FAST is a survival protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression - PubMed (original) (raw)
FAST is a survival protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression
Wei Li et al. Mol Cell Biol. 2004 Dec.
Abstract
The Fas-activated serine/threonine phosphoprotein (FAST) is tethered to the outer mitochondrial membrane, where it interacts with BCL-X(L) (17). Here we show that RNA interference-mediated knockdown of endogenous FAST results in apoptosis, whereas overexpressed recombinant FAST inhibits Fas- and UV-induced apoptosis, indicating that FAST is a survival protein. The antiapoptotic effects of FAST are regulated by interactions with the translational silencer TIA-1: a FAST mutant lacking its TIA-1-binding domain does not inhibit apoptosis, and overexpressed recombinant TIA-1 inhibits the antiapoptotic effects of FAST. Because the antiapoptotic effects of FAST require ongoing protein synthesis, we hypothesized that FAST might function by preventing TIA-1-mediated silencing of mRNAs encoding inhibitors of apoptosis. Consistent with this hypothesis, FAST promotes the expression of cotransfected reporter proteins, a process that requires its TIA-1-binding domain and is inhibited by overexpressed recombinant TIA-1. More compellingly, recombinant FAST increases the expression of endogenous cIAP-1 and XIAP, but not GAPDH, in transfected HeLa cells. Because FAST is released from mitochondria in cells undergoing Fas- or UV-induced apoptosis, we propose that FAST serves as a sensor of mitochondrial stress that modulates a TIA-1-regulated posttranscriptional stress response program.
Figures
FIG. 1.
Knocking down FAST results in apoptosis. (A) COS-7 cells were transfected with pMT2-HA-FAST together with a vector-based negative control RNAi (pSuppressor2-SiNC), FAST RNAi-1 (pSuppressor2-SiFAST-1), or FAST RNAi-2 (pSupressor2-SiFAST-2). After 48 h, cells were processed for Western blotting analysis to quantify the expression of recombinant HA-FAST. (B) HeLa cells were transfected with either negative control RNAi (SiNC) or FAST RNAi (SiFAST-1). After 48 h, cells were processed for Western blotting analysis to quantify the expression of endogenous FAST. (C) HeLa cells were transfected with pcDNA3-β-galactosidase together with either negative control RNAi (SiNC) or FAST RNAi (siFAST-1 or siFAST-2) before being processed for immunofluorescence microscopy using anti-β-galactosidase, anti-active caspase 3, or Hoechst dye. Arrows point out healthy transfected cells. Arrowheads point out transfected cells that are undergoing apoptosis. Size bar, 20 μm. (D) The mean percentages (±standard errors; n = 3) of transfected cells (revealed using anti-β-galactosidase) that exhibit active caspase-3 are presented as a bar graph. Calculated P values for selected comparisons are shown. β-gal, β-galactosidase.
FIG. 1.
Knocking down FAST results in apoptosis. (A) COS-7 cells were transfected with pMT2-HA-FAST together with a vector-based negative control RNAi (pSuppressor2-SiNC), FAST RNAi-1 (pSuppressor2-SiFAST-1), or FAST RNAi-2 (pSupressor2-SiFAST-2). After 48 h, cells were processed for Western blotting analysis to quantify the expression of recombinant HA-FAST. (B) HeLa cells were transfected with either negative control RNAi (SiNC) or FAST RNAi (SiFAST-1). After 48 h, cells were processed for Western blotting analysis to quantify the expression of endogenous FAST. (C) HeLa cells were transfected with pcDNA3-β-galactosidase together with either negative control RNAi (SiNC) or FAST RNAi (siFAST-1 or siFAST-2) before being processed for immunofluorescence microscopy using anti-β-galactosidase, anti-active caspase 3, or Hoechst dye. Arrows point out healthy transfected cells. Arrowheads point out transfected cells that are undergoing apoptosis. Size bar, 20 μm. (D) The mean percentages (±standard errors; n = 3) of transfected cells (revealed using anti-β-galactosidase) that exhibit active caspase-3 are presented as a bar graph. Calculated P values for selected comparisons are shown. β-gal, β-galactosidase.
FIG. 2.
FAST inhibits Fas- and UV-induced apoptosis. (A) HeLa cells were mock transfected or transfected with vectors encoding HA-FAST or β-galactosidase, cultured in the absence (untreated) or presence of anti-Fas antibody (FAS) before being processed for immunofluorescence microscopy using anti-HA, anti-β-galactosidase, anti-active caspase 3, and Hoechst dye. Arrows point out cells undergoing Fas-induced caspase-dependent apoptosis. Arrowheads point out FAST transfectants in which caspase 3 is not activated. Size bar, 20 μm. (B) HeLa cells were mock transfected or transfected with either FAST or β-galactosidase (β-gal), cultured in the presence of anti-Fas antibody, and then processed for TUNEL analysis (red) and immunofluorescence (anti-HA, green; anti-active caspase 3, blue) or Hoechst staining. Arrows point out untransfected cells or β-galactosidase-transfected cells. Arrowheads point out FAST transfectants. (C) The mean percentages (±standard errors; n = 3) of transfected cells (vector control, HA-FAST, HA-FASTN, or HA-FASTC; revealed using anti-HA or anti-β-galactosidase) cultured under the indicated conditions that exhibit active caspase 3 are presented as a bar graph. Calculated P values for selected comparisons are shown.
