By
From the * Manitoba Institute of Cell Biology and the § Department of Human Genetics, University of
Manitoba, Winnipeg, Manitoba, Canada; and the Department of Biochemistry, University of
Alberta, Edmonton, Alberta, Canada
Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3163) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3163 mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3163 protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.
The genetic regulation of cell death is thought to be a
central mechanism of cellular homeostasis and development (1). The Bcl-2 family of genes (1, 5), which are
related to ced-9 of Caenorhabditis elegans (6), were originally
identified as repressors of cell death. It is known that both
pro- and anti- apoptotic Bcl-2 homologues exist, however
their exact biochemical function has not been determined.
Recent studies suggests that Ced-9 and Bcl-2/Bcl-XL may
physically interact with proteins that are required for the
execution of apoptosis, Ced-3 and Ced-4 (7). Ced-3 is a
protease which in mammals is represented by a large family
of cysteine proteases which cleave after aspartic acid, now
called caspases (4, 10). In mammalian cells overexpression of bcl-2 prevents the processing and activation of caspase-3
(CPP32) (11, 12).
Bcl-2 family members bear COOH-terminal transmembrane domains that allow their association with the outer
mitochondrial membrane (13) and this mitochondrial localization is important for the suppressive function of Bcl-2
(14, 15). There is growing evidence that mitochondrial function is disturbed early in the apoptotic response and may be
important in mediating apoptosis (16). This is often seen
as the loss of mitochondrial membrane potential (16, 17)
and the release of cytochrome c (18), and cytochrome c has
been implicated in the activation of caspases (18). Bcl-2 can suppress the release of cytochrome c from mitochondria and prevent caspase activation (19, 20).
The adenovirus E1B 19K protein is functionally similar
to Bcl-2 as a survival factor (1). A two hybrid screen of
proteins that interact with E1B 19K in the yeast Saccharomyces cerevisiae identified several unique cDNAs named Nip1,
Nip2, and Nip3 (21). All three proteins interact with discrete conserved domains of E1B 19K protein and Bcl-2 that
are involved in suppression of cell death, although a function was not identified. We report here that Nip3 is a pro-apoptotic mitochondrial protein that both induces apoptosis and increases the sensitivity of cells to apoptosis induced
by drugs and granzyme B. Nip3 can overcome Bcl-2 suppression and may be related to pro-apoptotic members of
the Bcl-2 family.
Cell Cultures and Reagents.
Rat-1, Rat-1/Bcl-2, and MCF-7
cells used in this study were cultured in Cloning and Construction of Expression Plasmids.
After isolation
of a Nip3 cDNA, we utilized RACE to identify both 3
-minimal essential medium (
-MEM)1; (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (GIBCO). Murine monoclonal anti-HA 12CA5 and anti-T7 antibody were purchased
from Boehringer Mannheim (Laval, Quebec) and Novagen (Madison, WI), respectively. Rabbit anti-HSP60 antibody was donated by
Dr. Radhey Gupta (McMaster University, Hamilton, Ontario). FITC-conjugated goat anti-rabbit IgG (FITC; Sigma Chem. Co.,
St. Louis, MO) and Cy3-conjugated goat anti-mouse IgG (BioCan Ltd., Mississauga, Ontario) were used for fluorescence studies.
and 5
ends using a premade human brain marathon-ready cDNA according to the manufacturer's instructions (Clontech, Palo Alto,
CA). The Nip3 3
RACE was done using primer 5
CAGCGTTCCAGCCTCGGTTTCT3
while the 5
RACE was
done using three different primers 5
CAAAGGTGCTGGTGGAGGT3
, 5
TGTGGTGTCTGCGAGCGAGGT3
, and 5
TCCAGCAGTATTTTTTCCA3
. The appropriate RACE products
were TA cloned (Invitrogen, San Diego, CA) and several of the
resulting clones were analysed to obtain the described Nip3 sequence.
and 3
end of the Nip3
cDNA encoding only the open reading frame by PCR amplification and its sequence was confirmed by the T7 sequencing Kit
(Pharmacia LKB Biotechnology, Inc., Piscataway, NJ), then inserted in frame into the KpnI-BamHI sites of plasmid pKM1310
containing the 5
HA-tag. The recombinant cDNA 5
HA-Nip3
was then isolated by EcoRI digestion and blunt end ligated into
the plasmid pKG86 creating the plasmid pKG5HA-Nip3 under
the control of SP6 promoter which was used for in vitro transcription/translation of 5
HA-tagged Nip3 proteins. The plasmid
pKG5HA-Nip3163 was generated by PCR and constructed as described above.
