Correspondence to: Adi Kimchi, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel., lvkimchi{at}weizmann.weizmann.ac.il (E-mail), 972-8-9342428 (phone), 972-8-9344108 (fax)
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Abstract |
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Death-associated protein (DAP)kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)- and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by antiFas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-
, Fas, and FADD/MORT1induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.
Key Words:
DAP-kinase, tumor necrosis factor-, Fas, death domain, apoptosis
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Introduction |
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APOPTOSIS (programmed cell death) is an important regulatory mechanism that eliminates unwanted cells during development and maintenance of tissue homeostasis. Over the past few years several apoptotic genes have been identified. One approach consisted of the isolation of proteins that are recruited to the intracellular domains of cytokine receptors belonging to the tumor necrosis factor (TNF)1 family. The yeast two-hybrid system served as a major tool for the isolation of these cytoplasmic proteins that sequentially bind to the intracellular part of TNF- receptor (p55 TNF-R), the Fas receptor, and to each other. This includes a number of adaptor molecules (TRADD, FADD/MORT-1, RAIDD, MADD, TRAF-1 or -2, and FLIP), proteins with enzymatic activity (RIP, caspase 8 and 10, NIK), and death inhibitory molecules (c-IAPs) (for review see
Genetic screens performed in lower invertebrates provided a second fruitful approach for rescuing central apoptotic genes. The rescue of ced-3 from Caenorhabditis elegans and the identification of its mammalian homologues as cysteine proteases (named caspases, more than 14 family members) established that a major arm of the death-promoting pathways involves protease activation. In mammals, the activation of the proteolytic activity can be initiated by different mechanisms and at different intracellular sites. One takes place at the receptor proximal level through adaptor-mediated recruitment of the pro-caspases to the death-inducing signaling complex named DISC (e.g., by binding of pro-caspase 8 or 10 to FADD/MORT-1 that in turn binds to Fas) ( and
, MEKK-1, PITSLRE protein kinase, and the endonuclease CAD) (
A functional approach to gene cloning, based on transfections of HeLa cells with antisense cDNA libraries and subsequent isolation of the fragments that protected cells from interferon- (IFN-
)induced cell death, initiated an additional direction in the field (
CaM), had stronger cell death effects than the wild-type kinase, whereas a catalytically inactive mutant (K42A) was not cytotoxic to cells (
One of the surprising facets in the function of DAP-kinase relates to its antimetastatic activity, recently analyzed in animal model systems ( and Fas. The importance of the death domain in mediating the death-promoting function of DAP-kinase is documented here for the first time. Finally, by transfection-based functional analyses it is shown that DAP-kinase acts downstream to the DISC formation (i.e., FADD/MORT1 and caspase 8) and upstream of some other caspases, and that its death-promoting effects are counteracted by bcl-2.
