The strategy of functional gene cloning, used for the rescue of DAP-kinase, was designed with the intention of isolating genes that lie downstream to the IFN-γ early JAK/STAT signaling and, therefore, probably common to various apoptotic systems. This was achieved by introducing an IFN-stimulated responsive element into the transcription cassette that drives the antisense RNA expression. The latter step in the construction of the antisense cDNA library guaranteed that the selection will depend on intact JAK/STAT signaling from IFN-γ receptors, thus, increasing the probability of hitting genes that lie further downstream (Deiss and Kimchi 1991
; Deiss et al. 1995
). Indeed, the present finding that DAP-kinase mediates TNF-α– and Fas-induced cell death, strongly supports the notion that a central death effector gene has been rescued, upon which various types of receptor signaling cascades eventually converge.
The involvement of DAP-kinase in TNF-α– and Fas-induced cell death is supported here by several independent lines of evidence. First, expression of the antisense RNA fragment of DAP-kinase protected HeLa cells from Fas-induced cell death (). Second, the death domain of DAP-kinase (DD-DAPk) protected 293 human embryonic kidney cells as well as HeLa cells from apoptosis triggered by overexpression of p55-TNF-R and Fas death receptors or by the TNF-α ligand (). Also, the previous data that restoration of DAP-kinase expression in D122 Lewis lung carcinoma cells, which do not express endogenous DAP-kinase, accelerated significantly the appearance of the apoptotic phenotype in response to TNF-α support this line (Inbal et al. 1997
). Altogether, the data suggest that DAP-kinase functions as a positive mediator of these activated cytotoxic receptors belonging to the TNF receptor family.
The assays involving transfections with DD-DAPk support for the first time the notion that this region of the protein displays dominant negative features. Moreover, deletion of this region impaired the ability of DAP-kinase to induce cell death. In the yeast two-hybrid system, the death domain of DAP-kinase did not interact with itself (Feinstein, E., and A. Kimchi, unpublished data), suggesting that the death domain does not mediate homodimerization of DAP-kinase. Therefore, this domain could potentially mediate interactions with other proteins that are critical for the function of DAP-kinase in cell death, the nature of which is under current investigation.
The protection conveyed by the death domain of DAP-kinase was always partial (~50%) and remained so even when the amount of DNA used for the transient transfections were significantly increased (data not shown). The effects of DD-DAPk were, therefore, clearly milder than the effects of the DN-MORT obtained in the same assays. This is not surprising considering the different functional position along the death pathways of the two proteins. FADD/MORT-1 acts in the proximity of Fas and TNF-α receptors and, therefore, DN-MORT mutant blocks early receptor-generated events, such as the recruitment of caspase-8 to the receptor complex. As a consequence, it efficiently prevents most intracellular responses. DAP-kinase, in contrast, is not part of the DISC, but rather functions further downstream. The downstream position with respect to the DISC was based on two lines of evidence. One showed that DD-DAPk protected from FADD/MORT1–induced cell death ( a). The other illustrated that the death-promoting effect of the ΔCaM gain-of-function mutant of DAP-kinase was clearly resistant to the dominant negative components of the DISC (e.g., DN-MORT and DN-Caspase 8) ( b). Also, when assayed by the yeast two-hybrid system, the death domain of DAP-kinase did not bind to the death domain of the Fas receptor (Feinstein, E., and A. Kimchi, unpublished data). Beyond the receptor complex, the death pathways may diverge to several branches, and the partial protections conveyed either by antisense DAP-kinase RNA () or by DD-DAPk () imply that DAP-kinase functions along some but not all these branches. Also, the finding that DAP-kinase negative cell lines, such as MCF7 or D122 (Inbal et al. 1997
), can eventually be killed by TNF-α is consistent with the existence of DAP-kinase–dependent and –independent branches.
Virally produced inhibitors of caspases were used to show that members of the cysteine protease family are involved in DAP-kinase–induced cell death. Among the two inhibitors that were used, crmA is believed to be more specific to the subfamily of the interleukin 1β–converting enzyme (ICE)-like proteases, whereas p35 has a wider spectrum (for review see Villa et al. 1997
) In our experiments, both inhibitors suppressed ΔCaM-DAPk–induced cell death to a similar extent. These results suggest that ICE-like proteases mediate the effect of DAP-kinase. The caspase family in general and the ICE-like subgroup in particular include several proteases acting at different positions along death pathways. Therefore, it is hard to speculate, at the present time, about the specific proteases that mediate the effect of DAP-kinase and their defined substrates.
It is well established that the fast track of apoptosis (comprising a direct cascade of caspase activation) is not an exclusive pathway in the Fas-induced signaling (Scaffidi et al. 1998
). Mitochondrial-based events often provide a second route for caspase activation and cell death in these systems. In light of our findings that bcl-2 protected from cell death induced by the ΔCaM-DAPk mutant, one possibility is that DAP-kinase may be involved in one of these mitochondrial pathways. Alternatively, since DAP-kinase associates with the actin microfilament system (Cohen et al. 1997
), it might mediate signals converging into or emanating from the cytoskeleton. Should the protein substrates that are directly phosphorylated by DAP-kinase be identified, then the detailed mechanisms coupling the kinase to downstream targets may be deciphered. In any case, the multidomain structure of this enzyme predicts the formation of multiprotein complex around DAP-kinase. Taken together with its broad involvement in cell death induced by several different triggers, DAP-kinase appears to be a major player in apoptotic pathways.