Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Jan 5;96(1):145-50.
doi: 10.1073/pnas.96.1.145.

Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: implicated mechanisms for caspase activation

H Kanuka  1 , S Hisahara, K Sawamoto, S Shoji, H Okano, M Miura
Affiliations

Proapoptotic activity of Caenorhabditis elegans CED-4 protein in Drosophila: implicated mechanisms for caspase activation

H Kanuka et al. Proc Natl Acad Sci U S A. .

Abstract

CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A–F) The phenotypes of GMRced-4 transgenic flies are partially blocked by p35. (A and D) Wild-type compound eye. (B and E) Ectopic expression of one copy of GMRced-4 causes a reduction of eye size and disordered cell arrangement in the eye. (C and F) The size of the GMRced-4-expressing eye was partially rescued by the coexpression of p35. (A–C) Scanning electron microscope images. (D–F) Thin sections of adult eyes. Scale bar, 10 μm. (G–O) Ectopic expression of GMRced-4 induces cell death and caspase activation in eye discs from third-instar larvae. In this figure, anterior is to the right and posterior is to the left. (G, J, M) Wild-type. (H, K, M) Third-instar eye disc from GMRced-4 larvae. (I, L, O) GMR-p35; GMRced-4. (G–I) Acridine orange staining (AO) to detect dead cells. Scale bar, 100 μm. (J–L) Immunostaining by using anti-ELAV antibody. Scale bars, 100 μm. (M–O) In situ affinity labeling (IAL) of active caspases in third-instar larval eye discs. The dotted line indicates the outer shape of the eye disc. Scale bars, 80 μm. In each experiment, immunohistochemical analysis revealed the expression of CED-4 in larval eye discs, and CED-4-positive cells were distributed in the posterior of the morphogenetic furrow (data not shown).
Figure 2
Figure 2
Overexpression of CED-4 induces cell death and caspase activation in Drosophila S2 cells. (A) Overexpression of CED-4 kills Drosophila S2 cells. Cells were transfected with ced-4. At 24 h after heat-shock treatment, cells were fixed and stained with Hoechst 33342. Open arrowhead indicates condensed nuclei. Scale Bar, 10 μm. (B) Quantification of cell death in Drosophila S2 cells. The data (mean ± SD) are the percentage of cells with condensed chromatin in the total number of cells counted. (C and D) DEVD- and YVAD- cleaving activities of the cytoplasmic lysates of S2 cells expressing various proteins or treated with 1 μM staurosporine. S2 cells were transfected with various plasmids, and lysates were prepared 24 h after heat-shock treatment.
Figure 3
Figure 3
CED-4 activates DCP-1 and drICE in Drosophila S2 cells. (A) CED-4 enhanced the caspase activities of DCP-1 and drICE in S2 cells. S2 cells were transfected with ced-4 and either drICE or dcp-1, and the caspase activities (mean ± SD) were measured 24 h after heat shock. (B) CED-4 promoted cell death induced by Drosophila caspases (mean ± SD). S2 cells were transfected with the same combinations shown in (A), and the ratio of chromatin condensed cells (% of total cells: mean ± SD) were counted 24 h after heat-shock. (C) Activation of caspases as revealed by affinity labeling (see Materials and Methods). ced-4 was cotransfected with either empty vector, drICE, or dcp-1 in S2 cells, and cell lysates for the affinity labeling were prepared 24 h after heat-shock. (D) Expression of Drosophila caspases in S2 cells and Drosophila embryos (0–24 h). RT-PCR was performed by using specific primers for dcp-1, drICE, and dcp-2 (see Materials and Methods).
Figure 4
Figure 4
Direct interaction of CED-4 with drICE and requirement of ATP binding for caspase activation. (A) Schematic representation of CED-4 and its mutants. The amino acid sequence in CED-4 is numbered. Single-letter abbreviations for the amino acid residues are as follows; I, Ile; K, Lys; N, Asn; R, Arg. (B) CED-4 and its mutants were transfected into S2 cells, and their expressions were confirmed by Western blot (data not shown). CED-4 (K165R) suppressed wild-type CED-4-induced caspase activity in S2 cells. Caspase activities were measured and represented as mean ± SD. (C) ATPase inhibitor FSBA blocked CED-4-dependent caspase activity in S2 cells. Cytoplasmic lysates expressing CED-4 were preincubated with various amounts of FSBA, with or without 1 mM ATP at 37°C for 30 min. Caspase activities were then measured (mean ± SD). (D) CED-4 and CED-4 (K165R) coimmunoprecipitate with drICE. His6-tagged drICEmt (C211A) was cotransfected with ced-4 or ced-4 (K165R) into S2 cells, and cell lysates were prepared 24 h after heat shock, immunoprecipitation, and separation by 12.5% SDS/PAGE (see Materials and Methods). Expression of drICE, CED-4, or CED-4 (K165R) in the lysates used for immunoprecipitation experiments is shown (Lower).
Figure 5
Figure 5
A P-loop mutation of CED-4 converts the caspase activator to a suppresser. (A–C) The phenotypes of GMRced-4 transgenic flies are moderately inhibited by the ectopic expression of CED-4 (K165R). (A) GMR-ced4 (K165R)/+. (B) GMR-ced4/+. (C) GMR-ced4 (K165R)/+;GMR-ced4/+. (D) Overexpression of CED-4 (K165R) suppresses the caspase activation induced by cycloheximide (CHX), staurosporine (STS), and ecdysone (Ec). S2 cells were transfected with empty vector or ced-4 (K165R), then heat shocked, and cycloheximide (10 μg/ml), staurosporine (1 μM), or ecdysone (10 μM) was added. At 24 h after heat shock, the caspase activities were measured (mean ± SD).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Yuan J, Shaham S, Ledoux S, Ellis H M, Horvitz H R. Cell. 1993;75:641–652. - PubMed
    1. Yuan J, Horvitz H R. Development. 1992;116:309–320. - PubMed
    1. Hengartner M O, Ellis R E, Horvitz H R. Nature (London) 1992;356:494–499. - PubMed
    1. Ellis R E, Yuan J Y, Horvitz H R. Annu Rev Cell Biol. 1991;7:663–698. - PubMed
    1. Shaham S, Horvitz H R. Cell. 1996;86:201–208. - PubMed

Publication types

MeSH terms

LinkOut - more resources