Identification of p20. (A) Preparative SDS-PAGE of differentially solubilized protein from KB cells 60 h after infection with adenovirus pm1760/2072. Aliquots of fractions eluted from the gel were assayed by ³²P-Bcl-2Δc21/his6/HMK Far Western, and the radioactive bands corresponding to p20 and p21 Bax were detected and quantified by phosphorimaging. The levels relative to the maximal signal detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots from the same fractions were also subjected to analytical 12% SDS-PAGE, and the gels were stained with Coomassie brilliant blue (lower panel). The positions of molecular mass marker proteins are indicated. black, p20; gray, Bax. (B) Proteins eluting from preparative SDS-PAGE between 190 and 220 ml were concentrated and resolved by reverse-phase HPLC. The upper panel shows the A₂₈₀ profile. Equal aliquots from all fractions were assayed for ³²P-Bcl-2Δc21/his6/HMK–interacting protein by Far Western, for which only p20 was detected. Amounts relative to the maximal signal detected (set at 100) were plotted as a bar graph (lower panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjected to NH₂-terminal peptide sequence analysis. (C) Polypeptide sequence of p28 Bap31/CDM (single-letter code). Peptide sequencing of p20 revealed a perfect match with amino acids 2–11 of human Bap31(underlined; these sequence data available from GenBank/EMBL/ DDBJ under accession number X81817). This was the only detectable sequence in fraction 54, was detectable together with other sequences in fraction 53, and was not detected in fractions 52 and 55. Predicted TM segments are boxed and contain charged amino acids in TM1 and TM3 (asterisks). The predicted caspase recognition sites, AAVD·G, are highlighted, and cleavage is denoted by arrows following Asp at positions 164 and 238. A potential leucine zipper located between the caspase recognition sites is denoted by bold letters, as is the KKXX ER retention signal at the COOH terminus. (D) Comparison of putative death effector domain sequences for the indicated proteins. The sequences, given in the single-letter amino acid code, were obtained from GenBank/EMBL/DDBJ, and their relative positions in the molecule are shown in parentheses. Sequences were aligned using the PILEUP program of the GCG software package and were optimized by spacing (shown as dashes). Identical residues and conserved substitutions that were recorded for at least half of the sequences analyzed are shaded in gray.

Associations of p28 with Bcl-2, Bcl-X_L, and procaspase-8 (pro-FLICE). (A) GST (lane 1) or GST fused to p28 amino acids 165–246 (lane 2), 122–164 (lane 3), 1–164 (lane 4), and 1–246 (lane 5) were expressed in bacteria, purified, and transferred to nitrocellulose in duplicate after SDS-PAGE. One blot was stained with Ponceau S and the other probed by ligand blotting (Far Western) with ³²P-Bcl-2Δc22/his6/HMK, as indicated. Constructs and results are summarized below the blots. (B) Standard recombinant DNA manipulations were used to create cDNAs encoding Bcl-X_L tagged at the COOH terminus with the Myc epitope EQKLISEEDL (Chinnayan et al., 1997); pro-FLICE tagged at the COOH terminus with the hemagglutinin (HA) epitope, YPYDVPDYA (Chinnayan et al., 1997); Bcl-2 tagged at the NH₂ terminus with the HA epitope (Nguyen et al., 1994); and p28 tagged with the Flag epitope, in which the Flag sequence MDYKDDDDKA was inserted between Pro240 and Met241 of p28. The recombinant cDNAs, or Flag DNA alone (Control-Flag), were inserted into pcDNA 3 (Invitrogen) or RcRSV (Pharmacia Fine Chemicals) (HA-Bcl-2) and transfected into 293T cells, as indicated (pluses and minuses). After incubation of cell lysates with anti-Flag antibody, immunoprecipitates (ip) and lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and the blot developed with the indicated antibody and visualized by enhanced chemiluminescence. Ig HC, immunoglobulin heavy chain. (C) Same as in B, except that Bax was included in cotransfections, as indicated.

Induction of p28 cleavage and procaspase-3 (pro-CPP32) processing during apoptosis in vivo. (A) Cell extracts were obtained from KB cells that had either been infected for 60 h with adenovirus pm1716/2072 lacking expression of E1B 19K or had been mock infected (+ or − Apoptosis, respectively). After 12% SDS-PAGE and transfer to nitrocellulose, blots were incubated with affinity-purified chicken antibody against p28 amino acids 165–246 (α p28-C) or p28 amino acids 122–164 (α p28-M) and were developed with secondary antibody conjugated either to HRP and visualized by electrochemiluminescence (Amersham Intl., Arlington Heights, IL) (α p28-M) or to alkaline phosphatase and visualized with NBT/BCIP (Boehringer Mannheim Biochemicals, Indianapolis, IN) (α p28-C), according to the manufacturer's instructions. Bands corresponding to p28 are indicated. Arrows labeled a and b denote products whose sizes are consistent with cleavage of p28 at the sites designated a and b in the schematic. (B) KB cells expressing neomycin resistance either alone (minus Bcl-2, lanes 6–10) or together with Bcl-2 (plus Bcl-2, lanes 1–5) were infected with adenovirus pm1716/2072 lacking expression of E1B 19K and cell extracts prepared at 0, 24, 36, 48, and 60 h postinfection (p.i.; lanes 1 and 6, 2 and 7, 3 and 8, 4 and 9, and 5 and 10, respectively). Aliquots (15 μg protein) were subjected to 12% SDS-PAGE, transferred to nitrocellulose, and blots were probed with antibody against p28-M or against the 17-kD subunit of CPP32 (Boulakia et al., 1996), and the products were developed as described in A. The positions of p28 and the cleavage products a and b are indicated in the upper panels. The arrow denotes a cross-reacting product whose appearance is variable (e.g., it did not appear in A). The positions of full length pro-CPP32 and the processed 17-kD subunit (p17) and putative 29-kD processing intermediate (asterisk) are indicated in the lower panels.