FIG. 2.
FAST inhibits Fas- and UV-induced apoptosis. (A) HeLa cells were mock transfected or transfected with vectors encoding HA-FAST or β-galactosidase, cultured in the absence (untreated) or presence of anti-Fas antibody (FAS) before being processed for immunofluorescence microscopy using anti-HA, anti-β-galactosidase, anti-active caspase 3, and Hoechst dye. Arrows point out cells undergoing Fas-induced caspase-dependent apoptosis. Arrowheads point out FAST transfectants in which caspase 3 is not activated. Size bar, 20 μm. (B) HeLa cells were mock transfected or transfected with either FAST or β-galactosidase (β-gal), cultured in the presence of anti-Fas antibody, and then processed for TUNEL analysis (red) and immunofluorescence (anti-HA, green; anti-active caspase 3, blue) or Hoechst staining. Arrows point out untransfected cells or β-galactosidase-transfected cells. Arrowheads point out FAST transfectants. (C) The mean percentages (±standard errors; n = 3) of transfected cells (vector control, HA-FAST, HA-FASTN, or HA-FASTC; revealed using anti-HA or anti-β-galactosidase) cultured under the indicated conditions that exhibit active caspase 3 are presented as a bar graph. Calculated P values for selected comparisons are shown.
FIG. 3.
(A) Stress stimuli displace endogenous FAST from mitochondria. HeLa cells were left untreated or were treated with UV irradiation (10 mJ/cm2 followed by 0.5 or 1 h recovery) or anti-Fas antibody (1:200 dilution from culture supernatants) for 6 h before being processed to obtain pellets enriched in mitochondria (P20) and corresponding supernatants (S20). Individual fractions were processed for Western blotting analysis with anti-FASTN antibody or anti-BCL-XL antibody. (B) Fas ligation interrupts FAST/BCL-XL interaction. HeLa cell extracts prepared from cells cultured in the absence or presence of anti-Fas antibody were processed for coimmunoprecipitation with either rabbit anti-BCL-XL antibody or an isotype-matched control antibody (rabbit anti-Myc). Immunoprecipitates were then processed for Western blotting analysis with anti-FAST-N antibody or mouse anti-BCL-XL antibody.
FIG. 4.
Identification of FAST/TIA1 interaction sites. (A) Schematic depiction of HA-FASTN and HA-FASTC truncation mutants. MTD, mitochondrial tethering domain; BH3, BCL2 homology 3-related domain. (B) Schematic depiction of HA-TIA-1 truncation mutants. 1, 2, 3, RNA-recognition motifs; PRD, prion-related domain. (C) COS-7 cells were cotransfected with pcDNA3-FLAG-TIA1 and the indicated HA-FAST truncation mutants. After 28 h, cells were processed for coimmunoprecipitation analysis with mouse anti-HA antibody followed by Western blotting analysis with mouse anti-FLAG antibody, followed by mouse anti-HA antibody. Lysates from transfected cells were analyzed by Western blotting with anti-FLAG antibody (load). (D) COS-7 cells were cotransfected with pcDNA3-FLAG-FAST and the indicated HA-TIA1 truncation mutants. Lysates from transfected cells were immunoprecipitated with anti-HA antibody and then were sequentially blotted with anti-FLAG antibody and anti-HA antibody. Lysates from transfected cells were analyzed by Western blotting with anti-FLAG antibody (load). FL, FLAG.
FIG. 5.
TIA-1 inhibits the antiapoptotic effects of FAST. HeLa cells were transfected with the indicated constructs, cultured in the absence (untreated) or presence (Fas) of anti-Fas antibody, and then processed for immunofluorescence microscopy using anti-HA (or anti-β-galactosidase), anti-active caspase 3, or Hoechst dye. The mean percentages (±standard errors; n = 3) of transfected cells (revealed using anti-HA or anti-β-galactosidase) cultured under the indicated conditions that exhibit active caspase 3 are presented as bar graphs. Calculated P values for selected comparisons are shown.
FIG. 6.
FAST enhances the expression of cotransfected β-galactosidase. The left panel shows COS-7 cells cotransfected with a β-galactosidase reporter together with pMT2 alone, pMT2-HA-FAST, pMT2-HA-TIA-1, or pMT2-HA-TIA-1-PRD. After 48 h, cells were processed for Western blotting analysis to quantify the expression of β-galactosidase (β-gal). The right panel shows COS-7 cells cotransfected with a β-galactosidase reporter together with pMT2-HA-FAST and increasing amounts of pMT2-HA-TIA-1 (5:1, 2:1, 1:1). After 48 h, cells were processed for Western blotting analysis to quantify the expression of β-galactosidase and the expression of recombinant TIA-1 and FAST.
