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PNAS 97 (4): 1572-1577

Copyright © 2000 by the National Academy of Sciences.


A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Tal Raveh*, Hanna Berissi*, Miriam Eisenstein{dagger}, Taly Spivak*, and Adi Kimchi*,{ddagger}

*Departments of Molecular Genetics and {dagger}Chemical Services, Weizmann Institute of Science, Rehovot 76100, Israel

Accepted for publication November 30, 1999.

Received for publication October 3, 1999.

Abstract: Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-{gamma} to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the "linker" region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

{ddagger} To whom reprint requests should be addressed. E-mail: lvkimchi{at}

Communicated by Leo Sachs, Weizmann Institute of Science, Rehovot, Israel

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.020519497.

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