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Materials and Methods |
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Plasmids
All expression plasmids used in this work were constructed in pcDNA3 vector (Invitrogen Corp.). Construction of wild-type DAP-kinase and CaM mutant was described before (
DD and
CaM/
DD were constructed by truncation of wild-type and
CaM DAP-kinase, respectively, at the HindIII site, thus, deleting the 152COOH-terminal amino acids. DD-DAPk (amino acids 1,3011,431), Flag-tagged at the NH2 terminus, was constructed by PCR. The DD/L1337N mutation was constructed by in vitro mutagenesis. Amino acid numbers in DAP-kinase are according to Swissprot accession number P53355. The luciferase gene was subcloned into pcDNA3 from pGL3-luciferase (Promega), and bcl-2 from pBluescript-bcl-2. The previously described MORT1 and its dominant negative mutant (DN-MORT1), DN-Caspase-8 (also named MACH
-C360S), p55-TNF-R, and p55/Fas chimera cloned into pcDNA3, were used (
Cell Lines, Transfections, and Apoptotic Assays
The HeLa human epithelial carcinoma cells, 293 human embryonic kidney cells, and MCF7 human breast carcinoma cells were grown in DME (Biological Industries) with 10% FCS (Bio-Lab Scientific Ltd.). The HeLa-tTA clone ( and cycloheximide. All cells for transient transfection were seeded in a 6-well plate a day before transfection at density of 105 cells/well. Transfections were done by the calciumphosphate method. For each well, we used a mixture containing 0.5 µg of cell deathinducing plasmid (either p55-TNF-R, p55/Fas chimera, MORT1, or
CaM DAPk mutant), 1.5 µg of a plasmid to be tested for cell death protection (DN-MORT, DN-Caspase-8, DD-DAPk, CrmA, p35, or luciferase as a control), and 0.5 µg of GFP plasmid. Cells were counted and photographed 24 h after transfection. In each transfection four fields, each consisting of at least 100 GFP-positive cells, were scored for apoptotic cells according to their morphology. All the experiments were repeated at least four times. When indicated, cell lysates were prepared from the transient transfection at 24 h. For the experiments in Figure 3 e, cells were transfected solely with the DD-DAPk or DN-MORT and treated 24 h after transfection with human recombinant TNF-
(30 ng/ml; R&D Systems, Inc.) and cycloheximide (10 µg/ml) for 3 h. Stable transfections of HeLa cells and neutral red dye uptake assays were done as previously described (
(PeproTech) was added at 1,000 U/ml. For Fas killing of HeLa cells by agonistic antibodies, the cells were pretreated for 24 h with 25 U/ml of IFN-
(to increase Fas expression) and exposed to 50 ng/ml of antiFas/APO-1 antibodies (IgG3; P.H. Krammer). The percentage of viability was calculated as a fraction of the values measured in the absence of treatment. For poly (ADPribose) polymerase (PARP) cleavage experiments, protein A (5 µg/ml; Sigma Chemical Co.) was added concomitantly with the anti-Fas agonistic antibodies and cell extracts were prepared after 4 h.
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Immunoblot Analysis
Western analysis was done as described before (
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Results |
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DAP-kinase Is Involved in FAS and TNF-induced Apoptosis
The involvement of DAP-kinase in Fas-induced cell death was first analyzed in HeLa cells, and compared in the same assays to the well established involvement of DAP-kinase in IFN- responses. For this purpose, a previously described polyclonal population of HeLa cells, which stably expresses high levels of DAP-kinase antisense RNA from an Epstein-Barr virus-based vector (
as well as by the agonistic antibodies against Fas/APO-1, which trigger Fas signaling by inducing oligomerization of the receptors (Figure 1 a). The antisense DAP-kinase transfectants, however, displayed reduced cell death sensitivity to both IFN-
and Fas signaling. The extent of protection from IFN-
and Fas-induced cell death was similar and in both cases cell viability in treated cultures remained ~5055% (Figure 1 a). The difference in sensitivity between the two cell populations was also prominent when PRAP cleavage, indicative of caspase activation, was measured in response to increasing concentrations of the antiFas/APO-1 agonistic antibodies (Figure 1 b). Thus, a reduction in the levels of endogenous DAP-kinase protein by antisense RNA (
, the feature that served as the basis for the original selection, but also cell death responses to Fas. This suggests that DAP-kinase may be a common mediator in both cell death scenarios.
Another link between DAP-kinase and cytotoxic cytokines was found in our lab by a set of experiments that undertook an opposite approach. We reintroduced a DAP-kinase expression construct into DAP-kinase null cells and assayed whether it affected the cells' sensitivity to TNF-. Expression of DAP-kinase enhanced the number of apoptotic nuclei as compared with cells transfected with an empty vector (
induced cell death.
