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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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The death domain of DAP-kinase is important for its function in apoptosis. (a) Transient transfections of 293 cells with ΔCaM mutant, ΔCaM/ΔDD mutant, or with the DD-DAPk. Photographs were taken 24 h after transfection under fluorescent light microscope to visualize GFP positive cells. (b) Expression of ΔCaM and ΔCaM/ΔDD mutants in the transiently transfected 293 cells. Lane 1, expression of ΔCaM-DAPk; lane 2, expression of ΔCaM/ΔDD-DAPk; and lane 3, endogenous DAPk in cells transfected with a nonrelevant vector. Western blotting analysis was done with anti–DAP-kinase antibodies. (c) Schematic presentation of DAP-kinase mutant proteins used in these experiments. Kinase, kinase domain; CaM, calmodulin binding and regulatory domain; ankyrin, ankyrin repeats; and DD, death domain. Asterisks delineate the region that by deletion mapping was shown to be responsible for cytoskeletal binding. (d) Transfections as shown in a, with the indicated constructs or double transfections of ΔCaM mutant with the indicated constructs. The percentage of apoptotic cells was calculated as described in Materials and Methods.
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Figure 2: The death domain of DAP-kinase is important for its function in apoptosis. (a) Transient transfections of 293 cells with ΔCaM mutant, ΔCaM/ΔDD mutant, or with the DD-DAPk. Photographs were taken 24 h after transfection under fluorescent light microscope to visualize GFP positive cells. (b) Expression of ΔCaM and ΔCaM/ΔDD mutants in the transiently transfected 293 cells. Lane 1, expression of ΔCaM-DAPk; lane 2, expression of ΔCaM/ΔDD-DAPk; and lane 3, endogenous DAPk in cells transfected with a nonrelevant vector. Western blotting analysis was done with anti–DAP-kinase antibodies. (c) Schematic presentation of DAP-kinase mutant proteins used in these experiments. Kinase, kinase domain; CaM, calmodulin binding and regulatory domain; ankyrin, ankyrin repeats; and DD, death domain. Asterisks delineate the region that by deletion mapping was shown to be responsible for cytoskeletal binding. (d) Transfections as shown in a, with the indicated constructs or double transfections of ΔCaM mutant with the indicated constructs. The percentage of apoptotic cells was calculated as described in Materials and Methods.

Mentions: 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 (ΔCaM) 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 (Cohen et al. 1997). To quantitate the number of apoptotic cells, we cotransfected the ΔCaM 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 ΔCaM mutant of DAP-kinase resulted in massive apoptotic cell death (Fig. 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 ΔCaM mutant deleted of its death domain (Fig. 2 c, ΔCaM/ΔDD), apoptotic cells were much less abundant (23% apoptotic cells compared with 68% in ΔCaM transfections; see Fig. 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 (Fig. 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; Fig. 2 d). Therefore, it is concluded that the death domain contributes to the death-inducing function of DAP-kinase.


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)

The death domain of DAP-kinase is important for its function in apoptosis. (a) Transient transfections of 293 cells with ΔCaM mutant, ΔCaM/ΔDD mutant, or with the DD-DAPk. Photographs were taken 24 h after transfection under fluorescent light microscope to visualize GFP positive cells. (b) Expression of ΔCaM and ΔCaM/ΔDD mutants in the transiently transfected 293 cells. Lane 1, expression of ΔCaM-DAPk; lane 2, expression of ΔCaM/ΔDD-DAPk; and lane 3, endogenous DAPk in cells transfected with a nonrelevant vector. Western blotting analysis was done with anti–DAP-kinase antibodies. (c) Schematic presentation of DAP-kinase mutant proteins used in these experiments. Kinase, kinase domain; CaM, calmodulin binding and regulatory domain; ankyrin, ankyrin repeats; and DD, death domain. Asterisks delineate the region that by deletion mapping was shown to be responsible for cytoskeletal binding. (d) Transfections as shown in a, with the indicated constructs or double transfections of ΔCaM mutant with the indicated constructs. The percentage of apoptotic cells was calculated as described in Materials and Methods.
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Figure 2: The death domain of DAP-kinase is important for its function in apoptosis. (a) Transient transfections of 293 cells with ΔCaM mutant, ΔCaM/ΔDD mutant, or with the DD-DAPk. Photographs were taken 24 h after transfection under fluorescent light microscope to visualize GFP positive cells. (b) Expression of ΔCaM and ΔCaM/ΔDD mutants in the transiently transfected 293 cells. Lane 1, expression of ΔCaM-DAPk; lane 2, expression of ΔCaM/ΔDD-DAPk; and lane 3, endogenous DAPk in cells transfected with a nonrelevant vector. Western blotting analysis was done with anti–DAP-kinase antibodies. (c) Schematic presentation of DAP-kinase mutant proteins used in these experiments. Kinase, kinase domain; CaM, calmodulin binding and regulatory domain; ankyrin, ankyrin repeats; and DD, death domain. Asterisks delineate the region that by deletion mapping was shown to be responsible for cytoskeletal binding. (d) Transfections as shown in a, with the indicated constructs or double transfections of ΔCaM mutant with the indicated constructs. The percentage of apoptotic cells was calculated as described in Materials and Methods.
Mentions: 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 (ΔCaM) 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 (Cohen et al. 1997). To quantitate the number of apoptotic cells, we cotransfected the ΔCaM 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 ΔCaM mutant of DAP-kinase resulted in massive apoptotic cell death (Fig. 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 ΔCaM mutant deleted of its death domain (Fig. 2 c, ΔCaM/ΔDD), apoptotic cells were much less abundant (23% apoptotic cells compared with 68% in ΔCaM transfections; see Fig. 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 (Fig. 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; Fig. 2 d). Therefore, it is concluded that the death domain contributes to the death-inducing function of DAP-kinase.

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