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 prot ein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fr agments that could protect cells from apoptosis by acting in a dominant-neg ative 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 selec tion, and those in the sense orientation were examined further in a seconda ry screen for their ability to protect cells from DAP-kinase-dependent tumo r necrosis factor-cy-induced apoptosis, We isolated four biologically activ e peptides that mapped to the ankyrin repeats, the "linker" region, the dea th 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 co nstitutes a distinct substructure. The last fragment, spanning the C-termin al serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, wh ereas removal of this region from the complete protein caused enhancement o f the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted fu nctionally important regions in the protein and revealed an additional leve l of regulation of DAP-kinase apoptotic function that does not affect the c atalytic activity.