The Death Domain Is Essential for the Death-promoting Function of DAP-kinase and Displays Dominant Negative Features
To study the role of the death domain, it was first tested whether its deletion may reduce the death-inducing functions of DAP-kinase in transiently transfected 293 human embryonic kidney cells. A constitutively active mutant of DAP-kinase (CaM) in which the catalytic activity is no longer dependent on calcium/calmodulin was employed. This gain-of-function mutant was previously shown to be an effective inducer of cell death when transfected on its own into cells (
CaM mutant with a vector expressing the GFP protein. The latter was used as a marker to visualize the transfected cells and to assess the apoptotic frequency among the transfectants according to morphological alterations. Apoptotic cells were scored after 24 h. Overexpression of the
CaM mutant of DAP-kinase resulted in massive apoptotic cell death (Figure 2, a and d). Most of the GFP positive green cells rounded up and shrunk, some of them showed cytoplasmic blebs, and some were further fragmented into apoptotic bodies. In contrast, when the cells were transfected with the
CaM mutant deleted of its death domain (Figure 2 c,
CaM/
DD), apoptotic cells were much less abundant (23% apoptotic cells compared with 68% in
CaM transfections; see Figure 3 d). Similar results were obtained upon transfections of these constructs into MCF7 human breast carcinoma cells (data not shown). The two recombinant proteins were expressed to comparable levels in these transient transfection assays (Figure 2 b). Deletion of the death domain from the wild-type DAP-kinase, which as expected is a less effective killer than the constitutively active kinase, also reduced its ability to induce cell death (14.5% apoptotic cells compared with 32% in DAP-kinase transfections; Figure 2 d). Therefore, it is concluded that the death domain contributes to the death-inducing function of DAP-kinase.
Since death domains of other known proteins were shown to mediate proteinprotein interactions, we postulated that the death domain of DAP-kinase (DD-DAPk), which contains all the functionally conserved regions (CaM mutant of DAP-kinase it reduced significantly cell death induced by DAP-kinase overexpression (Figure 2 d). In contrast, a mutant death domain (DD/L1337N), which carries a mutation equivalent to the known inactivating lpr mutation in the Fas receptor death domain, failed to inhibit cell death induced by DAP-kinase (Figure 2 d). Thus, the DD-DAPk can be used as a dominant negative fragment that blocks the action of the full-length protein, and, therefore, might be suitable for checking the involvement of DAP-kinase in cell death induced by TNF-
or Fas.
The Death Domain of DAP-kinase Can Protect from TNF- and Fas-induced Cell Death
TNF- and Fas-induced cell death was triggered by overexpressing the corresponding receptors in 293 and HeLa cervical carcinoma cells in transient transfection assays. Both cell lines express the endogenous DAP-kinase protein (Figure 3 a). The receptor cDNAs were cotransfected with the vector expressing the GFP protein. Transfection of p55 TNF-R into 293 or HeLa cells resulted in massive cell death by 24 h (Figure 3 c). We also confirmed that the observed cell death in these transient assays was caused by p55 TNF-R activation, by using the previously described dominant negative mutant of FADD/MORT-1 (called DN-MORT or DN-FADD) that is deleted of its death effector domain (
by preventing the endogenous adaptor protein from forming the signaling complexes at the receptor level (
The death assays showed that in 293 and HeLa cells, the DD-DAPk inhibited TNF-induced cell death by ~50% (Figure 3 c). To further assess the specificity of DD-DAPk inhibitory effect toward DAP-kinase, another cell line, MCF7 breast carcinoma, was used in these transfections. MCF7 cells were chosen since they do not express the endogenous DAP-kinase gene (Figure 3 a), consistent with previous data (
We used the same method of cotransfections to investigate the involvement of DAP-kinase in Fas-induced cell death. For this purpose, we used a chimeric receptor composed of the extracellular portion of TNF-R and the intracellular portion of Fas, which was known to be more effective in inducing cell death by self oligomerization than wild-type Fas receptors (
Finally, in another type of assay, apoptosis was induced by adding an external ligand (instead of receptor overexpression). To this aim, HeLa cells were treated with a combination of TNF- and cycloheximide, which induced apoptosis in these cells, at 24 h after transfection with DD-DAPk, DN-MORT, or the luciferase control. DD-DAPk reduced apoptotic cell death by 60% (Figure 3 e) and DN-MORT was more potent in reducing cell death. This assay demonstrated again that DAP-kinase participates in TNF-induced cell death. It also indicated that its function is not dependent on de novo protein synthesis. Altogether, these transient transfection assays provide additional support for DAP-kinase being a positive mediator in both TNF-
and Fas-induced cell death.
