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DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain
1Department 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.
Insights
Death-associated protein (DAP)-kinase is crucial for tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Its death domain is vital for this function, placing DAP-kinase centrally in apoptosis pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Death-associated protein (DAP)-kinase is a serine/threonine kinase regulated by calcium/calmodulin.
- DAP-kinase possesses ankyrin repeats, a death domain, and is localized to the cytoskeleton.
- Its role in programmed cell death (apoptosis) was previously uncharacterized.
Purpose of the Study:
- To investigate the involvement of DAP-kinase in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis.
- To elucidate the function of the death domain of DAP-kinase in apoptotic signaling.
- To determine the position of DAP-kinase within the apoptotic cascade.
Main Methods:
- Expression of DAP-kinase antisense RNA to inhibit its function.
- Deletion of the death domain to assess its role.
- Overexpression of a fragment containing the death domain as a dominant-negative mutant.
- Inhibition of DAP-kinase activity using Bcl-2, crmA, and p35.
Main Results:
- DAP-kinase is involved in TNF-alpha and Fas-induced apoptosis.
- Antisense RNA targeting DAP-kinase protected cells from apoptosis.
- Deletion of the death domain abolished DAP-kinase's apoptotic function.
- Overexpression of the death domain fragment inhibited TNF-alpha, Fas, and FADD/MORT1-induced cell death.
- DAP-kinase's apoptotic function is inhibited by Bcl-2 and caspase inhibitors (crmA, p35).
- DAP-kinase functions downstream of the receptor complex and upstream of caspases.
Conclusions:
- DAP-kinase plays a central role in apoptosis induced by TNF-alpha and Fas.
- The death domain of DAP-kinase is essential for its apoptotic activity.
- DAP-kinase acts as a key mediator in the apoptotic pathway, upstream of caspase activation.