DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.

DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death.

A functional approach to gene cloning was applied to HeLa cells in an attempt to isolate cDNA fragments which convey resistance to gamma interferon (IFN-gamma)-induced programmed cell death. One of the rescued cDNAs, described in this work, was a fragment of a novel gene, named DAP-5. Analysis of a DAP-5 full-length cDNA clone revealed that it codes for a 97-kDa protein that is highly homologous to eukaryotic translation initiation factor 4G (eIF4G, also known as p220). According to its deduced amino acid sequence, this novel protein lacks the N-terminal region of eIF4G responsible for association with the cap binding protein eIF4E. The N-terminal part of DAP-5 has 39% identity and 63% similarity to the central region of mammalian p220. Its C-terminal part is less homologous to the corresponding region of p220, suggesting that it may possess unique functional properties. The rescued DAP-5 cDNA fragment which conveyed resistance to IFN-gamma-induced cell death was expressed from the vector in the sense orientation. Intriguingly, it comprised part of the coding region which corresponds to the less conserved C-terminal part of DAP-5 and directed the synthesis of a 28-kDa miniprotein. The miniprotein exerted a dual effect on HeLa cells. Low levels of expression protected the cells from IFN-gamma-induced programmed cell death, while high levels of expression were not compatible with continuous cell growth. The relevance of DAP-5 protein to possible changes in a cell's translational machinery during programmed cell death and growth arrest is discussed.

Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G0/G1-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical G0/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G0/G1-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor beta, reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced G0/G1-specific arrest requires at least these two receptor-generated events.

HeLa cell poly(A)- mRNA codes for a subset of poly(A)+ mRNA-directed proteins with an actin as a major product.

Poly(A)+ and poly(A)- mRNA from HeLa cells were separated and translated in heterologous messenger-dependent protein synthesizing systems. Two-dimensional electrophoretic analysis revealed three classes of polypeptides. At the level of detectability in the electropherograms, a small number (about 10) of proteins were detected only among the poly(A)- mRNA products, a larger number (about 40) were produced by both poly(A)- and poly(A)+ mRNA, and a large number of polypeptides were found exclusively in the poly(A)+ mRNA products. The major product of both poly(A)+ and poly(A)- mRNA was shown to be the beta form of actin.Previous cross hybridization measurements suggested little homology between poly(A)+ and poly(A)- mRNA populations. In view of the apparent identity of many poly(A)- products with those of poly(A)+, the homology between poly(A)+ and poly(A)- mRNA sequences was examined in greater detail. cDNA complementary to only the most abundant poly(A)+ message sequences was prepared. About 10% of this cDNA hybridized to abundant sequences in the poly(A)- fraction. This corresponded to only 2% of the total mass of poly(A)+ mRNA and accounted for the failure to detect cross hybridization in previous experiments. Thus, a small number of poly(A)+ sequences appear to be present in relatively high concentration in poly(A)- mRNA as evidenced by both the translation products and the cross hybridization results.

Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death.

Programmed cell death is often triggered by the interaction of some cytokines with their cell surface receptors. Here, we report that gamma interferon (IFN-gamma) induced in HeLa cells a type of cell death that had cytological characteristics of programmed cell death. In this system we have identified two novel genes whose expression was indispensable for the execution of this type of cell death. The rescue was based on positive growth selection of cells after transfection with antisense cDNA expression libraries. The antisense RNA-mediated inactivation of the two novel genes protected the cells from the IFN-gamma-induced cell death but not from the cytostatic effects of the cytokine or from a necrotic type of cell death. One of those genes (DAP-1) is expressed as a single 2.4-kb mRNA that codes for a basic, proline-rich, 15-kD protein. The second is transcribed into a single 6.3-kb mRNA and codes for a unique 160-kD calmodulin-dependent serine/threonine kinase (DAP kinase) that carries eight ankyrin repeats. The expression levels of the two DAP proteins were selectively reduced by the corresponding antisense RNAs. Altogether, it is suggested that these two novel genes are candidates for positive mediators of programmed cell death that is induced by IFN-gamma.

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.

Blocks in elongation and initiation of protein synthesis induced by interferon treatment in mouse L cells.

Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell-free extracts from interferon-treated mouse L cells, due to a "deficiency" in some specific tRNA species. A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met-tRNA(FMET) binding is inhibited. The block in initiation appears to be secondary to the block in elongation.

An interferon-induced phosphodiesterase degrading (2'-5') oligoisoadenylate and the C-C-A terminus of tRNA.

A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.

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 protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative 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 selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-alpha-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the "linker" region, the death 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 constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

The beta2-microglobulin mRNA in human Daudi cells has a mutated initiation codon but is still inducible by interferon.

The human Burkitt lymphoma cell line Daudi does not synthesize beta2-microglobulin (beta2m) and lacks the cell surface histocompatibility antigens. The cells, however, contain RNA hybridizing to a cloned human beta2m cDNA probe. cDNA from this Daudi beta2m RNA, was cloned and sequenced. By comparison with cDNA prepared from Ramos cells, which synthesized microglobulin, we determined the sequence of the 20 amino acid long leader peptide of pre-beta2m and show that in Daudi cells the initiator ATG has been mutated to ATC. Although Daudi beta2m RNA cannot be translated, interferon induces the beta2m RNA in Daudi cells as well as in normal human cells.