The cytoplasmic tail of HER-2 is cleaved by multiple caspases in vitro. Small pool expression cloning was used to identify caspase substrates from a prostate adenocarcinoma cDNA library. A, cDNA pools were transcribed and translated in vitro in the presence of [³⁵S]methionine and incubated with control (C) buffer or 25 ng of recombinant caspase-2 (C2), -3 (C3), or -8 (C8). Pool 158 contained a ∼76-kDa protein (indicated by an asterisk), which was cleaved by caspase-3 and -8. B, pool 158 was progressively subdivided and reanalyzed until a single cDNA (158-5E) encoding a ∼76-kDa protein, which was cleaved by caspase-3 into several fragments (indicated by arrows and labeled I-III), was isolated. 158-5E was identified as a partial human HER-2 cDNA encoding amino acids 762-1254. C, ³⁵S-labeled 158-5E protein was incubated with 2.5 or 25 ng of caspase-1, -2, -3, -5, -6, -7, -8, or -9 or buffer (C) for 1 h at 37 °C, and the cleavage products (labeled I-III) were identified.

HER-2 is cleaved by caspases at four sites in the cytoplasmic tail. Four putative caspase cleavage sites (Asp¹⁰¹⁶, Asp¹⁰¹⁹, Asp¹⁰⁸⁷, and Asp¹¹²⁵) were mutated to glutamic acid residues, and the mutant HER-2 proteins were examined for sensitivity to caspase proteolysis. A, MDA-MB-231 human breast cancer cells were transiently transfected with WT HER-2 or mutant HER-2 cDNAs as indicated. The next day, cells were preincubated with 100 nm epoxomicin for 1 h and then treated with 0.5 μg/ml TRAIL for 6 h. HER-2 was detected by immunoblotting using a carboxyl-terminal HER-2 mAb (cleavage products are indicated by arrows). B, schematic representation of HER-2 fragments (yellow) resulting from caspase proteolysis at each of the sites identified in A. The caspase cleavage-resistant D1016E/D1019E/D1087E/D1125E HER-2 mutant is designated 4× HER-2. The 25-kDa product (purple) is not detected by the carboxyl-terminal HER-2 Ab (red). C, ³⁵S-labeled 158-5E/WT HER-2 or 158-5E/4× HER-2 proteins were incubated with control (C) buffer or the indicated amounts (ng) of caspase-3 or -8.

The 47- and 25-kDa HER-2 products induce apoptosis by a caspase-dependent and Bcl-2-suppressible mechanism. A, schematic representation of the HER-2 products generated by caspase cleavage. B, MDA-MB-231 cells were co-transfected with cDNAs encoding FLAG-tagged HER-2 cleavage products (25, 22, or 47 kDa) or FLAG vector and pEGFP-N1 with or without a 1-h pretreatment with 100 μm Z-VAD-fmk. GFP-positive cells were scored for apoptotic nuclei 24 h later (mean ± S.E., n = 3). ^***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. C, immunoblot confirms expression of an amino-terminal FLAG-tagged 22-kDa HER-2 cDNA in transfected MDA-MB-231 cells. D, MDA-MB-231 cells were co-transfected with cDNAs encoding the 25-kDa HER-2 (left) or the 47-kDa HER-2 cleavage product (right), pEGFP-N1, and vector, dominant negative (DN) FADD, CrmA, Bcl-2, or p35 cDNA. Apoptosis was scored as in B. ^**, p < 0.01 versus vector by one-way ANOVA with Tukey's multiple comparison post-test.

Caspase-cleaved HER-2 contains a BH3-like domain and mimics the actions of Bad. A, sequence alignment of the 47- and 25-kDa HER-2 cleavage products and the BH3 domains of Bcl-2 family members. The absolutely conserved Leu and Asp residues in the BH3 domain that are required for proapoptotic activity are highlighted in yellow. B, the conserved Leu¹¹²⁰ and Asp¹¹²⁵ in human HER-2 were mutated to glutamic acid, either alone (L1120E or D1125E) or in combination (L1120E/D1125E, designated 2×E). MDA-MB-231 cells were co-transfected with cDNAs encoding the 25-kDa HER-2 product (WT or BH3 mutants) and pEGFP-N1. GFP-positive cells were scored for apoptotic nuclei 24 h later. C, apoptosis induced by the HER-2 products is Bax/Bak-dependent. WT or Bax/Bak DKO MEFs were infected with a bicistronic retrovirus encoding GFP and vector, GFP and HER-2 product (25 or 47 kDa), or GFP-tagged tBid for 48 h, and the percentage of GFP-positive cells with apoptotic nuclei was scored. D, 20-mer peptides (amino acids 1113-1132 of human HER-2) containing the WT HER-2 BH3 domain, a mutant 2×E HER-2 BH3 domain, or a Bid BH3 domain were incubated with isolated Jurkat cell mitochondria, and the released cytochrome c in the supernatant was analyzed by enzyme-linked immunosorbent assay. Readings were normalized to DMSO treatment. E, the 25-kDa HER-2 cleavage product binds to Bcl-x_L. MDA-MB-231 pools stably expressing Bcl-x_L or empty vector were immunoprecipitated (IP) with a rabbit Bcl-x_L antibody and immunoblotted with a mouse FLAG (left) or mouse Bcl-x antibody (right). F, the 25-kDa HER-2 acts synergistically with tBid to induce apoptosis, mimicking the actions of Bad. MDA-MB-231 cells stably expressing Bcl-x_L were transiently co-transfected with tBid and a bicistronic plasmid co-expressing GFP and vector, BAD, Noxa, or the 25-kDa HER-2 product. GFP-positive cells were scored for apoptotic nuclei 24 h later. G, the 25- and 47-kDa HER-2 products cooperate with Noxa to induce apoptosis. MCF-7 cells stably expressing caspase-3 were co-transfected with cDNAs encoding the 25- or 47-kDa HER-2 product, Bad (B), Noxa (N), or empty vector (alone and in combination) and pEGFP-N1. GFP-positive cells were scored for apoptotic nuclei 24 h later. H, MDA-MB-231 cells were transiently co-transfected with pEGFP-N1 and empty vector or cDNAs encoding full-length WT or mutant 2×E HER-2. Twenty-four h later, cells were treated with TRAIL for 24 h, and GFP-positive cells were scored for apoptotic nuclei. In B-D and F-H, data are the mean ± S.E. (n = 3). B and D,^*, p < 0.05; ^**, p < 0.01 for the indicated comparisons by two-tailed unpaired t test. C, ^***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. F, ^***, p < 0.001 versus tBid + vector by one-way ANOVA with Bonferroni post-test. G,^*, p < 0.05; ^**, p < 0.01 versus Noxa by one-way ANOVA with Bonferroni post-test. H, ^***, p < 0.001 versus WT HER-2 by two-way ANOVA with Bonferroni post-test.