Appearance of a Bcl-2–interacting polypeptide during E1A-induced apoptosis. KB cells expressing neomycin resistance, either alone (Neo) or together with Bcl-2, were infected with either adenovirus dl520E1B⁻ (expressing 12S E1A and no E1B products) or pm1760/ 2072 (expressing 12S and 13S E1A but not E1B 19K). At the indicated times after infection, samples of cells were either assessed for viability by exclusion of trypan blue (graph) or prepared for ligand blot (Far Western) analysis, as described in Materials and Methods, using ³²P-Bcl-2Δc21/his6/HMK as a probe (upper panel, Bcl- 2–expressing cells; lower panel, Neo control cells). Ligand blots were visualized by phosphorimaging. The radioactive band associated with a polypeptide of M_r 20 kD is labeled p20, whereas that which comigrates with Bax is designated p21 Bax. The latter was determined using a blot cut along the vertical midline of a protein lane and developing one half by immunoblot analysis with anti–human Bax (Chen et al., 1996) and the other by ligand blotting with ³²P-Bcl-2Δc21/ his6/HMK (results not shown).

Insertion of p28 into ER microsomes. Pre–β-lactamase (lanes 1–4) and p28 (lanes 5–8) mRNA was translated in a rabbit reticulocyte lysate system in the presence of [³⁵S]methionine, in the presence (lanes 2–4 and 6–8) or absence (lanes 1 and 5) of ribosome-stripped canine pancreas microsomes (Walter and Blobel, 1983). At the end of the reaction, microsomes were recovered and analyzed by SDS-PAGE and fluorography either directly (lanes 2 and 6) or after isolation of alkali-insoluble (NaCO₃, pH 11.5) product (lanes 3 and 7; Nguyen et al., 1993), or after treatment with proteinase K (lanes 4 and 8; McBride et al., 1992). The positions of p28, pre–β-lactamase (pre-β-L), and processed β-lactamase (β-L) are indicated, as is the gel front. c, marker translation product. The schematic shown below the fluorogram depicts the deduced topology of p28 in the ER membrane (see text).

In vitro cleavage of p28 by caspase-1 (ICE) and caspase-8 (FLICE) but not by caspase-3 (CPP23). (A) ³⁵S-labeled transcription–translation product of p28 cDNA was incubated with increasing concentrations of CPP32 or ICE, and the products were resolved by SDS-PAGE (Nicholson et al., 1995). Units of enzyme added per 25 μl reaction mixture were: none (lane 1), 0.0056 (lane 2), 0.98 (lane 3), 1.95 (lane 4), 3.9 (lane 5), 7.8 (lane 6), 15.6 (lane 7), 31.2 (lane 8), 62.5 (lane 9), and 125 (lane 10). The positions of polypeptide molecular mass markers are shown. The arrows designated a and b denote cleavage products whose sizes are consistent with cleavage of p28 at the sites indicated by a and b in the schematic at the bottom of the figure. (B) Same analysis as in A, except that p28 was incubated with purified ICE or FLICE, and the resulting p20 cleavage product was quantified using a Phosphorimager. 1 U of caspase enzyme activity is equivalent to 1 pmol aminomethylcoumarin liberated from fluorogenic tetrapeptide-AMC per min at 25°C at saturating substrate concentrations (Nicholson et al., 1995).

Ectopic expression of p20 induces apoptosis. (A) CHO LR73 cells expressing neomycin resistance, either alone (− Bcl-2) or together with Bcl-2 (+ Bcl-2), were cotransfected with a luciferase reporter plasmid and Rc/RSV expressing either full length p28 or p28 amino acids 1–164 (i.e., p20). After 2 d, cells were recovered, analyzed for luciferase activity, and the enzyme activity expressed relative to the values obtained in the presence of p28 (arbitrarily set at 100). The results shown are the average of two separate experiments. (B) CHO cells were transfected with the p28 and p20 expression plasmids together with pHook (Invitrogen). 24 h later, transfected cells were recovered with Capture-Tec beads, cultured on coverslips, stained with 4′,6′-diamidino-2-phenyl indole (DAPI), and visualized under a microscope.