A novel DAP5/p86 form appears in Fas- and p53-induced apoptosis. (A) Exponentially growing SKW B-lymphoma cells (3 × 10⁶, total cell number) were treated with agonistic anti-Fas antibodies for 0, 2, 5, and 8 h. Treatment was terminated by harvesting all cells and immediately boiling the pellets in Laemmli sample buffer. Immunoblots were reacted with anti-DAP5 polyclonal antibodies (top) and anti-PARP antibodies (bottom). Apoptotic cell death was assessed by detection of changes in the membrane composition by the annexin V fluorescence-activated cell sorting analysis. (B) HFB cells were exposed to anti-Fas agonistic antibodies, for the indicated time periods, in the presence or absence of CHX. The fate of DAP5 was followed by reacting the immunoblots with anti-DAP5 polyclonal antibodies. (C) LTR6/M1 cells, expressing a temperature-sensitive p53 mutant, were induced to undergo apoptosis by a temperature shift to 32°C. Apoptotic cell death was assessed by detection of changes in the membrane composition by annexin V fluorescence-activated cell sorting analysis. The fate of DAP5 was assessed by Western blotting. The sizes of protein markers (in kilodaltons) are shown on the right. The positions of DAP5/p97 and DAP5/p86 are marked by arrows (dashed arrows point to the minor rapidly migrating p97 and p86 bands). The asterisk marks a nonspecific band lightened by the anti-DAP5 antibodies.

DAP5/p97 is converted to DAP5/p86 by caspase cleavage. (A) SKW B-lymphoma cells were treated with anti-Fas agonistic antibodies for 5 h. Samples were fractionated on a 10% or 15% polyacrylamide gel, and DAP5 was assessed by reacting the Western blot (WB) with anti-DAP5 polyclonal or monoclonal antibodies (αDAP5 Abs), as indicated (see Fig. 8 for antibody epitopes). A nonspecific band is marked by an asterisk. (B) SKW B-lymphoma cells were treated with agonistic anti-Fas antibodies for 5 h after a 1-h preincubation period with caspase inhibitors (BD, DEVD, and YVAD), proteosome inhibitor (MG132), and calpain I inhibitor (ALLN). As a control, cells were preincubated with dimethyl sulfate solvent alone. Immunoblots were reacted with anti-DAP5 polyclonal antibodies (top) and anti-PARP antibodies (bottom). (C) HFB cells were transiently transfected with the following N-terminal Flag-tagged DAP5 constructs: wild-type DAP5 (DAP5/p97 [lanes 3 and 4]) and two constructs each carrying a potential disrupted caspase cleavage site (DAP5/p97 DETA⁷⁹⁰ [lanes 5 and 6]; DAP5/p97 DHVA⁸²⁴ [lanes 7 and 8]). An empty pECE vector served as a control (lanes 1 and 2); 24 h posttransfection the cells were exposed to anti-Fas agonistic antibodies for 12 h or to fresh medium alone. The exogenic DAP5 forms were pulled down by immunoprecipitation with anti-Flag antibodies followed by Western blotting with anti-DAP5 polyclonal antibodies.

Coimmunoprecipitation of eIF4A and eIF3 with the two DAP5 forms. (A) Growing SKW cells or SKW cells exposed for 2 or 5 h to anti-Fas agonistic antibodies were gently extracted in B buffer. Protein extract (1 mg) was subjected to immunoprecipitation with anti-DAP5 polyclonal antibodies. Coimmunoprecipitation of endogenous eIF4A was assessed by Western blotting the immunoprecipitates with anti-eIF4A antibodies (middle panel). Samples of 100 μg of total cell extracts were assessed for DAP5 and eIF4A levels by direct Western blotting (top and bottom panels, respectively). (B) 293 cells were transiently transfected with Flag-tagged DAP5 constructs in a pECE vector. The constructs included wild-type DAP5 (DAP5/p97), a mutant of DAP5 carrying a stop codon at position 790 (DAP5/p86), and an empty vector (pECE). At 48 h posttransfection the cells were extracted gently in B buffer. The ectopically expressed DAP5 was immunoprecipitated with anti-Flag antibodies and assessed with anti-DAP5 antibodies after resolution of the immunoprecipitates on gels (top); coimmunoprecipitation of endogenous eIF3 was assessed by Western blotting the immunoprecipitates with antibodies against the eIF3/p116 subunit (middle); total endogenous eIF3/p116 was measured by direct Western blotting (bottom).

