Functional role of death associated protein 3 (DAP3) in anoikis

Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, known as anoikis. Integrin stimulation protects cells from anoikis, but the responsible mechanisms are not well known. Here, we demonstrate that a pro-apoptotic GTP-binding protein, DAP3 (death associated protein 3), is critical for induction of anoikis. Downregulation of DAP3 expression by antisense oligonucleotides inhibited anoikis. Conversely, over-expression of DAP3 augmented cell death and caspase activation induced by cell detachment. Furthermore, association of DAP3 with FADD and activation of caspase-8 were induced by cell detachment. We also show that DAP3 is phosphorylated by kinase Akt (PKB), and active Akt can nullify apoptosis induction by DAP3. Mutation of a consensus Akt phosphorylation site in DAP3 renders it resistant to suppression by active Akt in cells. Integrin ligation stimulates Akt activation and phosphorylation of DAP3 in intact cells, as well as suppressing the ability of DAP3 over-expression to augment anoikis. Involvement of DAP3 in anoikis signaling demonstrates a novel mechanism role for this GTP-binding protein in apoptosis induction caused by cell detachment.


INTRODUCTION
The process of apoptosis is critical for the development and maintenance of all multicellular metazoans. Dysregulated apoptosis is germane to many human diseases, including cancer, immunodeficiency, and neurodegeneration. At least two major pathways for apoptosis induction have been identified, commonly known as the extrinsic and intrinsic pathways (reviewed in (1,2)). The extrinsic pathway is triggered by Tumor Necrosis Factor (TNF)-family cytokines, and involves the adapter protein FADD, which recruits pro-caspases-8 and -10 to death receptor complexes, resulting in activation of these cell death proteases and initiation of the apoptosis process. The intrinsic pathway, in contrast, is triggered by myriad stimuli that impinge on mitochondria, causing these organelles to release apoptogenic proteins into the cytosol, such as cytochrome c, which binds caspase-9-activator Apaf-1, initiating the apoptosis mechanism.
DAP3 (death associated protein 3) was identified as a pro-apoptotic protein during a functional screen based on tumor cell transfection with an antisense cDNA expression library and screening for rescue from cytokine-induced apoptosis (reviewed in (3,4)). It was subsequently shown that DAP3 antisense protects tumor cells from by guest on March 24, 2020 http://www.jbc.org/ Downloaded from findings provide novel insights into the mechanisms responsible for anoikis regulation, and thus may have relevance to pathophysiological situations where cell detachment from matrix is involved, such as tumor metastasis and wound healing.

Anoikis Assays
Anoikis assays were performed using established procedures (13). For cell viability assays or caspase 8 assays, cells were detached and maintained in suspension as described above in HEMA-coated culture plates. After 48 hours, cells were harvested and the percentage of dead cells was assayed by trypan blue staining and caspase 8 activity of cell lysates was assayed by hydrolysis of fluorigenic substrate Ac-IETD-AFC (Calbiochem, Inc.), as described below.

Caspase 8 Activity Assays
Caspase 8 activity was measured in cell lysates as described (7) at 37°C using a fluorometric plate reader (Perkin-Elmer, LS50B) in the kinetic mode with excitation and emission wave lengths of 400 and 505 nm, respectively. Activity was measured by the release of 7-amino-4-trifluoromethyl-coumarin (AFC) (RFU) from the synthetic substrate Ac-IETD-AFC (PharMingen) after 30 min incubation.

Immunoprecipitation Assays
HEK293 cells (1X10 6 ) in 10 cm plates were transiently transfected with 10 µg (total) of plasmid DNA. Cells were cultured in attached or detached (in suspension culture) conditions as described above. After 24 or 48 hours, cells were suspended in lysis buffer containing 0.1% NP-40, 20 mM Tris-HCl pH 7.5, 2 mM MgCl 2 , 1 mM EGTA, 130 mM NaCl and protease inhibitors (Boehringer Mannheim). After pre-clearing with 10 µl protein A or protein G-agarose, immunoprecipitations were performed using 10 µl of anti-FLAG antibody M2-conjugated agarose (Sigma) or each antibody at 4°C for 4 hours.
After extensive washing by lysis buffer, immune complexes were analyzed by SDS-PAGE or immunoblotting using indicated antibodies, followed by HRPase-conjugated antibodies and detection using an enhanced chemiluminescence (ECL) system (Amersham, Inc.).

