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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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Related in: MedlinePlus

Functional positioning of DAP-kinase along TNF/Fas cell death pathways. Schematic representation of the results is depicted above each bar graph. (a) Transient transfections of 293 cells with vectors encoding FADD/MORT1 together with vectors encoding the indicated proteins. GFP plasmid is present in each of the transfections. (b and c) Transfections as in a, except that the ΔCaM mutant of DAP-kinase was used instead of FADD/MORT1 together with vectors encoding the indicated proteins.
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Figure 4: Functional positioning of DAP-kinase along TNF/Fas cell death pathways. Schematic representation of the results is depicted above each bar graph. (a) Transient transfections of 293 cells with vectors encoding FADD/MORT1 together with vectors encoding the indicated proteins. GFP plasmid is present in each of the transfections. (b and c) Transfections as in a, except that the ΔCaM mutant of DAP-kinase was used instead of FADD/MORT1 together with vectors encoding the indicated proteins.

Mentions: To place DAP-kinase along the apoptotic pathways of TNF-α 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 protein–FADD/MORT1 that recruits proteins such as caspase-8 to the vicinity of TNF-R and Fas. In agreement with results from other laboratories (Boldin et al. 1995; Chinnaiyan et al. 1995), overexpression of FADD/MORT1 efficiently induced cell death. Cotransfection of FADD/MORT1 with its previously mentioned dominant negative mutant DN-MORT1, significantly reduced cell death (Fig. 4 a). As expected, a dominant negative mutant of caspase-8 (DN-Caspase-8), in which the cysteine in the active site was substituted for serine (MACHα-C360S in Boldin et al. 1996), reduced cell death induced by MORT1. Interestingly, cotransfection of DD-DAPk together with FADD/MORT1 reduced very similarly the number of apoptotic cells, thus, placing DAP-kinase downstream to MORT1.


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)

Functional positioning of DAP-kinase along TNF/Fas cell death pathways. Schematic representation of the results is depicted above each bar graph. (a) Transient transfections of 293 cells with vectors encoding FADD/MORT1 together with vectors encoding the indicated proteins. GFP plasmid is present in each of the transfections. (b and c) Transfections as in a, except that the ΔCaM mutant of DAP-kinase was used instead of FADD/MORT1 together with vectors encoding the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199731&req=5

Figure 4: Functional positioning of DAP-kinase along TNF/Fas cell death pathways. Schematic representation of the results is depicted above each bar graph. (a) Transient transfections of 293 cells with vectors encoding FADD/MORT1 together with vectors encoding the indicated proteins. GFP plasmid is present in each of the transfections. (b and c) Transfections as in a, except that the ΔCaM mutant of DAP-kinase was used instead of FADD/MORT1 together with vectors encoding the indicated proteins.
Mentions: To place DAP-kinase along the apoptotic pathways of TNF-α 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 protein–FADD/MORT1 that recruits proteins such as caspase-8 to the vicinity of TNF-R and Fas. In agreement with results from other laboratories (Boldin et al. 1995; Chinnaiyan et al. 1995), overexpression of FADD/MORT1 efficiently induced cell death. Cotransfection of FADD/MORT1 with its previously mentioned dominant negative mutant DN-MORT1, significantly reduced cell death (Fig. 4 a). As expected, a dominant negative mutant of caspase-8 (DN-Caspase-8), in which the cysteine in the active site was substituted for serine (MACHα-C360S in Boldin et al. 1996), reduced cell death induced by MORT1. Interestingly, cotransfection of DD-DAPk together with FADD/MORT1 reduced very similarly the number of apoptotic cells, thus, placing DAP-kinase downstream to MORT1.

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