HER-2 is proteolyzed by caspases in HER-2-overexpressing breast cancer cells during the induction of apoptosis. A, HER-2-overexpressing SKBR3 human breast cancer cells were treated with 2 μg/ml TRAIL and 1 μg/ml cycloheximide for 0-16 h with or without pretreatment with 50 μm Z-VAD-fmk for 1 h. HER-2 was detected by immunoblotting using a HER-2 mAb that recognizes the carboxyl terminus. B, SKBR3 cells were preincubated with 100 nm epoxomicin and 50 μm Z-VAD-fmk for 1 h as indicated and then treated with 2 μg/ml TRAIL and 1 μg/ml cycloheximide for 0-16 h. C, SKBR3 cells were preincubated with 100 nm epoxomicin and 50 μm Z-VAD-fmk for 1 h as indicated and then treated with 200 μm etoposide for 0-16 h. In A-C, the HER-2 cleavage products are indicated by arrows. Apoptotic nuclei were scored at each time point in parallel experiments, and the percentage of apoptotic cells is indicated.

Cleavage-resistant and truncated HER-2 confer greater protection against apoptosis than WT HER-2. A, immunoblot analysis of MDA-MB-231 pools stably expressing vector, WT, caspase-truncated (amino acids 1-1016, designated Trun), or caspase cleavage-resistant 4× HER-2. B, FACS analysis of cell surface expression of HER-2 proteins in MDA-MB-231 pools. C, MDA-MB-231 pools were treated with 0-5 μg/ml TRAIL for 24 h, and apoptotic nuclei were scored (mean ± S.E., n = 3). ^**, p < 0.01; ^***, p < 0.001 for the indicated comparisons by two-way ANOVA with Bonferroni post-test. D, immunoblot analysis of MDA-MB-231 pools treated with TRAIL (0 or 5 μg/ml) for 24 h. E, MDA-MB-231 pools were serum-starved for 24 h, treated with 100 ng/ml EGF for 10 min, and examined for phospho-Akt (serine 473) and total Akt levels by immunoblotting. F, MDA-MB-231 pools were treated with 5 μg/ml TRAIL for 24 h. Phospho-Akt (serine 473), total Akt levels, phospho-p42/p44 MAPK (Thr²⁰²/Tyr²⁰⁴), and total p42/p44 MAPK were determined by immunoblotting. A positive control lysate for phospho-Akt (serine 473; Cell Signaling) was also immunoblotted.

The 47- and 25-kDa HER-2 products localize to mitochondria and induce cytochrome c release. A, confocal images of MDA-MB-231 cells transfected with cDNAs encoding FLAG-tagged 25- or 47-kDa HER-2 cleavage products. Transfected MDA-MB-231 cells were immunostained with FLAG mAb (green). Mitochondria were labeled with Mitotracker Deep Red 633 (red), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Colocalization (yellow) is shown in the merged image. Bar, 10 μm. B, immunoblot analysis of cytochrome c levels in cytosolic and mitochondrial fractions of MDA-MB-231 cells transfected with vector or HER-2 products. C, confocal analysis of cytochrome c release. MDA-MB-231 cells stably expressing pBABE vector (left) or pBABE-Bcl-x_L (right) were co-transfected with the FLAG-tagged 25- or 47-kDa HER-2 product (or tBid) and pEGFP-N1 in the presence of Z-VAD-fmk. GFP-positive cells (green), cytochrome c immunostaining (red), and 4′,6-diamidino-2-phenylindole staining (DAPI, blue) are shown. In the MDA-MB-231/pBABE cells (left), cells with cytochrome c release (indicated by an arrow in the GFP panels) exhibit diffuse or nearly absent cytochrome c staining. In the MDA-MB-231/pBABE-Bcl-x_L (right), cells with intact mitochondrial cytochrome c (indicated by an arrow in the GFP panels) show punctate staining. D, quantitation of the cytochrome c release data shown in C (mean ± S.E., n = 5). ^***, p < 0.001 versus vector by two-way ANOVA with Bonferroni post-test. E, immunoblots of cytosolic and mitochondrial fractions from HER-2-overexpressing SKBR3 cells treated with 2.5 μg/ml TRAIL and 1 μg/ml cycloheximide for the indicated number of hours.