cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity

1994). These findings led to the concept that exposure of Ofer Cohen, Elena Feinstein and cell cultures to a specific cytokine may provide a wellAdi Kimchi1 controlled in vitro system for the isolation of genes that Department of Molecular Genetics, The Weizmann Institute of mediate PCD. Yet, the great potential that resides in this Science, Rehovot 76100, Israel approach began to be exploited only recently. One line of 1Corresponding author research was directed towards cloning genes that code for proteins that interact with the intracellular domain of O.Cohen and E.Feinstein contributed equally to this work death-inducing receptors such as p55 TNF receptor 1 and the Fas/APO-1 receptor. This approach employed the yeast DAP-kinase was initially identified as a gene whose two-hybrid selection system for the rescue of proteins that anti-sense-mediated reduced expression protected interact directly with the death domain that constitutes the HeLa cells from interferon-γ-induced programmed cell functional intracellular regions of these receptors. It led death. It was cloned in our laboratory by a functional to the isolation of a few novel death-domain-containing gene selection approach. According to its amino acid proteins, FADD/MORT-1, RIP and TRADD, that function sequence, this 160 kDa protein was predicted to be a as critical mediators of PCD (Boldinet al., 1995; novel type of calmodulin-regulated serine/threonine Chinnalyanet al., 1995; Heuet al., 1995; Stanger et al., kinase which carries ankyrin repeats and the death 1995; Tewari and Dixit, 1996). A second approach, domain. In this work we have shown that the kinase was pioneered in our laboratory, employed a functional selecautophosphorylated and capable of phosphorylating an tion strategy of gene cloning (Deiss and Kimchi, 1991). exogenous substrate in a Ca 2F/calmodulin-dependent Using this approach, several novel genes, named DAP manner. We proved that calmodulin binds directly to genes (for Death Associated Proteins), that function as the recombinant kinase, and generated a constitutively potential positive mediators of IFNγ-induced cell death, active kinase mutant by the deletion of the calmodulinwere isolated and initially characterized (Deiss et al., regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is 1995; Kissil et al., 1995). Other laboratories adapted localized to the cytoskeleton, in association with the similar functional selection strategies and candidate genes microfilament system, and mapped a region within that mediate cell death induced by IL-3 deprivation or by the protein which is responsible for binding to the T-cell receptor activation were cloned including Requiem cytoskeleton. Several assays attributed a cell death (Gabiget al., 1994), ALG-2 and ALG-3 (Vitoet al., 1996). function to the gene. Ectopic expression of wild-type Our method, called Technical Knock Out (TKO), was DAP-kinase induced the death of target cells, and the based on random inactivation of genes via introduction of killing property depended strictly on the status of the anti-sense cDNA expression libraries, prepared from a intrinsic kinase activity. Conversely, a catalytically mixture of non-treated and IFNγ-treated cells. The genes inactive mutant that carried a lysine to alanine substituof interest were selected and cloned by virtue of the tion within the kinase domain, displayed dominantdefined phenotypic change—reduced susceptibility to the negative features and protected cells from interferoncytokine-induced cell death—which resulted from their γ-induced cell death. DAP-kinase is therefore a novel inactivation. HeLa cells, transfected with Epstein–Barr cytoskeletal-associated cell death serine/threonine virus (EBV)-based vectors carrying anti-sense cDNA kinase whose activation by Ca2F/calmodulin may be libraries, were subjected to positive selection of cells that linked to the biochemical mechanism underlying the survived in the continuous presence of IFNγ. The rescued cytoskeletal alterations that occur during cell death. plasmids that were positively scored in a second round of