Death associated proteins (DAPs): from gene identification to the analysisof their apoptotic and tumor suppressive functions

The process of apoptosis (programmed cell death) has become the subject of intensive and extensive research over the past few years. Various approache s are being used to identify and study genes which function as positive med iators of apoptosis, Here, we address a novel approach of gene cloning aime d at isolating intracellular death promoting genes by utilizing a functiona l screen. This method, called TKO, was based on transfection of cells with an anti-sense cDNA library, followed by the selection of transfectants whic h survived in the continuous presence of a killing cytokine - interferon-ga mma. It led to the identification of five novel apoptotic genes and to the finding that a known protease-cathepsin D, is actively recruited to the dea th process. The five novel apoptotic genes (named DAP genes for: Death Asso ciated Proteins) code for proteins which display a diverse spectrum of bioc hemical activities. The List comprises a novel type of calcium/calmodulin-r egulated kinase which carries ankyrin repeats and a death domain (DAP-kinas e), a nucleotide-binding protein (DAP-3), a small proline-rich cytoplasmic protein (DAP-1), and a novel homolog of the eIF4G translation initiation fa ctor (DAP-5), Extensive studies proved that these genes are critical for me diating cell death initiated by interferon-gamma, and in some of the tested cases also cell death induced by Fas/APO-1, TNF-alpha, and a detachment fr om extracellular matrix. Moreover, one of these genes, DAP-kinase, was rece ntly found to display strong tumor suppressive activities, coupling the con trol of apoptosis to metastasis. The advantage of functional approaches of gene cloning is that they select the relevant rate limiting genes along the death pathways in a complete unb iased manner. As a consequence, novel targets and unpredicted mechanisms em erged, A few examples illustrating this important point will be discussed, One relates to the calcium/calmodulin-dependent DAP-kinase, which is locali zed to the actin microfilaments. It was found that the correct localization of DAP-kinase to the microfilament network was critical for the execution of the apoptotic process, and more specifically for the disruption of the s tress fibers-a typical hallmark of apoptosis, Another important breakthroug h step in our understanding of apoptotic processes relates to the identific ation and analysis of the DAP-5 gene. The structure/ function features of t his novel translation regulator resemble the proteolytically cleaved eIF4G which appears in cells upon infection with some RNA viruses and which direc ts cap-independent translation, Thus, the rescue of DAP-5 highlighted the i mportance of regulation of protein translation in certain apoptotic systems , Finally, the isolation of cathespin D by our method suggests that lysosom al proteases are recruited during apoptosis, in addition to the well known caspase family of proteases, and that a unique pattern of regulation affect ing the processing of this protease takes place. The major challenge now is to analyse how these diverse DAP gene activities constitute biochemical pa thway(s) leading to programmed cell death, and what is their functional pos ition with respect to other known positive mediators and suppressors of apo ptosis such as the Bcl2 and caspase family members.