V体育官网入口 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The VSports app下载. gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2007 Jan 10;26(1):221-31.
doi: 10.1038/sj.emboj.7601460. Epub 2006 Dec 7.

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling

Christine Feig  1 , Vladimir Tchikov, Stefan Schütze, Marcus E Peter
Affiliations

Palmitoylation of CD95 facilitates formation of SDS-stable receptor aggregates that initiate apoptosis signaling

Christine Feig et al. EMBO J. .

Abstract

Apoptosis signaling through CD95 (Fas/APO-1) involves aggregation and clustering of the receptor followed by its actin-dependent internalization. Internalization is required for efficient formation of the death-inducing signaling complex (DISC) with maximal recruitment of FADD, caspase-8/10 and c-FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS-stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS-stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase-8 activation. hiDISCs are found both inside and outside of detergent-resistant membranes. The formation of SDS-stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS-stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase-8 VSports手机版. Our data demonstrate that SDS-stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases. .

PubMed Disclaimer

Figures (V体育平台登录)

Figure 1
Figure 1
After stimulation, CD95 aggregates into SDS-stable forms, which preferentially associate with FADD and caspase-8. (A) Postnuclear lysates of unstimulated or LzCD95L (10 min)-stimulated SKW6.4 cells were subjected to immunoprecipitation with anti-caspase-8 (C15) and analyzed for association with the DISC-components CD95, c-FLIPL, FADD and caspase-10. Cleavage fragments of caspase-8 (p18), c-FLIPL (p43) and caspase-10 (p41) are shown. Note: CD95 is expressed in multiple glycosylated forms in these cells, of which only one participates in CD95hi formation. (B) SKW6.4 and H9 cells were stimulated with LzCD95L for the indicated times and caspase-8 was immunoprecipitated from the postnuclear lysates. (C) SKW6.4 and H9 cells were stimulated with LzCD95L for 30 min and FADD was immunoprecipitated from the postnuclear lysates.
Figure 2
Figure 2
CD95 forms a high molecular weight DISC (hiDISC) containing only SDS-stable CD95 aggregates. (A) Postnuclear lysates from SKW6.4 cells were separated on a continuous sucrose gradient (10–50% (w/w)). Equal volumes of each fraction were analyzed on SDS–PAGE. (B) Postnuclear lysates from SKW6.4 cells stimulated for 120 min with 1 μg/ml anti-APO-1-3 and separated on a continuous sucrose gradient. (C) Postnuclear lysates from SKW6.4 cells stimulated for 120 min with 1 μg/ml anti-APO-1-2 and separated on a continuous sucrose gradient. The stippled box indicates a CD95 species sedimenting at low buoyancy. The double lined box labels a 95 kDa species of SDS-stable CD95.
Figure 3
Figure 3
The hiDISC does not contain the internalized stimulating antibody. (A) Postnuclear lysates from SKW6.4 cells were separated on a continuous sucrose gradient (10–50% (w/w)). Equal volumes of each fraction were analyzed on SDS–PAGE. (B) Postnuclear lysates from SKW6.4 cells stimulated for 10 min with 1 μg/ml biotinylated anti-APO1-3 were separated on a continuous sucrose gradient. (C) Postnuclear lysates from SKW6.4 cells stimulated for 120 min with 1 μg/ml biotinylated anti-APO-1-3 were separated on a continuous sucrose gradient. Biotinylated heavy and light chains of the stimulating anti-APO-1 antibody were detected using streptavidin–HRP. No biotinylated IgG could be detected in high-density fractions not even after long exposure (data not shown). The stippled boxes indicate a CD95 species sedimenting at low buoyancy.
Figure 4
Figure 4
Caspase-8 only associates with CD95 in the hiDISC. (A) SKW6.4 cells were stimulated with LzCD95L for 30 min. Postnuclear lysates were subjected to three consecutive rounds of immunoprecipitation with 10 μg mouse IgG2b control antibody and separated on a continuous sucrose gradient. Equal volumes of each fraction were analyzed on SDS–PAGE. (B) SKW6.4 cells were stimulated with LzCD95L for 30 min. Postnuclear lysates were subjected to three consecutive rounds of immunoprecipitation with 10 μg anti-caspase-8 antibody and separated on a continuous sucrose gradient. The stippled box indicates a CD95 species sedimenting at low buoyancy.
Figure 5
Figure 5
After stimulation, SDS-stable CD95hi forms inside and outside of DRMs. Total cell extracts from unstimulated (A) or stimulated (20 min with LzCD95L) (B) SKW6.4 cells were loaded on a discontinuous gradient to isolate DRMs. Equal volumes of each fraction were analyzed for lipid raft markers and DISC-proteins on an SDS–PAGE.
Figure 6
Figure 6
CD95 is palmitoylated on cysteine 199. (A) Domain structure of CD95 and sequence comparison of the juxtamembrane region between human and murine CD95. CRD, cysteine-rich domain; TM, transmembrane domain. Basic amino acids that are part of the putative palmitoylation motif are shown in green. (B) Formation of CD95hi is sensitive to the palmitoylation inhibitor 2-bromo-palmitic acid (2-BrPA) and alkaline treatment. (C) Endogenous CD95 is palmitoylated. SKW6.4 cells were metabolically labeled with 0.5 mCi/ml [9,10(n)3H]palmitic acid for 3 h and CD95 was immunoprecipitated with the C20 antibody. (D) CD95 is palmitoylated on cysteine 199. Flag-tagged CD95 wt and C199S mutant were expressed in 239T, metabolically labeled with 0.25 mCi/ml [9,10(n)3H]palmitic acid for 1 h and CD95 was immunoprecipitated with an anti-Flag antibody.
Figure 7
Figure 7
Inhibition of palmitoylation impairs formation of CD95hi, CD95 internalization and activation of caspase-8. (A) Palmitoylation-deficient CD95 mutants show reduced CD95 aggregation. Flag-tagged CD95 wt, and the CD95 mutants C199A (CA), C199S (CS) and Y291F (YF) were expressed in 239T, and exogenous CD95 was immunoprecipitated with an anti-Flag antibody. (B) SKW6.4 cells were pretreated for 1 h with 100 μM 2-BrPA or DMSO as a control followed by incubation with the biotin-labeled agonistic anti-CD95mAb (anti-APO-1-3) coupled to magnetic streptavidin microbeads. Total cell lysates or magnetic fractions derived after 0, 3, 10, 30 and 60 min of anti-CD95 mAb treatment were analyzed for CD95, caspase-8 and signature proteins of endosomal maturation (Rab4) and lysosomes (CatD) by Western blotting. A small amount of CD95hi in unstimulated cells was likely formed instantly after the addition of anti-CD95 to the sample. (C) Inhibition of palmitoylation inhibits CD95 internalization. SKW6.4 cells were pretreated for 1 h with 100 μM 2-BrPA (c, d) or left untreated as a control (a, b) followed by incubation with biotin-labeled anti-APO-1-3 as indicated. CD95 was visualized by staining with streptavidin Alexa Flour 488. Nuclei were visualized by DAPI staining. (D) SKW6.4 cells were pretreated with 50 μM of 2-BrPA for 1 h before addition of 1 μg/ml FITC-conjugated anti-APO-1 on ice. The temperature was raised to 37°C for 30 min. Noninternalized receptor in all samples was visualized by adding Texas Red-labeled goat anti-mouse antibody, and CD95 internalization was quantified as described (Algeciras-Schimnich et al, 2002). Three fields with 50–100 cells were counted for each condition. The mean values and with standard deviation are shown. (E) Inhibition of palmitoylation impairs caspase-8 processing. SKW6.4 cells were pretreated for 1 h with 100 μM 2-BrPA and then stimulated with LzCD95L for the indicated times. (F) NIH3T3 cells were transiently transfected with either empty vector (vec), wild-type CD95 (wt) or CD95 C199S (CS). At 16 h after transfection, cells were treated with anti-APO-1-3 for 120 min and apoptosis was monitored using a fluorogenic caspase-3/7 substrate. The insert shows expression levels of CD95 in transfected cells.
See this image and copyright information in PMC

V体育平台登录 - References

    1. Algeciras-Schimnich A, Peter ME (2003) Actin dependent CD95 internalization is specific for Type I cells. FEBS Lett 546: 185–188 - PubMed
    1. Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME (2002) Molecular ordering of the initial signaling events of CD95. Mol Cell Biol 22: 207–220 - VSports在线直播 - PMC - PubMed
    1. Barnhart BC, Alappat EC, Peter ME (2003) The CD95 type I/type II model. Semin Immunol 15: 185–193 - PubMed (VSports)
    1. Barnhart BC, Legembre P, Pietras E, Bubici C, Franzoso G, Peter ME (2004) CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J 23: 3175–3185 - PMC - PubMed
    1. Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM, Ricci JE, Edris WA, Sutherlin DP, Green DR, Salvesen GS (2003) A unified model for apical caspase activation. Mol Cell 11: 529–541 - PubMed

Publication types

MeSH terms

VSports最新版本 - LinkOut - more resources