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.
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-alpha-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.
Raveh
T
T
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Berissi
H
H
Eisenstein
M
M
Spivak
T
T
Kimchi
A
A
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D005820:Genetic Testing; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D020800:Receptor, Nerve Growth Factor / Q000737:chemistry; D016415:Sequence Alignment; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D010446:Peptide Fragments; D020800:Receptor, Nerve Growth Factor; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases