QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 June 2008
doi: 10.1242/jcs.021394

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lin, Y.-M. Articles by Chen, R.-H. Search for Related Content PubMed PubMed Citation Articles by Lin, Y.-M. Articles by Chen, R.-H. Social Bookmarking                         What's this?

eIF3k regulates apoptosis in epithelial cells by releasing caspase 3 from keratin-containing inclusions

¹ Institute of Biochemical Sciences, National Taiwan University, Taiwan
² Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
³ Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taiwan
⁴ Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan
⁵ Institute of Molecular and Cellular Biology and Department of Life Science, National Taiwan University, Taiwan

View larger version (29K): [in this window] [in a new window]   Fig. 1. Characterization of the eIF3k antiserum. (A) Western blot analyses on lysates from untransfected HeLa cells or HeLa cells transfected with FLAG-tagged eIF3k (eIF3k-Flag), or on GST-eIF3k purified from bacteria (GST-eIF3k). Antibodies used are eIF3k antiserum, anti-Flag and eIF3k antiserum pre-absorbed with GST-eIF3k. (B) Immunoprecipitation analysis with the eIF3k antiserum or a control antiserum. Lysates of HEK 293T cells were used for immunoprecipitations with antibodies as indicated, followed by western blot with eIF3k antiserum. (C) Expression of eIF3k in various cells as indicated. All cell lines are from human origin except for NIH3T3 (3T3) and Cos-1 (Cos).

View larger version (75K): [in this window] [in a new window]   Fig. 2. Characterization of the subcellular distribution of eIF3k. (A) eIF3k colocalizes with K8 and K18. HeLa and HaCaT epithelial cells were fixed, double stained with anti-eIF3k antiserum and antibody to K8 or K18, and examined by epifluorescence or confocal microscopy as indicated. One confocal section corresponding to each individual labeling is shown. The overlay of green and red images is shown in the `Merge' panel. (B) eIF3k does not colocalize with F-actin or microtubules. HeLa cells were fixed, double stained with eIF3k antiserum and either rhodamine-phalloidin or anti--tubulin, and examined by confocal microscopy. (C) Immunofluorescence analysis of eIF3k distribution in cells without keratin filaments. SW13 and NIH3T3 cells were immunostained with the eIF3k antiserum and then examined by epifluorescence microscopy. (D) The eIF3k antiserum does not crossreact with an epitope in K18. The eIF3k antiserum was pre-absorbed with 100 µg recombinant K18. HeLa cells were double stained by the pre-absorbed eIF3k antiserum (red) and anti-K18 antibody (green), and were examined by epifluorescence microscopy. (E) HeLa cells stably expressing eIF3k siRNA and GFP (see Fig. 5A for description) were mixed with parental HeLa cells and then examined by immunostaining with eIF3k antiserum. (F) Distribution of eIF3k in both the detergent-soluble and -insoluble compartments of cells. HeLa cells were extracted with lysis buffer containing 0.5 % Triton X-100 (see Materials and Methods). The detergent-soluble and -insoluble fractions were collected and one fifteenth of each fraction was separated by SDS-PAGE followed by western blot analyses with antibodies as indicated. CrkII and K8 were used as the reference protein for soluble and insoluble fractions, respectively. The percentages indicate the amount of eIF3k in each fraction. Scale bars: 5 µm.

View larger version (102K): [in this window] [in a new window]   Fig. 3. Distributions of eIF3k and K8 in various tissues. Sections of indicated mouse tissues were double stained with anti-eIF3k and -K8 antibody (Troma-1), and then analyzed by confocal microscopy. Boxed regions are magnified in the bottom panel. The asterisk indicates a goblet cell. The concentration of staining at the apical pole of the enterocytes is marked by arrowheads. L, lumen; LP, lamina propria. Scale bars: 25 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Interaction of eIF3k with K18. (A) Yeast two-hybrid assay. Yeast strain L40 was co-transformed with LexA- and Gal4-based vectors as indicated. The transformants were tested for β-galactosidase activity (β-gal; upper panel) and for their ability to grow in medium without histidine (middle panel) as described in the Materials and Methods. Yeast grown on the –His plate and that stained positive for β-galactosidase activity indicate the interaction between proteins encoded by the two plasmids. (B) eIF3k interacts with K18 endogenously. HeLa cells were lysed with Empigen BB lysis buffer as described in the Materials and Methods, and then subjected to immunoprecipitations with antibodies to K8 and K18 (1:1 mixture of anti-K8 and -K18 antibodies; upper panel), eIF3k (lower panel) or a control IgG (both panels). The immunoprecipitates and cell lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated. The positions of immunoglobulin light chain and eIF3k are indicated in the bottom panel. (C,D) Mapping of the domains in eIF3k (C) and K18 (D) that are responsible for their interaction. Yeast strain L40 was transformed with LexA- and Gal4-based vectors as indicated and analyzed as in A. Domain organizations of eIF3k, K18 and mutants used in this study are shown in the top panel. Numbers refer to amino acid positions.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Downregulation of eIF3k protects cells from apoptosis. (A) Generation of eIF3k siRNA stable transfectants. HeLa cells were co-transfected with plasmid expressing eIF3k-specific siRNA or control siRNA and the plasmid pEGFP/Neor (carrying both GFP and the neomycin-resistant gene) at a ratio of 4:1. Cells were selected with G418. For transfectants carrying the control vector, all G418-resistant clones were pooled. eIF3k-siRNA transfectants were first tested for the expression of eIF3k and seven clones displaying a reduced level of eIF3k were pooled. The two pools of stable transfectants were lysed and subjected to western blot with antibodies to eIF3k and tubulin. (B-D) HeLa cells stably expressing eIF3k siRNA or control siRNA as described in A were assayed for apoptosis by flow-cytometry analysis of cells with fragmented DNA (sub-G1 DNA). The percentage of cells with fragmented DNA is plotted. In B and D, cells were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (B) or 1 µM staurosporine (STS, D), respectively, for various durations. In C, cells were irradiated with UV at 0.015 J/cm2 and then cultured for various durations as indicated. (E) Clonogenic survival of UV-irradiated cells carrying eIF3k siRNA or control siRNA. HeLa cells as in A were irradiated with UV at the indicated doses. Cells were cultured for 8 days and then stained by crystal violet. Colony numbers were quantitated by ImageJ software. The percentage of clonogenic survival was determined by the ratio of colony numbers derived from the treated population to those from the untreated population. (F) Expression of the siRNA-resistant eIF3k (eIF3kR) construct reverses the apoptosis-modulating effect of eIF3k siRNA. HeLa cells transfected with constructs as indicated were assayed for apoptosis induced by UV or TNF (bottom panel), or the level of eIF3k (upper panel). For B-F, data shown are mean ± s.d. from three independent experiments. *P<0.05; **P<0.005.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Colocalization of eIF3k and K8/K18 in cytoplasmic inclusions of apoptotic cells. (A) HeLa cells were irradiated with UV at 0.015 J/cm2 and then cultured for 7 hours. Cells were fixed, triple stained with Hoechst 33258 and antibodies to eIF3k and K8 or K18, and then examined by epifluorescence or confocal microscopy as indicated. One confocal section corresponding to each individual labeling is shown. The overlay of green and red images is shown in the `Merge' panel. Apoptotic cells are marked by arrows. (B) Cells as in A were double stained with antibody to eIF3k and with M30, and were examined by confocal microscopy. (C) HeLa cells were treated with 10 ng/ml of TNF together with 2.5 ng/ml of Actinomycin D for 5 hours, or 1 µM staurosporine for 7 hours. Cells were stained as in A and examined by epifluorescence microscopy. Scale bars: 5 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 7. eIF3k regulates apoptosis in a K8/K18-dependent manner. (A) Generation of eIF3k-siRNA stable transfectants. EBNA-SW13 cells were co-transfected with a plasmid expressing eIF3k siRNA or control siRNA and a plasmid carrying the puromycin-resistant gene, and then selected with puromycin. Pools of stable transfectants were established as described in Fig. 5 and then used to analyze the expression of eIF3k by western blot. (B-D) Pools of stable transfectants as described in A were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (B), or 1 µM staurosporine (D) for various durations, as indicated. Alternatively, cells were irradiated with UV at 0.015 J/cm2 and then cultured for the indicated durations (C). (E) EBNA-SW13 cells carrying eIF3k siRNA or control siRNA as shown in A were co-transfected with pDR2-K8 and pDR2-K18. The transfectants were selected by hygromycin and then lysed for western blot analysis with antibodies as indicated. Lysate of the parental EBNA-SW13 cells was included to reveal the lack of K8/K18 expression in this cell line. (F,G) SW13-K8/K18 cells carrying eIF3k siRNA or control siRNA as shown in E were treated with 10 ng/ml TNF together with 2.5 ng/ml Actinomycin D (F) or 1 µM staurosporine (G) for various durations as indicated. Apoptotic cells were analyzed as in Fig. 5. Data shown are mean ± s.d. from three independent experiments. *P<0.05; **P<0.005.

