Rab4 and EEA-1, markers for endosomal trafficking, were readily detected within CD95-containing vesicles very early after stimulation and peaking at 10 min (Figure 4A). Already detectable at 3 min and peaking at 30 min, CD95-containing vesicles also had lysosomal characteristics as evidenced by the appearance of cathepsin D (CatD), suggesting rapid association/fusion of CD95-containing receptosomes with CatD-containing lysosomal compartments. While a low level of FADD was detected in CD95 containing membrane structures at basal levels, its appearance in magnetic vesicles peaked at 30 min. Similar to FADD, caspase-8 and its intermediate cleavage products peaked at 10 min and could be detected as late as 3 h following stimulation (data not shown), and suggested that most of the caspase-8 activation occurred while inside the cells located on endosomal and even lysosomal vesicles. A similar kinetics for association of caspase-10 within the CD95-containing vesicles was also observed. Figure 4.Internalization and endosomal maturation of CD95 DISC complexes. Time course of intracellular CD95-receptosome trafficking in SKW6.4 (A) and Jurkat (B) cells. Total cell lysates or magnetic fractions derived after 0, 3, 10, 30 and 60 min of anti-APO-1 mAb treatment were analyzed for signature proteins of endosomal maturation (Rab4 and EEA-1), lysosomes (CatD), actin, CD95 and DISC proteins. Note: twice as much lysate proteins were loaded to visualize Rab4, EEA-1 and CatD in the Jurkat cells. Time course of intracellular CD95-receptosome trafficking in ACHN (C) and HCT15 (D) cells. Total cell lysates or magnetic fractions derived after anti-APO-1 mAb treatment were analyzed as described in A and B. Download figure Download PowerPoint In contrast to type I SKW6.4 cells, no significant increase in Rab4, EEA-1 or CatD was observed in type II Jurkat cells, suggesting a lack of directional movement of CD95 into endosomal vesicles (Figure 4B). Consistent with the delayed and lower amounts of DISC component assembly in type II cells, FADD, caspase-8 and -10 were detected at lower levels and at later time points than in type I cells. Since differences observed between type I SKW6.4 and II Jurkat cells might be due to the lower levels of CD95 expression on type II cells or limited to only lymphoid cells (Huang et al, 2000), we analyzed type I ACHN cells that express lower levels of CD95 than type II HCT15 cells (Algeciras-Schimnich et al, 2003). Rab4 was detected within CD95-containing vesicles with maximal association at 5 min in ACHN cells, indicating that receptor internalization had already begun (Figure 4C). EEA-1 appeared at 5 min and peaked at 30 min, consistent with maturation to endosomal vesicles. CatD was also detected at 5 min with further increases to 60 min indicative of movement of CD95 and its associated proteins into the lysosomal compartment. Analysis of DISC components revealed that recruitment of FADD, caspase-8 and -10 as well as caspase-8 activation peaked at 30 min, a time point at which most of the receptor had moved into an EEA-1-containing compartment. In contrast to type I ACHN cells, Rab4, EEA-1 and CatD demonstrated minimal increases following stimulation in type
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