The Caenorhabditis elegans sex determination protein FEM-1 is a CED-3 substrate that associates with CED-4 and mediates apoptosis in mammalian cells.

Sex-specific elimination of cells by apoptosis plays a role in sex determination in Caenorhabditis elegans. Recently, a mammalian pro-apoptotic protein named F1Aalpha has been identified. F1Aalpha shares extensive homology throughout the entire protein with the C. elegans protein, FEM-1, which is essential for achieving all aspects of the male phenotype in the nematode. In this report, the role of FEM-1 in apoptosis was investigated. Overexpression of FEM-1 induces caspase-dependent apoptosis in mammalian cells. FEM-1 is cleaved in vitro by the C. elegans caspase, CED-3, generating an N-terminal cleavage product that corresponds to the minimal effector domain for apoptosis. Furthermore, CED-4 associates with FEM-1 in vitro and in vivo in mammalian cells and potentiates FEM-1-mediated apoptosis. Similarly, Apaf-1, the mammalian homologue of CED-4 was found to associate with F1Aalpha. These data suggest that FEM-1 and F1Aalpha may mediate apoptosis by communicating directly with the core machinery of apoptosis.

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms (1). Genetic studies of apoptosis in the nematode Caenorhabditis elegans have identified four genes that define the core machinery of apoptosis. ced-3, ced-4, and egl-1 promote and ced-9 inhibits apoptosis (2,3). The direct association of CED-4 with both CED-9 and pro-CED-3 was demonstrated by co-immunoprecipitation assay in mammalian cells and in vitro pull-down assays (4 -6). Furthermore, CED-4 was also shown to oligomerize and facilitate the proteolytic activation of CED-3 in mammalian cells (7,8). It has been proposed that the EGL-1 protein activates apoptosis by binding to and thereby negatively regulating CED-9 (9). The binding of EGL-1 to CED-9 was subse-quently shown in the mammalian system to disrupt the association between CED-9 and CED-4 resulting in the CED-4dependent activation of CED-3 (10).
By isolating mutations that transform the entire animal from one sex to the other, a group of genes that controls the sexual fate of C. elegans has been identified (11)(12)(13)(14). Three fem genes, fem-1, fem-2, and fem-3 (15) are essential for male development. Loss-of-function mutations in any one of the fem genes prevent all aspects of male development and transform the animals that are genetically males into females (16,17). The predicted product of the fem-1 gene is an intracellular protein that contains ankyrin repeats, which in many other proteins mediate specific protein-protein interaction (17). However, despite the vital role of FEM-1 in sex determination, no biochemical function has yet been ascribed to the protein.
A human homologue of FEM-1, called F1A␣ or FEM1␤ (18,19), has recently been identified. F1A␣ is pro-apoptotic and shares high homology with FEM-1 (18). The percentage identity between the two proteins ranges from 22 to 47% depending on the regions of the proteins that are compared (Fig. 1A). The degree of homology between FEM-1 and F1A␣ is comparable with that between several of the functionally conserved components of apoptosis in C. elegans and mammals. For example, the amino acid identity between CED-9 and Bcl-2 is 23% (20) and that between CED-3 and caspase-3 is 34% (21). In this study we evaluated the possible molecular functions of FEM-1 in apoptosis using a mammalian cell line as a model system.

EXPERIMENTAL PROCEDURES
Reagents-Antibodies against the Myc epitope (A14, 9E10) and HA epitope (Y11, F7) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. NIH3T3 and L929 were originally from American Type Culture Collection (ATCC). The tumor necrosis factor-sensitive MCF7 breast carcinoma cells were provided by Dr. V. Dixit, University of Michigan. SH-SY5Y human neuroblastoma cells that are routinely used in the laboratory (22,23) were originally provided by Dr. Wolfgang Sadee, University of California, San Francisco. Cell lines were grown according to the directions provided by suppliers. All media were supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.) and antibiotics (100 mg of streptomycin/ml and 100 IU of penicillin/ml, Life Technologies, Inc.). The peptide caspase inhibitor ZVAD-fmk 1 was purchased from Enzyme System Products.