Antibody Production
A rabbit antiserum to DAP3 phosphopeptides was raised against a purified peptide, CRVRNA(T)DAVGIV, in which threonine (T) was phosphorylated. The first cysteine(C) residue was bound to the maleimide-activated carrier proteins (Pierce, Inc.) through its sulfhydryl group. The antiserum was purified using a phophopeptide-conjugated column.

DAP3 is required for anoikis.
To explore the requirement of DAP3 for anoikis, we used antisense oligodeoxynucleotide-based gene silencing. Two different antisense (AS) oligodeoxynucleotides (ODN) targeting the human DAP3 mRNA, AS1 and AS2, were effective at reducing levels of endogenous DAP3 protein when transfected into HEK293 cells, whereas various control ODNs (including a sense sequence) did not ( Figure 1A). We then tested the effects of transfected ODNs on cell death induced by cell detachment from matrix. AS-ODNs reduced cell death by approximately half, compared to control untreated cells or sense ODN-treated cells ( Figure 1B). Thus, expression of endogenous DAP3 appears to be required, at least in part, for cell death induction by cell detachment.

Cell detachment promotes DAP3 interaction with FADD.
Previously we showed that DAP3 associates with FADD (7). We therefore explored the effects of cell detachment on interaction of DAP3 with FADD, using co-immunoprecipitation assays.
In addition, we compared control-transfected cells with cells transfected with plasmids encoding constitutively active mutants of the protein kinase, Akt, because of its by guest on March 24, 2020 http://www.jbc.org/ Downloaded from previously documented ability to suppress anoikis (15).
In attached cells, little endogenous DAP3 associated with endogenous FADD.
In contrast, when cells were suspended, then association of DAP3 and FADD increased, as determined by co-immunoprecipitation assay ( Figure 2A). In cells expressing the active forms of Akt, namely myristoylated Akt or Akt (E40K), less FADD was recovered with immunoprecipitated DAP3, suggesting that Akt suppresses the interaction of these proteins. Levels of FADD and DAP3 were not altered by either cell detachment or Akt activity, excluding differences in protein expression as a trivial explanation for these findings ( Figure 2A).
As expected by a previous report (16), cell detachment correlated with elevations in caspase-8 protease activity, as measured in cell lysates by a fluorogenic substrate, Ac-IETD-AFC ( Figure 2B). Akt suppressed caspase-8 activation induced by cell detachment, consistent with prior reports (16).

Akt phosphorylates DAP3 and regulates the pro-apoptotic activity of DAP3.
The ability of Akt to suppress DAP3 association with FADD in suspended cells prompted us to explore the possibility that DAP3 might be a direct or indirect substrate of this protein by guest on March 24, 2020 http://www.jbc.org/ Downloaded from kinase. Akt phosphorylates proteins on serine or threonine residues within the sequence motif RXRXX(S/T) (17). Interestingly, we found that DAP3 contains a site that conforms to the consensus Akt phosphorylation motif at Thr237 (RVRNAT).
To examine whether DAP3 is directly phosphorylated by Akt, active Akt was . Unlike cytochrome c, however, there is no evidence thus far that DAP3 must be released from mitochondria to perform its cell death function (10). Rather, incomplete import of DAP3 into mitochondria seems to generate an extra-mitochondrial pool of this pro-apoptotic protein, the amount of which varies among cell types and cell lines (9). Cytochrome c and DAP3 also differ in that the former resides in the space between the inner and outer mitochondrial membranes, while the latter resides in the matrix (23 (7). Anoikis has been shown to be dependent on extrinsic pathway participants FADD and caspase-8, but not on components of the intrinsic pathway (16,26). Thus, a role for DAP3 in anoikis is consistent with evidence that this pro-apoptotic protein participates in the extrinsic pathway. Interestingly, we observed that cell detachment resulted in an increase in the association of DAP3 with FADD, without altering the total levels of either of these proteins. This observation suggests an inducible protein interaction, which could be related to the recent observation that