In Vitro Translation. 35S-labeled Nip3 protein and mutants were prepared by in vitro transcription and translation of cDNA cloned in pcD-3HA vector using TnT coupled Reticulocyte Lysate System (Promega Corp., Madison, WI) according to manufacturer's instructions.
Two-dimensional Peptide Mapping. [35S]methionine-labeled in vitro translated full-length Nip3 and Nip3163 proteins were immunoprecipitated using mouse monoclonal anti-Nip3 antibody. The proteins were then recovered from unfixed dried SDS-polyacrylamide gels by homogenizing the gel slices, then precipitated with trichloroacetic acid and oxidized with performic acid as described previously (22). The oxidized proteins were then resuspended in 25 ml of 50 mM NH4HCO3 and digested overnight at 37°C with 10 mg TPCK-treated trypsin. An additional 5 mg of trypsin was then added, and digestion continued for at least another 4 h. After digestion, samples were repeatedly lyophilized (four times) using a Speedvac concentrator. Tryptic digests of the methionine-labeled Nip3 proteins were subjected to electrophoresis using pH 1.9 buffer (15% acetic acid, 5% formic acid) followed by ascending chromatography with Scheidtmann buffer (isobutyric acid/pyridine/acetic acid/butanol/water (65:5:3:2:29) as described previously (23). [35S]methionine-labeled peptides were visualized by autoradiography.
Transient Transfection and Apoptosis Assays. Rat-1 cells, Rat-1/Bcl-2 or MCF-7 cells were transfected with Nip3 and mutants in the expression plasmid pcDNA3 using LipofectAMINE Reagent (GIBCO BRL). Cells (3-8 × 105) were plated onto 100 × 20 mm tissue culture dishes (Nunc, Roskilde, Denmark) in 12 ml of complete medium 24 h before transfection. The DNA-LipofectAMINE mixture was allowed to incubate in a tissue culture incubator for 3 h for Rat-1 or 5 h for MCF-7 cells.
Aliquots of 5 × 105 cells were Western blotted as described previously using mouse monoclonal anti-HA antibody 12CA5 or anti-T7 antibody using an ECL detection kit according to manufacturers instructions (Amersham, Amersham, UK). For some experiments, cells were treated with granzyme B and perforin as previously described (24), etoposide or camptothecin, then stained with anti-T7 antibody with FITC-conjugated rabbit anti-mouse antibody and apoptotic cells were enumerated by altered DNA morphology with Hoechst dye staining, counting between 200 and 300 cells per sample (24). For subcellular localization, cells were grown on cover slips, transfected with HA-Nip3 then fixed with 4% formaldehyde in PBS. Cells were double stained with mouse monoclonal anti-HA antibody 12CA5 and anti-HSP60 antibody and visualized with FITC-conjugated goat anti-mouse IgG secondary antibody and Cy3-conjugated goat anti-rabbit antibody. Fluorescence was visualized using a Zeiss Axiophot microscope equipped with a cooled CCD camera driven by IPLabs Spectrum H-SU2 (version 3.0; Signal Analytics Corp., Vienna, VA) and Multiprobe 1.1 E (Signal Analytics) software.RNA Isolation and Northern Hybridization Analysis.
Total RNA
from mice tissues was kindly provided by Dr. Robert Shiu (University of Manitoba). For Northern blot analysis 30 µg of total
RNA was hybridized with nick-translated Nip3 probe (1-5 × 108 cpm µg DNA) at 42°C for 16-20 h. After hybridization and
washing blots were exposed to Kodak XAR film at 70°C with
an intensifying screen.
