Apoptosis is characterized by a translation switch from capdependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and deathassociated protein 5 (DAP5) proteins (eIF4GI M-FAG͞p76 and DAP5͞ p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5͞p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG͞p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5͞p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.

The death-associated protein (DAP) kinase family includes three protein kinases, DAP kinase, DAP kinaserelated protein 1, and ZIP kinase, which display 80% amino acid identity within their catalytic domains and are functionally linked to common subcellular changes occurring during cell death, such as the process of membrane blebbing. Here we show physical and functional cross talk between DAP kinase and ZIP kinase. The two kinases display strong synergistic effects on cell death when coexpressed and physically bind each other via their catalytic domains. Furthermore, DAP kinase phosphorylates ZIP kinase at six specific sites within its extracatalytic C-terminal domain. ZIP kinase localizes to both the nucleus and the cytoplasm and fractionates as monomeric and trimeric forms. Significantly, modification of the DAP kinase phosphorylation sites influences both the localization and oligomerization status of ZIP kinase. A mutant ZIP kinase construct, in which the six serine/threonine residues were mutated to aspartic acid to mimic the phosphorylated state, was found predominantly in the cytoplasm as a trimer and possessed greater cell death-inducing potency. This suggests that DAP kinase and ZIP kinase function in a biochemical pathway in which DAP kinase activates the cellular function of ZIP kinase through phosphorylation, leading to amplification of death-promoting signals.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIa and subunit IV of DNA polymerase a is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F. Oncogene (2001) 20, 1379 ± 1387.

DAP5 (Death Associated Protein 5), also named p97 and NAT1, is a member of the eIF4G family that lacks the eIF4E binding site. Its function was linked to programmed cell death (PCD) based on the seminal finding that a fragment of DAP5/ p97 protein, which acted in a dominant-negative manner, protected against IFN-gamma (IFN-g)-induced cell death. Subsequently, it was found that DAP5 protein is activated during cell death by caspase cleavage, yielding a C-terminaltruncated protein of 86 kDa. The p86 form promotes internal ribosome entry site (IRES)-dependent translation of several mRNAs including Apaf-1, c-myc, XIAP, HIAP2 and DAP5 itself, which carries an IRES element in its 5 0 UTR. The activation of DAP5 IRES creates a positive feedback loop, which results in sustained translation of DAP5 protein during PCD under conditions of reduced cap-dependent translation. DAP5 deficiency prevents embryonic stem (ES) cell differentiation, suggesting a wider range of DAP5 mRNA targets and additional mechanisms that might activate the protein.