Plasmid Constructions-cDNA for fem-1 was from Dr. Andrew Spence, University of Toronto, Canada; cDNAs for ced-3, ced-4, and ced-9 were from Dr. Robert Horvitz, MIT. cDNA for apaf-1 and caspase-8 were from Dr. Xiaodong Wang, Howard Hughes Medical Institute, Dallas, TX and Dr. Marcus E. Peter, German Research Center, Heidelberg, Germany, respectively. Expression plasmids for CED-3C/S (CED-3C358S) (24), FEM-1, FEM-1-D320A, and FEM-1-D344A were generated by site-directed mutagenesis using the Transformer TM Site-Directed Mutagenesis kit (CLONTECH). DNA fragments for epitope-tagged constructs containing CED-3C/S, CED-4, CED-9, Apaf-1, FEM-1D320A, FEM-1D344A, FEM-1, and its deletion mutants were obtained by polymerase chain reaction amplification using pfu polymerase (Stratagene). Appropriate restriction sites and epitope tags were included in the primers. Amplification products were inserted into the pXJ40 mammalian expression vector driven by the CMV promoter (25). All epitope tags are at the N termini. DNA fragments generated by polymerase chain reaction and the junctions of insertion were confirmed by sequencing. pEGFP-FEM-1 was constructed by releasing the BamI/KpnI fragment from pXJHAFEM-1 and inserting it into the BglII and KpnI sites of the pEGFPC1 vector (CLONTECH). Construction of the pXJHAF1A␣ has been described (18).
In Vitro Cleavage Analysis-FEM-1 and its mutants were [ 35 S]methionine-labeled using the TnT TM T7 coupled reticulocyte lysate system (Promega). 1-2 l of the in vitro translated product was incubated with 20 l of recombinant CED-3 enzyme at 30°C for 2 h. For the control reactions, the in vitro translated products were mixed with 20 l of Ni-NTA resin slurry that had been incubated with lysate from bacteria containing the expression vector alone.
Cell Death Assays-Mammalian cells at approximately 60 -70% confluency were transfected with 2 g of expression plasmids of FEM-1 or its mutants together with 0.5 g of pCMV-␤-Gal using LipofectAMI-NE TM (Life Technologies, Inc.). At defined times after transfection, ␤-Gal positive cells were counted and the apoptotic cells were scored as described (18).
Nuclear Staining of EGFP-expressing Cells-SH-SY5Y cells on glass coverslips at 70% confluency were transfected with pEGFP or pEGFP-FEM-1. 24 h after transfection, the cells were fixed, rinsed with phosphate-buffered saline and then incubated for 2 min with Hoescht 33342 dye (Molecular Probes Inc.) to enable nuclear staining. The cells were subsequently visualized using a Zeiss Axioplan microscope.
Co-immunoprecipitation and in Vitro Binding-For expression constructs of GST fusion proteins, sequences encoding FEM-1 and F1A␣ were excised from the respective pXJHA constructs as BamHI-KpnI fragments and cloned in-frame into GST fusion protein vector pGEX-TK4E. Co-immunoprecipitation and in vitro binding analyses were performed as described (18).

Overexpression of FEM-1 Induces Caspase-dependent Apoptosis in Mammalian Cells-SH-SY5Y
human neuroblastoma cells were co-transfected with expression vectors encoding fulllength FEM-1 or control vector together with pCMV-␤-Gal as a marker for transfected cells. At 24 h post-transfection, a significant percentage of total ␤-galactosidase-positive (blue) cells co-transfected with FEM-1 became shrunken and rounded, displaying morphological features typical of cells undergoing apoptosis (Fig. 1B). Nuclear condensation, another hallmark of apoptosis, was also observed in cells expressing EGFP-FEM-1, a fusion protein of FEM-1 and the green fluorescent protein (GFP) (Fig. 1C). In the round cell apoptosis assay, extended incubation up to 30 h resulted in a progressive increase in the percentage of total blue cells that exhibited the round cell morphology (Fig. 1D). Treatment with the broad spectrum caspase inhibitor, ZVAD-fmk, abrogated the apoptotic activity of FEM-1 (Fig. 1D) suggesting that FEM-1 mediates apoptosis through caspase-dependent pathways in SH-SY5Y cells. Overexpression of F1A␣ in SH-SY5Y cells yielded similar results (Fig. 1D). The apoptotic effect of FEM-1 overexpression is not restricted to SH-SY5Y cells as a similar degree of apoptosis was observed when the protein was overexpressed in L929 fibrosarcoma cells (data not shown). However, FEM-1 was unable to induce apoptosis in the breast carcinoma MCF-7 and NIH3T3 cells (data not shown) suggesting that the apoptotic function of FEM-1 may be cell type-dependent. We (18) and others (19) have noted that F1A␣ is a member of a gene family. It would be interesting to determine if other members of the F1A gene family may also have a role in apoptosis and whether this activity is cell type-dependent.