We isolated several Nip3
cDNAs from a human EBV transformed peripheral B lymphocyte library (Clonetech) by PCR and RACE of both 5
and 3
ends, and identified the longest as a 1,535-bp sequence (see Genebank accession number AF002697 for
complete sequence). This is similar to the cDNA identified
earlier (21) except in the 3
UTR where we find no evidence of a homologous calbindin region in several independent experiments. The RNA has a long 3
UTR and
shorter 5
UTR, and the encoded protein contains 194 amino acids and has a predicted molecular mass of ~21.54
kD with a pI of 6.08. There is a putative transmembrane domain between amino acids 164 to 184 (Fig. 1 A). Nip3
mRNA is expressed in human breast carcinoma MCF-7 as
a major transcript of ~1.7 kb and two minor transcripts of
1.5 and 1.3 kb. The level of Nip3 mRNA in mouse tissues
was also examined to determine how widely the gene is expressed. Two transcripts of 2.5 and 1.7-kb were identified with the 1.7-kb transcript found in all tissues examined and
the larger transcript only in certain tissues such as brain,
heart, kidney, liver, and submaxillary gland (Fig. 1 B). The
identification of more than one Nip3 transcript in human
and murine cells may indicate that the gene is alternately
spliced or another closely related gene exists.
Nip3 Protein Is Expressed as a Dimer and Monomer.
We prepared [35S]methionine-labeled Nip3 by in vitro transcription and translation and examined the protein on SDS-PAGE. Nip3 was seen as a major band at ~60 kD and a smaller band of 30 kD (Fig. 2 A). Nip3 has a predicted molecular mass of ~21.54 kD, much smaller than the size of the two bands estimated from SDS-PAGE, indicating that the protein runs anomalously. The two bands were seen under both reducing and nonreducing conditions. However, when the COOH terminus from amino acid 163 was removed (Nip3163) (Fig. 1 A), the large molecular mass form was absent and a major band of 28 kD and a minor band of 27 kD were detected. These data suggest that the 30-kD protein is a Nip3 monomer while the 60-kD protein is a dimer that was formed by interaction at the COOH terminus of the protein. To examine more directly if Nip3 is a homodimer, we compared tryptic peptide fragments of the 60-kD Nip3 to the 28-kD Nip3163. After in vitro translation, [35S]methionine-labeled Nip3 and Nip3163 were purified by immunoprecipitation with mouse mAb to Nip3 and SDS PAGE. Tryptic peptides were prepared and fragments separated by 2D electrophoresis and chromatography and detected by radioautography as described in Materials and Methods. Three peptide fragments of Nip3 and Nip3163 contain methionine, while the COOH-terminal fragments including the truncated region of Nip3163 do not. Fig. 2 B illustrates that the two proteins have three major [35S]methionine-labeled peptides and their positions are very similar after 2D mapping. No other major peptides are detected in the 60-kD Nip3 band. Minor proteins are also similar and likely represent partially digested Nip3.
The ability of Nip3 to homodimerize was next examined in the yeast two hybrid system by inserting the Nip3 cDNA in the GAL4 binding and activating domains which were then simultaneously expressed in yeast cells (KGY37). This was compared to Nip3 interaction with non specific protein controls. A strongly positive interaction was found with Nip3 in both vectors indicating that the protein was capable of binding to itself (Table 1). In contrast, the Nip3163 was unable to interact with Nip3 or with itself (Fig. 2 C) indicating that the COOH terminus was required for homodimerization.
|
Boyd et al. (21) reported that HA-Nip3 localizes in discrete cytoplasmic patches which are typical of a mitochondrial distribution. To confirm the mitochondrial localization of Nip3 and to test the hypothesis that it depends on the putative transmembrane domain, we transfected Rat-1 cells with HA-Nip3 or HA-Nip3163 then stained with FITC-conjugated anti-HA antibody. Cells were simultaneously treated with anti-HSP60 antibody which primarily stains mitochondria (15) and Cy3-conjugated second antibody. Both the green Nip3 and red HSP60 fluorescence appeared in a similar punctate pattern, and when the images were combined, a uniform yellow staining pattern indicated virtually complete coincidence of the two stains (Fig. 3, A-C). In contrast, HA-Nip3163 stained diffusely throughout the cytoplasm rather than the punctate distribution of the intact Nip3, however, a small amount of HA staining appeared to colocalize with HSP60 by double fluorescence (Fig. 3, D-F).
Overexpression of Nip3 but not Nip3163 Induces Apoptosis.
T7 epitope-tagged Nip3 was transiently expressed in Rat-1 or MCF-7 cells. Cells were stained with both anti-T7 antibody to identify cells expressing Nip3, and Hoechst dye to quantitate apoptosis. Over time, progressively more Nip3 expressing cells became apoptotic starting around 12 h and reaching completion by 48 to 60 h (Fig. 4 A). Nip3 protein measured by anti-T7 Western blot were highest between 12 and 24 h after transfection then progressively decreased with time paralleling the induction of apoptosis (Fig. 4 B). To determine if the transmembrane domain of Nip3 which is necessary for mitochondrial localization was also necessary to induce apoptosis, the experiment was repeated using the Nip3163. The mutant was unable to induce apoptosis (Fig. 4, A and B). Of additional interest, Nip3163 protein levels decreased very rapidly with time despite the inability of the mutant to induce apoptosis, thus indicating that the loss of protein in the cell lysates was not related to the death of cells.
Nip3 Sensitizes Rat-1 Cells to Apoptosis.
To determine
whether Nip3 can influence apoptosis induced by other
cell death signals, Rat-1 cells were transiently transfected with T7-Nip3 for 12 h then treated with increasing doses
of the cytotoxic lymphocyte protease granzyme B, or the
topoisomerase I and II inhibitors etoposide or camptothecin. The 12-h transfection was chosen because of minimal
effects of Nip3 on cell survival at this time. Apoptotic cells
were then quantitated by Hoechst dye in cells either expressing or not expressing T7-Nip3 protein within the
same samples, using immunofluorescence staining by anti-T7 antibody to detect Nip3. The frequency of apoptotic
cells was clearly higher in Nip3-expressing cells (Fig. 5). A
5-10-fold increase in sensitivity to the drugs and a 2-3-fold
increase in granzyme B apoptosis was observed in three independent experiments.
Nip3 Overcomes Bcl-2 Suppression of Apoptosis.
Since it had been demonstrated that Bcl-2 binds Nip3 (21), we next determined if Bcl-2 was capable of suppressing Nip3-induced apoptosis. Rat-1 and Rat-1/Bcl-2 cells (Fig. 6 C) were transfected with T7-Nip3 and apoptotic cells appearing in both populations was determined over time. Apoptotic Rat-1 cells expressing Nip3 were detected within 12 h and reached maximum levels by 48 h, while Nip3 did not induce cell death in Rat-1/Bcl-2 cells until 24 h after transfection. However, by 48 h virtually all cells expressing Nip3 were apoptotic despite Bcl-2 overexpression (Fig. 6 A). The Rat-1 cells expressing Bcl-2 were highly resistant to granzyme B and perforin (Fig. 6 A). We noticed that Nip3 protein level was considerably higher in Rat-1/Bcl-2 cells than in Rat-1 cells by 12 h after transfection and remained higher throughout the time course. However, a significant decrease in Nip3 level was detected by 48-60 h in Rat-1/ Bcl-2 cells at the same time that recovery of apoptosis was detected (Fig. 6 B).
In the present study we have identified the E1B 19-kD/ Bcl-2-binding protein Nip3 as a pro-apoptotic protein that localizes to mitochondria. Nip3 protein is unusual in several respects. It migrates on SDS-PAGE at a molecular mass that is much higher than the 21.54-kD predicted by the primary amino acid sequence and appears in vivo after transfection and in vitro after transcription and translation as 60- and 30-kD proteins. The 60-kD band does not dissociate after reduction or reduction and alkylation, nor after treatment with 6M urea (Chen, G., and A.H. Greenberg, unpublished data). Thus Nip3 appears to interact with a second protein very strongly and the evidence favors homodimerization. Three experiments support this conclusion. First, truncation of the COOH-terminal 31 amino acids results in the loss of the 60-kD Nip3 band and only a lower molecular weight protein is observed. Second, [35S]methionine-labeled tryptic peptide fragments of the 60-kD Nip3 were identical to the 28-kD Nip3163, and finally, Nip3 exhibits strong interaction with itself in the yeast two hybrid system. The inability to bind Nip3163 to interact with Nip3 or itself indicates that it cannot dimerize and is consistent with the requirement of the COOH-terminal portion of Nip3 for homodimerization.
The COOH-terminal domain of Nip3 not only influences dimer formation but also directs its expression to mitochondria. Nip3 colocalized with HSP60 which is primarily expressed in mitochondria (25) while the mutant Nip3163 lacking the transmembrane domain was mostly expressed as a free cytosolic protein. A minor component of the Nip3163 protein was colocalized with the mitochondrial markers suggesting that it may interact by a mechanism other than membrane insertion, perhaps by binding to other mitochondrial proteins. Nip3163 does not interact with Nip3 or form a homodimer in vitro so it likely does not homodimerize with endogenous Nip3 in vivo.
Nip3 protein expression progressively decreases over time, which may reflect the increasing death of Nip3 expressing cells. Partial Bcl-2 suppression of Nip-3 induced apoptosis and a significant increase in Nip3 protein levels suggests that at least part of the Nip3 is lost by cell death. However, a progressive decrease in protein levels was also observed with the Nip3163 mutant which does not induce apoptosis, therefore another mechanism is affecting protein turnover. Nip3 contains PEST sequences suggesting that the protein may be susceptible to rapid degradation by proteases. PEST sequences commonly contain high local concentrations of amino acids P, E, S, T, and D flanked by charged amino acids (26) and these are abundantly present in Nip3. Thus the posttranslational control of Nip3 expression through rapid protein degradation may constitute a mechanism for regulating the intracellular levels of a potentially lethal protein.
Nip3 was originally identified as an E1B 19K interacting protein, but the structure of Nip3 does not closely resemble other death agonists of the Bcl-2 family. Bcl-2 family agonists such as Bax act by heterodimerizing with anti-apoptotic family members and mutations in BH1 and BH2 domains in Bcl-2 or Bcl-XL result in loss of binding and anti-apoptotic activity (27, 28). However, other studies suggest that pro- and anti-apoptotic Bcl-2 family members can act independently (28) or through other family members such as BAD (29, 30). Nip3 does not contain either a BH1 or BH2 domain, although it may have a BH3 domain in which leucine110, aspartic acid115, and isoleucine117 are conserved based on the critical amino acids of the BH3 domain of BAK that determines BAK-Bcl-XL heterodimerization (31). Other BH3 domain-only Bcl-2 family members that are death agonists have been described such as Bik/Nbk (32, 33), Bid (34) and, more recently, Hrk (35). Bik and Hrk have putative transmembrane domains while Bid is a cytoplasmic factor that is hypothesized to be recruited to membrane-associated Bcl-2 or Bax. Thus it is possible that Nip3 is a death factor of the Bcl-2 family related to Bik/Nbk, Bid, or Hrk.
Many Bcl-2 family members are integral membrane proteins that bear COOH-terminal regions that allow localization to the mitochondrial outer membrane, nuclear envelope and endoplasmic reticulum (15, 36, 37). Localization of Bcl-2 to mitochondria also appears to be important in its suppression of cell death as mutants lacking the transmembrane domain are ineffective when overexpressed (14, 38). Nip3 shares the ability to localize to mitochondria and recent work suggests that mitochondria may play an important role in the regulation of apoptosis. They are necessary for the apoptotic activity of cytosolic extracts of Xenopus laevis oocytes (39), and can initiate nuclear destruction when taken from cells undergoing apoptosis in in vitro reconstituted cell systems (16). Nip3 localization in mitochondria places it in a position to influence mitochondrial function early in the apoptotic response. Nip3 can overcome Bcl-2 suppression of apoptosis so it might act on Bcl-2 or other anti-apoptotic family members that regulate mitochondrial permeability transition pores (16, 17) or cytochrome c release (18). Nip3 preferentially forms homodimers even under reducing conditions and could provide a stable link between Bcl-2 homo- and/or heterodimers and other proteins perhaps forming larger complexes or altering their interaction with regulators such as BAD (30).
In conclusion, we have identified the E1B 19K/Bcl-2 binding protein Nip3 as a homodimer localized to mitochondrial membrane. Nip3 expression in mitochondria induces apoptosis and can overcome Bcl-2 suppression of cell death, indicating that Nip3 is a novel pro-apoptotic protein.
Address correspondence to Arnold H. Greenberg, Manitoba Institute of Cell Biology, University of Manitoba, 100 Olivia St., Winnipeg, MB Canada R3E OV9. Phone (204) 787-2112; Fax: (204) 787-2190; E-mail: agreenb{at}cc.umanitoba.ca
Received for publication 15 May 1997 and in revised form 3 September 1997.
1 Abbreviations used in this paper:We thank Dr. Radhey Gupta for his gift of anti-HSP60 antibody.
This work was supported by grants from the National Cancer Institute and MRC of Canada. A.H. Greenberg is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.
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