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DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E, Kimchi A - J. Cell Biol. (1999)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
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)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/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-alpha, Fas, and FADD/MORT1-induced 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.

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Expression of DD-DAPk protects from TNF-α– and Fas-induced cell death. (a) Expression of endogenous DAP-kinase in cell lines. Western blotting analysis of extracts of indicated cells, using anti–DAP-kinase antibodies. (b) Expression of recombinant DD-DAPk. Extracts from cells transfected with DD-DAPk construct were immunoprecipitated by anti-Flag antibodies and analyzed by Western blotting using anti-Flag. (c) Transient transfection of 293, HeLa, or MCF7 cells with vectors encoding p55-TNF-R, GFP, and either luciferase (Luc), death domain of DAP-kinase (DD-DAPk), or dominant negative mutant of FADD/MORT1 (DN-MORT). The percentage of apoptotic cells was calculated as described in Materials and Methods. (d) Same as c, except that p55/Fas chimera was used instead of p55-TNF-R. (e) Transient transfection of HeLa cells with vectors encoding GFP and either luciferase, DD-DAPk, or DN-MORT. Cells were treated 24 h after transfection with a combination of TNF-α and cycloheximide. The number of apoptotic cells was scored under fluorescent microscopy 3 h after treatment.
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Figure 3: Expression of DD-DAPk protects from TNF-α– and Fas-induced cell death. (a) Expression of endogenous DAP-kinase in cell lines. Western blotting analysis of extracts of indicated cells, using anti–DAP-kinase antibodies. (b) Expression of recombinant DD-DAPk. Extracts from cells transfected with DD-DAPk construct were immunoprecipitated by anti-Flag antibodies and analyzed by Western blotting using anti-Flag. (c) Transient transfection of 293, HeLa, or MCF7 cells with vectors encoding p55-TNF-R, GFP, and either luciferase (Luc), death domain of DAP-kinase (DD-DAPk), or dominant negative mutant of FADD/MORT1 (DN-MORT). The percentage of apoptotic cells was calculated as described in Materials and Methods. (d) Same as c, except that p55/Fas chimera was used instead of p55-TNF-R. (e) Transient transfection of HeLa cells with vectors encoding GFP and either luciferase, DD-DAPk, or DN-MORT. Cells were treated 24 h after transfection with a combination of TNF-α and cycloheximide. The number of apoptotic cells was scored under fluorescent microscopy 3 h after treatment.

Mentions: 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 (Gossen and Bujard 1992) was used for transient transfections because of its high transfectability and the fact that it undergoes apoptotic death in response to TNF-α 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 calcium–phosphate method. For each well, we used a mixture containing 0.5 μg of cell death–inducing plasmid (either p55-TNF-R, p55/Fas chimera, MORT1, or ΔCaM 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 Fig. 3 e, cells were transfected solely with the DD-DAPk or DN-MORT and treated 24 h after transfection with human recombinant TNF-α (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 (Deiss et al. 1995). Human recombinant IFN-γ (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-γ (to increase Fas expression) and exposed to 50 ng/ml of anti–Fas/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 (ADP–ribose) 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.


DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E, Kimchi A - J. Cell Biol. (1999)

Expression of DD-DAPk protects from TNF-α– and Fas-induced cell death. (a) Expression of endogenous DAP-kinase in cell lines. Western blotting analysis of extracts of indicated cells, using anti–DAP-kinase antibodies. (b) Expression of recombinant DD-DAPk. Extracts from cells transfected with DD-DAPk construct were immunoprecipitated by anti-Flag antibodies and analyzed by Western blotting using anti-Flag. (c) Transient transfection of 293, HeLa, or MCF7 cells with vectors encoding p55-TNF-R, GFP, and either luciferase (Luc), death domain of DAP-kinase (DD-DAPk), or dominant negative mutant of FADD/MORT1 (DN-MORT). The percentage of apoptotic cells was calculated as described in Materials and Methods. (d) Same as c, except that p55/Fas chimera was used instead of p55-TNF-R. (e) Transient transfection of HeLa cells with vectors encoding GFP and either luciferase, DD-DAPk, or DN-MORT. Cells were treated 24 h after transfection with a combination of TNF-α and cycloheximide. The number of apoptotic cells was scored under fluorescent microscopy 3 h after treatment.
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Related In: Results  -  Collection

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Figure 3: Expression of DD-DAPk protects from TNF-α– and Fas-induced cell death. (a) Expression of endogenous DAP-kinase in cell lines. Western blotting analysis of extracts of indicated cells, using anti–DAP-kinase antibodies. (b) Expression of recombinant DD-DAPk. Extracts from cells transfected with DD-DAPk construct were immunoprecipitated by anti-Flag antibodies and analyzed by Western blotting using anti-Flag. (c) Transient transfection of 293, HeLa, or MCF7 cells with vectors encoding p55-TNF-R, GFP, and either luciferase (Luc), death domain of DAP-kinase (DD-DAPk), or dominant negative mutant of FADD/MORT1 (DN-MORT). The percentage of apoptotic cells was calculated as described in Materials and Methods. (d) Same as c, except that p55/Fas chimera was used instead of p55-TNF-R. (e) Transient transfection of HeLa cells with vectors encoding GFP and either luciferase, DD-DAPk, or DN-MORT. Cells were treated 24 h after transfection with a combination of TNF-α and cycloheximide. The number of apoptotic cells was scored under fluorescent microscopy 3 h after treatment.
Mentions: 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 (Gossen and Bujard 1992) was used for transient transfections because of its high transfectability and the fact that it undergoes apoptotic death in response to TNF-α 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 calcium–phosphate method. For each well, we used a mixture containing 0.5 μg of cell death–inducing plasmid (either p55-TNF-R, p55/Fas chimera, MORT1, or ΔCaM 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 Fig. 3 e, cells were transfected solely with the DD-DAPk or DN-MORT and treated 24 h after transfection with human recombinant TNF-α (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 (Deiss et al. 1995). Human recombinant IFN-γ (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-γ (to increase Fas expression) and exposed to 50 ng/ml of anti–Fas/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 (ADP–ribose) 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
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)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/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-alpha, Fas, and FADD/MORT1-induced 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.

Show MeSH
Related in: MedlinePlus