FEM-1 Is a Substrate of CED-3-F1A␣ has been demonstrated to be a substrate of caspase-3 in vitro (18). Although the caspase-3 cleavage site in F1A␣, Asp-Asn-Ile-Asp 342 , is not completely conserved in FEM-1 in which the corresponding site is Ala-His-Thr-Asp 344 (Fig. 2B), alternate caspase cleavage site(s) may be present in FEM-1. We tested the ability of the C. elegans caspase, CED-3, to cleave FEM-1 in vitro. Incubation of 35 S-labeled FEM-1 with recombinant CED-3 generated a prod-uct of approximately 37 kDa ( Fig. 2A). The estimated size of the full-length FEM-1 including the N-terminal hemagglutinin (HA) epitope tag was 74 kDa; therefore, the cleavage might have occurred in the center of the protein generating two fragments of similar size. To facilitate the visualization of the cleavage products, a radiolabeled N-terminal deletion mutant of FEM-1, FEM-1-(44 -656), was subjected to cleavage analysis. A 26-kDa and a 37-kDa fragment was generated ( Fig. 2A) supporting the idea that CED-3 cleaved FEM-1 in the central region of the molecule.
To determine the cleavage site, the aspartic acid (Asp) residue of two potential caspase cleavage sites (Fig. 2B), Asp 320 and Asp 344 were independently mutated to alanine (Ala) in FEM-1. 35 S-Labeled FEM-1 and its point mutants were then subjected to cleavage analyses. Whereas the D344A mutant was cleaved as efficiently as FEM-1, the D320A mutant appeared to be resistant to cleavage by CED-3 (Fig. 2C), suggesting that CED-3 cleaves FEM-1 at Asp 320 .
Mapping the Effector Domain of Apoptosis in FEM-1-As FEM-1 was cleaved by CED-3, it was of interest to determine if the cleavage products would be as active as the wild type protein in mediating apoptosis. Overexpression of the N-terminal cleavage product, FEM-1-(1-320) induced apoptosis in SH-SY5Y cells (Fig. 3). The C-terminal fragment, FEM-1-(321-

FEM-1 Is CED-4-associating Protein That Mediates Apoptosis 17926
656), on the other hand, was apoptotically inactive (Fig. 3). FEM-1-(1-320) appeared to be more potent in inducing apoptosis than the wild type protein as cell death could be observed at an earlier (18 h) time point when FEM-1-(1-320) was overexpressed (Fig. 3). Similar to wild-type FEM-1, the apoptotic activity of FEM-1-(1-320) was inhibited by the caspase inhib-itor, ZVAD-fmk (data not shown). Deletion of the C-terminal region up to the border of the ankyrin repeat cluster (FEM-1-(1-257)) rendered the molecule apoptotically inactive (Fig. 3) indicating that the region immediately distal to the ankyrin repeat cluster up to amino acid 320 is important for apoptotic activity. Deletion of the first 83 amino acids of FEM-1 as in FEM-1-(84 -656), also abrogated the apoptotic activity of FEM-1 (Fig. 3). The lack of activity of the deletion mutants was not attributable to the lack of protein expression in SH-SY5Y cells. The wild type as well as the deletion mutants of FEM-1 were expressed at similar levels except for FEM-1-(1-320), which was expressed at a relatively low level (data not shown). These data suggest that the minimal effector domain of apoptosis resides in the N-terminal region of FEM-1 (amino acids 1-320).
While overexpression of wild-type FEM-1 and the D344A mutant were able to induce apoptosis in SY5Y cells to a similar degree, the cleavage-resistant D320A mutant had no apoptotic activity (Fig. 3). As deletion analysis indicated that the effector domain for apoptosis resides in the N terminus of FEM-1, these results suggest that the C-terminal domain of FEM-1, similar to F1A␣, might serve a regulatory role in suppressing its intrinsic apoptotic activity.