Disappearance of intact eIF4GI during cell death. (A) SKW B-lymphoma cells were treated with anti-Fas agonistic antibodies for 0, 2, and 5 h. The fate of eIF4GI was assessed by Western blotting (WB) with polyclonal antibodies generated against eIF4GI N terminus (left panel) or C terminus (two right panels). The position of full-length eIF4GI is indicated by an arrow. Bands exclusively recognized by either anti-eIF4G antibody in the Fas-treated cells are marked by their approximated molecular sizes (in kilodaltons) (p110, p76, p50, and p40). The asterisks mark nonspecific bands lightened by the anti-eIF4GI antibodies. (B) SKW B-lymphoma cells, either nontreated or pretreated for 2 h with CHX or with anti-Fas agonistic antibodies, were incubated in methionine-free medium for 1 h, labeled with [³⁵S]Met for 1.5 h, and harvested thereafter (the treatment with CHX or with anti-Fas continued during the methionine starvation and the radioactive pulse). The level of insoluble radioactivity incorporated per microgram of protein extract was set as 100% in control cells. The results represent the average of four independent experiments.

Preferential translation of DAP5 during cell death. SKW cells were treated for 3 h with anti-Fas agonistic antibodies (Abs) or were left untreated. During the last hour, cells were transferred to methionine-depleted medium supplemented with their corresponding treatments; they were then pulse-labeled with 80 μCi [³⁵S]Met per ml for 1.5 h and harvested. Samples of cytoplasmic extracts containing 1.5 × 10⁶ cpm were subjected to immunoprecipitation with anti-DAP5 polyclonal antibodies (top) and with anti-β-tubulin antibodies (bottom). The first lane in each panel represents nonspecific background bound to the Sepharose beads. The asterisks mark protein bands that reacted with the beads or the antibodies nonspecifically. Values from the densitometric analysis of the DAP5 and β-tubulin bands are shown at the right. The translation rate of each protein in the growing cells was set as 100%, and the translation rate of each protein following Fas treatment was calculated accordingly. Similar results were obtained in three additional independent experiments.

DAP5's 5′UTR possesses an IRES which directs cap-independent translation and is selectively sustained during cell death. (A) 5′UTRs of DAP5 and of BiP were inserted between the two cistrons of a basic bicistronic LS vector, in which the LUC reporter gene is translated in a cap-dependent manner from the first cistron and SeAP is translated in a cap-independent manner from the second cistron, to generate LS-DAP5 and LS-BiP respectively. 293 or HFB cells were transfected with a vector lacking an insert in the intercistronic region (LS), LS-DAP5, or LS-BiP and further assessed 48 h posttransfection. For each experiment, the SeAP/LUC ratio obtained by the LS vector was designated 1, and the relative fold increase in SeAP/LUC ratio in the other vectors was calculated. The results represent the average of three independent experiments. In 293 cells, the average values (in arbitrary units) of the reporter activities for LS, LS-BiP, and LS-DAP5 vectors, respectively, were 995, 784, and 1,167 for LUC and 14, 56, and 189 for SeAP. In HFB cells, the average values (in arbitrary units) of the reporter activities for LS and LS-DAP5 vectors, respectively, were (98 and 85) for LUC and (27 and 286) for SeAP. The inserts correspond to Northern blots of nontransfected (left lane of each) and LS-DAP5 transfected (right lane of each) 293 or HFB cells, respectively, probed by the SeAP cDNA. (B) HFB cells were transfected with LS-DAP5 vector. After 36 h, the medium was replaced by medium containing or lacking anti-Fas agonistic antibodies; 12 h later, the enzymatic activity of each reporter enzyme was determined. For each experiment, the SeAP or LUC value obtained for the control cells was designated 1 and served for normalization of the corresponding reporter activity under Fas-stimulated conditions. These results represent the average of seven independent experiments. The raw data from control and Fas-treated cells, respectively, were 197 and 63, 124 and 24, 767 and 246, 192 and 70, 133 and 35, 606 and 253, and 1290 and 229 for LUC and 138 and 135, 111 and 85, 178 and 148, 147 and 119, 296 and 227, 112 and 105, and 383 and 281 for SeAP. (C) DAP5's 5′UTR was inserted into a bicistronic vector in which CAT is translated from the first cistron and LUC is translated from the second cistron, generating CL-DAP5. Insertion of DAP5's 5′UTR into a bicistronic vector in which CAT, the first cistron, is preceded by a stable hairpin generated hpCL-DAP5. These constructs were transcribed and translated in vitro in the presence of [³⁵S]methionine. The intensity of each band was determined by phosphorimager analysis.

