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Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulum¹

Max-Planck-Institute for Biochemistry, Martinsried, Germany

²Correspondence: Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. E-mail: sgrimm{at}biochem.mpg.de

SPECIFIC AIMS

BH3 domain-only proteins constitute a protein family that mediates specific cell death signals. We wanted to investigate a novel BH3 domain protein and its involvement in apoptosis induction.

PRINCIPAL FINDINGS

1. Spike (for small protein with inherent killing effect) is a BH3-only protein that induces apoptosis
A cDNA that was named Spike was isolated in a screen for proapoptotic genes. A sequence comparison with known apoptosis-inducing genes revealed that Spike contains a BH3 domain in the carboxyl-terminal part of the protein. This domain was also highly conserved in the Caenorhabditis elegans and Drosophila melanogaster Spike proteins, which likewise induced cell death. To further elucidate the proapoptotic signal of Spike, we constructed progressive carboxyl-terminal deletions. We observed that the truncation of the very carboxyl terminus resulted in a slight increase of apoptosis induction, whereas deletion of the BH3 domain almost completely abrogated its proapoptotic activity. In addition, point mutants of the BH3 domain efficiently reduced cell killing by Spike. This demonstrates that Spike’s BH3 domain is necessary for cell killing.

2. Spike has some uncommon characteristics
BH3-only proteins are supposed to exert their proapoptotic activity by interacting with other Bcl-2 family members. Consequently, we wanted to test whether Spike’s apoptosis induction correlated with such an interaction. However, we did not observe a coimmunoprecipitation of Spike with all tested Bcl-2 family members such as Bax, Bcl-XL, or Bcl-2. We also tested Spike and several deletion mutants for the ability to self-associate. Although this activity has only rarely been observed for BH3-only proteins, Spike could be shown to efficiently self-associate. In addition, in contrast to most proteins of this family, Spike was not found to be associated with mitochondria. Immunofluorescence microscopy revealed a considerable overlap of endogenous Spike distribution with Bap31, which is an adapter protein for procaspase-8 and Bcl-XL, localized to the endoplasmic reticulum (ER). A complete colocalization was observed with an ER-specific dye (ER-tracker). The overexpressed Spike could also be found associated with the ER.

3. Spike competes with Bcl-XL for its interaction with Bap31
As our studies had pointed to a colocalization of Spike with Bap31, we explored whether it can interact with Bap31. Immunoprecipitation experiments in 293T cells revealed that Bap31 associates with Spike (Fig. 1 A). Using different deletion constructs of Spike, we observed that all mutants that could induce apoptosis also interact with Bap31 (Fig. 1B ). In addition, an immunoblot showed that Spike led to the specific cleavage of a BAP31-YFP fusion protein, which contains the two caspase-8 cleavage sites at the carboxyl-terminal moiety of BAP31. In contrast, Bax was mostly inactive under these conditions, despite a stronger apoptosis induction (Fig. 1C ). As we could observe a diminished interaction between Bcl-XL and Bap31 on coexpression of Spike (Fig. 1A ), we tested whether Spike functions to prevent the complex formation between Bap31 and Bcl-XL. Figure 1D shows that the association of Bap31 and Bcl-XL was significantly reduced when different amounts of Spike were transfected. At the same time, a progressively stronger interaction between Spike and Bap31 could be observed. This was also observed with all Spike deletion mutants that induce apoptosis. That led us to assume that Spike is able to prevent the antiapoptotic complex between Bcl-XL and Bap31 and to form a proapoptotic complex with Bap31. Accordingly, Bcl-XL turned out to be a very potent suppressor of Spike-mediated cell death.

View larger version (63K): [in this window] [in a new window]   Figure 1. Spike interacts with Bap31 and can inhibit the complex between Bap31 and Bcl-XL. A) Spike interacts with Bap31. The indicated constructs were transfected into 293T cells, and immunoprecipitations (IP) were performed. HA,; myc,. B) The interaction between Bap31 and different Spike deletions. The associations of the indicated constructs were probed with immunoprecipitations. WT, Wild-type. C) Spike leads to the specific cleavage of a BAP31-YFP fusion protein. Expression plasmids for Luciferase (Luc), Spike, or Bax together with a fusion construct of BAP31-YFP and an expression construct for YFP were transfected into HeLa cells. The BAP31-YFP construct comprised the two caspase-8 cleavage sites at the carboxyl terminus of the BAP31 moiety. After 18 h, lysates were probed with an antibody against YFP. Equal loading was verified by an immunoblot against ß-actin (middle panel). Apoptosis induction was checked by a fluorescein-activated cell sorter (FACS) analysis of aliquots of the same transfection (bottom panel). GFP, Green fluorescent protein. D) Spike and its proapoptotic deletions can inhibit the complex between Bap31 and Bcl-XL. Increasing amounts of Spike or its deletion constructs that are still active for cell death were transfected into 293T cells. The interactions among Bap31, Bcl-XL, and Spike were tested in immunoprecipitations.