Functional Position of DAP-kinase with Respect to the DISC formation, bcl-2, and Terminator Caspases
To place DAP-kinase along the apoptotic pathways of TNF- and Fas, several known components of the system were assayed in cotransfection assays. First the 293 cells were transfected with a vector encoding the adaptor proteinFADD/MORT1 that recruits proteins such as caspase-8 to the vicinity of TNF-R and Fas. In agreement with results from other laboratories (
-C360S in
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The reciprocal approach was to test whether DN-MORT1, DN-Caspase-8, or DD-DAPk could rescue death imposed by CaM-DAPk overexpression. Both DN-MORT1 and DN-Caspase-8 did not reduce cell death induced by activated DAP-kinase, whereas DD-DAPk served as a positive control to the experiment (Figure 4 b). Thus, DAP-kinase functions downstream to FADD/MORT1 as well as to caspase-8, which are both recruited to the DISC formed at the cytoplasmic portions of the TNF-R or Fas. The inability of DN-MORT1, which is composed of the death domain of MORT1, to protect from DAP-kinaseinduced death can serve as a control for specificity of overexpressed death domains. To address pathways involving mitochondria, the ability of bcl-2 to rescue death by activated DAP-kinase was tested. It was found that bcl-2 reduced death from 86 to 32% (Figure 4 c), suggesting some functional interaction with mitochondrial-based events.
Finally, the possibility that other caspases may function as downstream mediators of DAP-kinase was tested by using two natural caspase inhibitors: CrmA, which is encoded by the cowpox virus genome; and p35, which is a baculovirus encoded protein (for review see or Fas-induced cell death (
CaM mutant of DAP-kinase with either one of these inhibitors decreased significantly cell death (32% for CrmA, 26% for p35 compared with 86% without inhibitors) (Figure 4 c). These results functionally place some members of the caspase family, probably other than caspase-8, downstream to DAP-kinase, along pathway(s) leading to cell death.
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Discussion |
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The strategy of functional gene cloning, used for the rescue of DAP-kinase, was designed with the intention of isolating genes that lie downstream to the IFN- early JAK/STAT signaling and, therefore, probably common to various apoptotic systems. This was achieved by introducing an IFN-stimulated responsive element into the transcription cassette that drives the antisense RNA expression. The latter step in the construction of the antisense cDNA library guaranteed that the selection will depend on intact JAK/STAT signaling from IFN-
receptors, thus, increasing the probability of hitting genes that lie further downstream (
and Fas-induced cell death, strongly supports the notion that a central death effector gene has been rescued, upon which various types of receptor signaling cascades eventually converge.
The involvement of DAP-kinase in TNF- and Fas-induced cell death is supported here by several independent lines of evidence. First, expression of the antisense RNA fragment of DAP-kinase protected HeLa cells from Fas-induced cell death (Figure 1). Second, the death domain of DAP-kinase (DD-DAPk) protected 293 human embryonic kidney cells as well as HeLa cells from apoptosis triggered by overexpression of p55-TNF-R and Fas death receptors or by the TNF-
ligand (Figure 3). Also, the previous data that restoration of DAP-kinase expression in D122 Lewis lung carcinoma cells, which do not express endogenous DAP-kinase, accelerated significantly the appearance of the apoptotic phenotype in response to TNF-
support this line (
The assays involving transfections with DD-DAPk support for the first time the notion that this region of the protein displays dominant negative features. Moreover, deletion of this region impaired the ability of DAP-kinase to induce cell death. In the yeast two-hybrid system, the death domain of DAP-kinase did not interact with itself (Feinstein, E., and A. Kimchi, unpublished data), suggesting that the death domain does not mediate homodimerization of DAP-kinase. Therefore, this domain could potentially mediate interactions with other proteins that are critical for the function of DAP-kinase in cell death, the nature of which is under current investigation.
The protection conveyed by the death domain of DAP-kinase was always partial (~50%) and remained so even when the amount of DNA used for the transient transfections were significantly increased (data not shown). The effects of DD-DAPk were, therefore, clearly milder than the effects of the DN-MORT obtained in the same assays. This is not surprising considering the different functional position along the death pathways of the two proteins. FADD/MORT-1 acts in the proximity of Fas and TNF- receptors and, therefore, DN-MORT mutant blocks early receptor-generated events, such as the recruitment of caspase-8 to the receptor complex. As a consequence, it efficiently prevents most intracellular responses. DAP-kinase, in contrast, is not part of the DISC, but rather functions further downstream. The downstream position with respect to the DISC was based on two lines of evidence. One showed that DD-DAPk protected from FADD/MORT1induced cell death (Figure 4 a). The other illustrated that the death-promoting effect of the
CaM gain-of-function mutant of DAP-kinase was clearly resistant to the dominant negative components of the DISC (e.g., DN-MORT and DN-Caspase 8) (Figure 4 b). Also, when assayed by the yeast two-hybrid system, the death domain of DAP-kinase did not bind to the death domain of the Fas receptor (Feinstein, E., and A. Kimchi, unpublished data). Beyond the receptor complex, the death pathways may diverge to several branches, and the partial protections conveyed either by antisense DAP-kinase RNA (Figure 1) or by DD-DAPk (Figure 3) imply that DAP-kinase functions along some but not all these branches. Also, the finding that DAP-kinase negative cell lines, such as MCF7 or D122 (
is consistent with the existence of DAP-kinasedependent and independent branches.
Virally produced inhibitors of caspases were used to show that members of the cysteine protease family are involved in DAP-kinaseinduced cell death. Among the two inhibitors that were used, crmA is believed to be more specific to the subfamily of the interleukin 1ßconverting enzyme (ICE)-like proteases, whereas p35 has a wider spectrum (for review see CaM-DAPkinduced cell death to a similar extent. These results suggest that ICE-like proteases mediate the effect of DAP-kinase. The caspase family in general and the ICE-like subgroup in particular include several proteases acting at different positions along death pathways. Therefore, it is hard to speculate, at the present time, about the specific proteases that mediate the effect of DAP-kinase and their defined substrates.
It is well established that the fast track of apoptosis (comprising a direct cascade of caspase activation) is not an exclusive pathway in the Fas-induced signaling (CaM-DAPk mutant, one possibility is that DAP-kinase may be involved in one of these mitochondrial pathways. Alternatively, since DAP-kinase associates with the actin microfilament system (
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Acknowledgements |
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We thank D. Wallach (Weizmann Institute of Science), M. Tewari (Temple University, Philadelphia, PA), R. Stein (Tel-Aviv University), C. Kahana (Weizmann Institute of Science), and S.J. Korsmeyer (Washington University, Seattle, WA) for providing the different plasmids and P. Krammer (DKFZ) for antiAPO-1 agonistic antibodies.
This work was supported by the Israel Foundation, which is administered by the Israel Academy of Science and Humanities, and by QBI Enterprises. A. Kimchi is the incumbent of the Helena Rubinstein Chair of Cancer Research.
Submitted: March 19, 1999; Revised: May 28, 1999; Accepted: June 4, 1999.
1.used in this paper: CAM, deletion of calmodulin regulatory domain; CARD, caspase-recruiting domain; DAP, death-associated protein; DD, death domain; DISC, death-inducing signaling complex; DN-MORT/FADD, dominant negative mutant of MORT/FADD; GFP, green fluorescent protein; ICE, interleukin 1ßconverting enzyme; IFN-
, interferon-
; PARP,; poly (ADPribose) polymerase; TNF, tumor necrosis factor; TNF-R, TNF-
receptor
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