Translation via the DAP5 IRES in vitro is mediated by DAP5 protein. Capped transcripts of the bicistronic vectors LS-DAP5 or LS-EMCV were translated in vitro in the presence of [³⁵S]methionine. The translation reactions were supplemented with bacterially produced GST-260, using GST supplement as a control (A), or with Flag-DAP5/p97 or Flag-DAP5/p86 immunoprecipitated from transiently transfected 293 cells, using immunoprecipitates from nontransfected 293 cells a control (B and C). Intensities of the LUC and SeAP bands were quantified with a phosphorimager. The SeAP/LUC ratio in the control reaction was set as 1, and that of the DAP5-supplemented reactions was calculated accordingly. (A) LS-DAP5 transcripts were translated as described above, supplemented by equivalent amounts of GST-260 or GST proteins. A typical autoradiogram of the resulting translation products is presented (right). Corresponding amounts of RRL and GST-260, as present in the translation reactions, were separated on a 12% gel and immunoblotted with anti-DAP5 polyclonal antibodies (left). (B) Capped LS-DAP5 transcripts were translated as described above, supplemented with anti-Flag immunoprecipitates from transiently transfected 293 cells overexpressing DAP5 protein forms. The resulting autoradiogram of the translation products is presented at the right. The same translation reactions were separated on 10% gel and immunoblotted with anti-DAP5 polyclonal antibodies (left). The solid and dashed arrows on the left indicate endogenous DAP5 within the RRL; the dashed arrow indicates the minor fast-migrating DAP5 form, present both in cells (Fig. 1) and in RRL. Exogenous DAP5 forms from the immunoprecipitations are marked by the arrows to the right. (C) An experiment similar to that in panel B was performed on both LS-DAP5 and LS-EMCV transcripts in parallel, supplemented by equivalent amounts of exogenous DAP5 proteins (compare within bar pairs) as follows: bar 1, control; bar 2, DAP5/p86; bars 3 to 5, increasing amounts of DAP5/p97. The quantity of supplemented exogenous DAP5 proteins was determined by densitometry of immunoblots of the translation reactions, reacted with anti-DAP5 antibodies. The level of endogenous DAP5 in the RRL was scored as 1, and the calculated relative levels of exogenous DAP5 are presented below each bar pair. The raw band intensity data of the LS-DAP5 translation reactions presented in the graph are detailed in the form of bar no. (luciferase, SeAP, SeAP/LUC ratio): 1 (894, 417, 0.47), 2 (1207, 948, 0.79), 3 (1222, 617, 0.50), 4 (1131, 812, 0.72), and 5 (937, 661, 0.71).

Amino acid homology alignment of eIF4GI and DAP5. eIF4GI and DAP5 are divided into regions based on the high homology of the central region of eIF4GI with the N-terminal region of DAP5. The shaded boxes indicate binding sites of translation initiation factors: 4E, eIF4E, the cap binding protein; 4A, eIF4A, an ATP-dependent RNA helicase; and 3, eIF3, a ribosome adapter. The site of cleavage of eIF4GI by 2A protease is indicated by a dashed line at position 490. The identified caspase cleavage site of DAP5 is indicated by a dashed line at position 790. DAP5 protein fragments corresponding to amino acids 488 to 742 and 672 to 830 were used for the production of polyclonal and monoclonal antibodies, respectively, against DAP5. Regional homologies at the amino acid level of the conserved N-terminal region and of the less homologous DAP5 miniprotein region between DAP5 and eIF4GI are indicated. Alignment was performed as previously published (21), and homology was determined by using the Dayhoff PAM250 residue weight table. ID, identity; SIM, similarity.