4. Spike is involved in apoptosis by specific death receptors
As the Bap31 complex has been implicated in regulating death receptor-mediated apoptosis, we wanted to investigate Spike’s role in this signaling pathway. Consequently, we used the dominant-negative Spike mutant N19 (Fig. 2 A, B), a dominant-negative Fas associated death domain protein (FADD) mutant, and the apoptosis repressor Bcl-XL to transfect HeLa cells. After stimulation with an antagonistic antibody against the Fas receptor, we measured apoptosis induction by FACS analysis. The dominant-negative Spike mutant was able to repress apoptosis almost as efficiently as the Bcl-XL gene and the dominant-negative FADD (Fig. 2D , left panel). In contrast, although Bcl-XL was a potent repressor for TNF-induced apoptosis, this stimulus could not be affected by the Spike mutant N19 (Fig. 2D , right panel). In addition, HeLa cells transfected with the Spike antisense oligonucleotide (Fig. 2C ) and a control oligonucleotide were stimulated with an anti-Fas antibody for apoptosis induction. Figure 2E shows that apoptosis was repressed when Spike was down-regulated. In contrast, apoptosis induced by TNF was not significantly altered by the antisense oligonucleotide.

View larger version (39K): [in this window] [in a new window]   Figure 2. Spike mediates a death receptor signal. A) Identification of a dominant-negative Spike variant. WT Spike and different deletions of Spike that are inactive for apoptosis induction were cotransfected with WT Spike, and cell death was determined 24 h later by FACS analysis; data are means ± SD from three experiments. B) The Spike deletion mutant N19 can interact with WT Spike. 293T cells were transfected with the indicated constructs, and an immunoprecipitation was performed. C) Test of an antisense Spike oligonucleotide for down-regulation of the endogenous Spike. Western blots, after transfection of a Spike antisense (AS) oligonucleotide and the same oligonucleotide mutated in four positions, are shown. The mitochondrial protein Tim23 is used as a loading control. D) The dominant-negative (dn) Spike N19 protects the cells against Fas but not tumor necrosis factor (TNF)-induced apoptosis. HeLa cells transfected with the indicated constructs were treated with Fas (100 ng/ml) or TNF (50 ng/ml) for 8 or 24 h, respectively; data are means ± SD from three experiments. E) The Spike antisense oligonucleotide reduces Fas-induced cell death. Antisense (2 µg) or a control oligonucleotide was transfected into HeLa cells, and apoptosis was quantified after stimulation with Fas (100 ng/ml) or TNF (50 ng/ml); data are means ± SD minus background apoptosis from three experiments.

CONCLUSIONS

Recently, a novel, procaspase-containing protein complex at the ER was identified, consisting of p28 Bap31, Bcl-2/Bcl-XL, and procaspase-8L. As Spike is able to interfere with the complex formation between Bap31 and Bcl-XL, we propose a model shown in Figure 3 . Accordingly, the complex on Bap31 is held in check by Bcl-XL, as long as they are associated. Displacement of Bcl-XL from Bap31 by Spike (and possibly by additional but as yet unidentified components) leads to the formation of a proapoptotic complex. Our data therefore also point to a novel activity of antiapoptotic Bcl-2 proteins. Rather than protecting the cell solely by interacting with other Bcl-2 family members, they also might shield adapter proteins such as Bap31 against the association with proapoptotic BH3-only proteins. This adds a new perspective to the way BH3-only and antiapoptotic Bcl-2 proteins function to regulate cell death. Our study further establishes the ER as an important organelle in the induction of apoptosis and could lead to a deeper understanding of the signals that are received by and emanate from the Bap31 complex and regulate cell death.

View larger version (17K): [in this window] [in a new window]   Figure 3. Model for the proapoptotic activity of Spike. BAP31 and Bcl-XL interact at the ER and form a protein aggregate that is incompetent for cell death induction. Spike can also associate with BAP31. This inhibits the binding of the antiapoptotic Bcl-XL protein to the BAP31 complex and forms a proapoptotic complex.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0657fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0657fje

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