FIG. 7.
FASTC increases reporter gene expression and is regulated by BCL-XL. (A) COS-7 cells were cotransfected with a β-galactosidase reporter together with pcDNA3 vector and pMT2 alone, pMT2-HA-FAST, pMT2-HA-FASTN, pMT2-HA-FASTC, or pMT2-HA-FASTCΔBH3. After 48 h, cells were processed for Western blotting analysis to quantify the expression of β-galactosidase. (B) COS-7 cells were cotransfected with a β-galactosidase reporter together with pcDNA3-BCL-XL and pMT2 alone, pMT2-HA-FAST, pMT2-HA-FASTN, or pMT2-HA-FASTC.
FIG. 8.
Effects of siFAST-1 on β-galactosidase expression. COS-7 cells were cotransfected with a β-galactosidase (β-gal) reporter together with pMT2-HA-FAST, pMT2-HA-FASTN, or pMT2-HA-FASTC and either pSupressor2-siNC (lanes 1 to 3) or pSupressor-2-siFAST-1 (lanes 4 to 6). After 48 h, cells were processed for Western blotting analysis to quantify the expression of β-galactosidase (A) or individual HA-FAST constructs (B). Molecular size markers are shown at the left. Bands corresponding to β-galactosidase, HA-FAST, and HA-FASTC are indicated by arrows.
FIG. 9.
Fas ligation modulates the expression of β-galactosidase. (A) HeLa cells were cotransfected with a β-galactosidase reporter together with pMT2 vector, pMT2-HA-FAST, or pMT2-HA-FASTC, and then they were cultured in the absence or presence of anti-Fas antibody before being processed for Western blotting analysis to quantify the expression of β-galactosidase (β-gal) (upper panel), HA-FAST, and HA-FASTC (lower panel). (B) HeLa cells were transfected with negative control siRNA or siFAST-1 and cultured for 28 or 36 h in the presence of z-VAD (100 nM; Enzyme Systems Products), and then they were treated with anti-Fas antibody for 6 h or overnight, as indicated, before being processed for immunoblotting analysis to quantify the expression of β-galactosidase and endogenous GAPDH.
FIG. 10.
The antiapoptotic effects of FAST require active NF-κB and ongoing protein synthesis. HeLa cells were transfected with the indicated constructs and then were cultured in the absence (untreated) or presence of anti-Fas antibody (Fas), with or without cycloheximide (CHX; 0.025 μg/ml), before being processed for immunofluorescence microscopy using anti-HA (or anti-β-galactosidase), anti-caspase 3, or Hoechst dye. The mean percentages (±standard errors; n = 3) of transfected cells (revealed using anti-HA or anti-β-galactosidase) that exhibit active caspase-3 are presented as bar graphs. Calculated P values for selected comparisons are shown.
FIG. 11.
FAST promotes the expression of cIAP-1 and XIAP in HeLa transfectants. (A) HeLa cells were transfected with cDNAs encoding a vector control or recombinant FAST. After 48 h, cells were processed for Western blotting to quantify the expression of endogenous cIAP-1, XIAP, and GAPDH. These blots are representative of three independent experiments. (B) The expression of cIAP-1 and XIAP was quantified by densitometry. Protein expression in vector transfectants was assigned a value of 1. The relative increase in expression observed in FAST transfectants is indicated as means ± standard errors (n = 3). P values were calculated using the Student's t test.
FIG. 12.
Proposed mechanism by which FAST inhibits Fas-induced apoptosis.
References
- Anderson, P., and N. Kedersha. 2002. Stressful initiations. J. Cell Sci. 115:3227-3234. - PubMed
- Barber, G. N. 2001. Host defense, viruses and apoptosis. Cell Death Differ. 8:113-126. - PubMed
- Barnhart, B., E. Alappat, and M. E. Peter. 2003. The CD95 Thpe I/type II model. Semin. Immunol. 15:185-193. - PubMed
- Cheng, E. H., M. C. Wei, S. Weiler, R. A. Flavell, T. W. Mak, T. Lindsten, and S. J. Korsmeyer. 2001. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell 8:705-711. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- AR 051472/AR/NIAMS NIH HHS/United States
- R56 AI033600/AI/NIAID NIH HHS/United States
- R01 AI033600/AI/NIAID NIH HHS/United States
- AI 50167/AI/NIAID NIH HHS/United States
- R01 AI050167/AI/NIAID NIH HHS/United States
- R01 AR051472/AR/NIAMS NIH HHS/United States
- AI 33600/AI/NIAID NIH HHS/United States
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Research Materials
Miscellaneous