CED-4 Associates with FEM-1 in Vitro and in Vivo in
Mammalian Cells-As in most physiological pathways, many cell death signaling molecules function by interacting with other proteins in the pathway. To investigate the possibility that FEM-1 may communicate with the core components of the cell death pathway through protein-protein interactions, co-immunoprecipitation analyses were performed. HA-tagged FEM-1 was co-expressed with Myc-tagged CED-3C/S, a catalytically inactive mutant of CED-3 (24), CED-4, or CED-9 in 293T cells. HA-FEM-1 was immunoprecipitated, and the associated Myctagged proteins were detected with anti-Myc antibody. CED-4 but not CED-3C/S or CED-9 appeared to interact with FEM-1 (Fig. 4A, lanes 4, 2, and 6). HA-FEM-1 also co-immunoprecipitated with Myc-CED-4 in the reciprocal experiment in which the Myc-tagged proteins were immunoprecipitated (data not shown). FEM-1 expressed as a GST fusion protein was able to specifically pull down in vitro translated 35 S-labeled CED-4 ( Fig. 4C) suggesting that the proteins were in direct physical contact. Parallel experiments were performed with F1A␣ and Apaf-1, which are the mammalian homologues of FEM-1 and CED-4, respectively (18,26). As predicted, the HA-tagged F1A␣ co-immunoprecipitated with the Myc-tagged Apaf-1 (Fig. 4B,  lane 8), and the association appeared to be direct since in vitro translated 35 S-labeled Apaf-1 was specifically pulled down by GST-F1A␣ (Fig. 4C).
CED-4 Potentiates the Apoptotic Activity of FEM-1-The association of CED-4 with FEM-1 in the cells raised the possibility that CED-4 might have a role in modulating the function of FEM-1 or vice versa. In SH-SY5Y cells, overexpression of FEM-1 induced apoptosis in a dose-dependent and saturable manner at a defined time point (data not shown). When the amount of FEM-1 expression plasmid was reduced from 2 to 0.5 g, no significant apoptosis was observed at the 24-h time point (Fig. 4D). Consistent with a previously reported observation (6), overexpression of CED-4 itself did not induce apoptosis in mammalian cells (Fig. 4, D and E). However, CED-4 co-expression significantly enhanced FEM-1-mediated but not caspase-8-mediated apoptosis (Fig. 4D). Furthermore, CED-4 increased the rate of FEM-1-mediated apoptosis (Fig. 4E), and the effect appeared to be specific because the rate of cell death induced by caspase-8 overexpression in these cells was unaffected by CED-4 co-expression (data not shown). The levels and kinetics of FEM-1 protein expression were not affected by CED-4 co-  (18). Arrows indicate the aspartic acid residues in the tetrapeptides Glu-Leu-Leu 320 -Asp and Ala-His-Thr 344 -Asp that were systematically replaced with alanine residues in subsequent cleavage analyses. C, FEM-1 is cleaved by CED-3 at Asp 320 in vitro. 35 S-Labeled FEM-1, FEM-1D320A, or FEM-1D344A was incubated with CED-3, and the cleavage products were fractionated by SDS-PAGE and detected by autoradiography. expression (data not shown) suggesting that CED-4 did not affect the turnover of FEM-1. Whether the effect of CED-4 on the apoptotic activity of FEM-1 depends on direct interaction between the two proteins remains to be investigated.
The mammalian counterpart of CED-4, Apaf-1 (26), which was non-apoptotic when overexpressed, was also able to potentiate the apoptotic activities of FEM-1 and F1A␣ in SH-SY5Y cells (data not shown). This result together with our previous observation that the dominant negative mutant of caspase-9 blocks F1A␣ apoptotic activity (18) suggest that F1A␣ is in the Apaf-1-caspase-9 signaling pathway.
The Potential Role of FEM-1 in Apoptosis-A recent study has linked the sex determination pathway directly to the core machinery of cell death in the hermaphrodite-specific neurons of C. elegans (27). TRA-1A, the terminal regulator in the C. elegans sex determination pathway, is found to repress the transcription of the cell death activator gene, egl-1, leading to the survival of hermaphrodite-specific neurons during hermaphrodite development. Since FEM-1 is a negative regulator of TRA-1A in the genetic pathway of sex determination, the question arises as to whether the observed apoptotic activity of FEM-1 in mammalian cells is mediated through a TRA-1A-like activity propagating a signal to the cell death pathway. However, tra-1 is known to be one of the most rapidly diverging genes when the homologues in C. elegans and Caenorhabditis briggsae are compared (28); it is questionable whether a mammalian protein similar to TRA-1 exists. Our data suggest that FEM-1 is able to communicate directly with the signaling components that constitute the core cell death machinery in C. elegans.
Despite its critical role in mediating the male phenotype, FEM-1 protein can be detected in the soma and germ cells of both sexes of C. elegans throughout development and during adulthood (29). It is therefore possible that FEM-1 might be involved in apoptosis during sexual development as well as in other developmental and/or physiological pathways.