View larger version (61K): [in this window] [in a new window]   Fig. 8. Knockdown of eIF3k promotes keratin-dependent sequestration of active caspase 3 in the insoluble compartment of cells. SW13-K8/K18 (A) or SW13 (B) cells stably expressing eIF3k siRNA or control siRNA as described in Fig. 7E and Fig. 7A, respectively, were irradiated with or without UV at 0.015 J/cm2 and then cultivated for 7 hours. Cells were extracted with lysis buffer containing 0.5% Triton X-100, and soluble and insoluble fractions were separately collected as described in the Materials and Methods. Equal volumes of the soluble and insoluble fractions were mixed as the total lysates (Total). One fifteenth of each fraction and total lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated. CrkII and K8 (or Lamin A) were used as the reference protein for soluble and insoluble fractions, respectively.

View larger version (50K): [in this window] [in a new window]   Fig. 9. Downregulation of eIF3k promotes the sequestration of active caspase 3 in K8/K18-residing cytoplasmic inclusions. (A) HeLa cells stably expressing eIF3k-specific siRNA (si eIF3k) or control siRNA (si control) as described in Fig. 5 were irradiated with UV and then cultivated for 7 hours. Cells were fixed, double stained with antibody to active caspase 3 and M30 antibody, and examined by confocal microscopy. (Top) The projection view of stacked confocal images, which were created using 19 (for control-siRNA-expressing cells) or 20 (for eIF3k-siRNA-expressing cells) overlaying 0.42-µm z-sections is shown (see Movies 1, 2 in supplementary material for 3D-reconstructed images). (Bottom) The images of a single z-section are shown. Scale bar: 5 µm. (B) Measurement of the correlation coefficient of the M30 (green) and active caspase 3 (red) images shown in A. Confocal images from one section were used for colocalization analysis with the LSM510 software. A total of 14 randomly selected cells in each group were analyzed. (C) Measurement of the percentage of M30-positive dots in a cell with active-caspase-3 signal. M30 dots with a diameter greater than 0.2 µm were counted and the projection view of 3D images from ten randomly selected cells in each group was analyzed. Data shown are mean ± s.d. (D) Interaction of active caspase 3 with K18. HeLa cells irradiated with UV at 0.015 J/cm2 were lysed for pull-down analysis with GST, GST-K18 (K18), and GST-K18 head-domain deletion mutant (H). Bound proteins and lysates of HeLa cells with or without UV irradiation were analyzed by western blot with antibody to active caspase 3. The relative amounts of various GST fusion proteins used for pull-down analysis are shown on the bottom panel. (E) Downregulation of eIF3k increases the association of active caspase 3 with K18. HeLa cells expressing eIF3k siRNA or control siRNA were irradiated with UV at 0.015 J/cm2 and then cultivated for 12 hours. Cells were lysed with Empigen lysis buffer and lysates were used for immunoprecipitation with anti-K18 antibody. The immunoprecipitates and cell lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated. The positions of the19 kDa and 17 kDa fragments of the active caspase 3 as well as the full-length protein (48 kDa) and caspase-3-cleaved K18 (26 kDa) are indicated.

View larger version (26K): [in this window] [in a new window]   Fig. 10. The effect of eIF3k on the cleavage of caspase 3 nuclear and cytosolic substrates. (A) Downregulation of eIF3k diminishes the cleavage of ICAD and PARP. SW13 or SW13-K8/K18 cells expressing eIF3k siRNA or control siRNA were irradiated with UV at 0.015 J/cm2 and then cultivated for 6 hours (for SW13 cells) or 7 hours (for SW13-K8/K18 cells). Cells were then lysed for western blot analyses with antibodies as indicated. The positions of full-length (116 kDa) and truncated (85 kDa) PARP are indicated. The bands shown in the ICAD blot represent full-length ICAD. (B) Model of eIF3k function during apoptosis. eIF3k is associated with K8/K18 filaments in non-apoptotic cells. Upon induction of apoptosis, procaspase 3 is concentrated in the K8/K18 network (Lee et al., 2002). This leads to a local activation of caspase 3, which in turn cleaves K18. After disintegration of the keratin filaments, cytoplasmic inclusion bodies are formed, which sequester K8, K18, eIF3k and several apoptotic-promoting factors (omitted). In this study, we show that eIF3k inhibits the association of active caspase 3 with K18 and therefore releases this caspase to the cytosol.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter