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Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.

Beclin 1, an essential autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members. The dissociation of Beclin 1 from the Bcl-2 inhibitors is essential for its autophagic activity, and therefore is tightly controlled. We recently revealed a novel phosphorylation-based mechanism by which death-associated protein kinase (DAPk) regulates this process. We found that DAPk phosphorylates Beclin 1 on T119, a critical residue within its BH3 domain, and thus promotes Beclin 1 dissociation from Bcl-X(L) and autophagy induction. Here we report that T119 phosphorylation also reduces the interaction between Beclin 1 and Bcl-2, in line with the high degree of structural homology between the BH3 binding pockets of Bcl-2 and Bcl-X(L) proteins. Our results reveal a new phosphorylation-based mechanism that reduces the interaction of Beclin 1 with its inhibitors to activate the autophagic machinery.

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)-α 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-α, 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.

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.

Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein.

DRP-1 and ZIPk are two members of the Death Associated Protein Ser/Thr Kinase (DAP-kinase) family, which function in different settings of cell death including autophagy. DAP kinases are very similar in their catalytic domains but differ substantially in their extra-catalytic domains. This difference is crucial for the significantly different modes of regulation and function among DAP kinases. Here we report the identification of a novel alternatively spliced kinase isoform of the DRP-1 gene, termed DRP-1β. The alternative splicing event replaces the whole extra catalytic domain of DRP-1 with a single coding exon that is closely related to the sequence of the extra catalytic domain of ZIPk. As a consequence, DRP-1β lacks the calmodulin regulatory domain of DRP-1, and instead contains a leucine zipper-like motif similar to the protein binding region of ZIPk. Several functional assays proved that this new isoform retained the biochemical and cellular properties that are common to DRP-1 and ZIPk, including myosin light chain phosphorylation, and activation of membrane blebbing and autophagy. In addition, DRP-1β also acquired binding to the ATF4 transcription factor, a feature characteristic of ZIPk but not DRP-1. Thus, a splicing event of the DRP-1 produces a ZIPk like isoform. DRP-1β is highly conserved in evolution, present in all known vertebrate DRP-1 loci. We detected the corresponding mRNA and protein in embryonic mouse brains and in human embryonic stem cells thus confirming the in vivo utilization of this isoform. The discovery of module conservation within the DAPk family members illustrates a parsimonious way to increase the functional complexity within protein families. It also provides crucial data for modeling the expansion and evolution of DAP kinase proteins within vertebrates, suggesting that DRP-1 and ZIPk most likely evolved from their ancient ancestor gene DAPk by two gene duplication events that occurred close to the emergence of vertebrates.

Death-associated protein kinase (DAPk) is a Ser/Thr kinase whose activity is necessary for different cell death phenotypes. Although its contribution to cell death is well established, only a handful of direct substrates have been identified; these do not fully account for the multiple cellular effects of DAPk. To identify such substrates on a large scale, we developed an in vitro, unbiased, proteomics-based assay to search for novel DAPk substrates. Biochemical fractionation and mass spectrometric analysis were used to purify and identify several potential substrates from HeLa cell lysate. Here we report the identification of two such candidate substrates, the ribosomal protein L5 and MCM3, a replication licensing factor. Although L5 proved to be a weak substrate, MCM3 was efficiently and specifically phosphorylated by DAPk on a unique site, Ser160. Significantly DAPk phosphorylated this site in vivo upon overexpression in 293T cells. Activation of endogenous DAPk by increasing intracellular Ca2+ also led to increased phosphorylation of MCM3. Importantly short hairpin RNA-mediated knockdown of endogenous DAPk blocked both basal phosphorylation and Ca2+-induced phosphorylation, indicating that DAPk is both necessary and sufficient for MCM3 Ser160 phosphorylation in vivo. Identification of MCM3 as an in vivo DAPk substrate indicates the usefulness of this approach for identification of physiologically relevant substrates that may shed light on novel functions of the kinase.

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.

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.

Cell cultures treated by interferon become unable to support viral replication. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis [l-3]. Interferon induces several biochemical mechanisms which could mediate this inhibition: (i) A double-stranded RNA (dsRNA) and ATP-dependent phosphorylation of initiation factor eIF2 and possibly other ribosome-associated proteins [4-71; (ii) The dsRNA ATP-dependent synthesis of an unusual oligo-adenylate isomer with 2’-5 phosphodiester bonds [7-91; (iii) A mechanism which affects polypeptide chain elongation, that does not require dsRNA and is reversed by tRNAs [lo-l 21. We have reported [3,9] the separation and isolation, from extracts of interferon-treated L cells, of the dsRNA ATP-activated protein kinase PKi, and of the (2’-5’) oligo-isoadenylate synthetase E. Here, we demonstrate that the oligonucleotide produced by E requires, to inhibit mRNA translation, an additional protein F already present constitutively in untreated cells. The purified F is shown to be an oligo-isoadenylate-dependent ribonuclease which degrades the mRNA template. Our data show that the dsRNA ATP-dependent nuclease activity reported [ 13,141 to be increased in extracts of interferon-treated cells, can be explained as the activation of a constitutive ribonuclease, F, by the product of the interferon-induced and dsRNA-dependent oligo-isoadenylate synthetase E. This is in

GENE expression may be controlled during translation by ribosomal selection of mRNAs or even individual cistrons. Escherichia coli initiation factors associated with ribosomes affect the binding of ribosomes to mRNA1,2; initiation factor IF3, for instance, influences the specificity of mRNA-ribosome interaction3,4. IF3 activity has been separated into several fractions which show various specificities for different mRNA cistrons4–9. An important problem is the possibility of intracellular changes in IF3 activity10–12. From uninfected E. coli, we have now isolated a protein which changes the specificity of IF3 toward different mRNAs; we call this interference factor i. Pure factor i binds to IF3 and specifically affects the translation of T4 and MS2 RNA. Whereas the initiation of translation of the MS2 coat protein cistron is inhibited by factor i, the synthetase cistron—when available—is more rapidly initiated in the presence of factor i. The overall translation of T4 mRNA appears unchanged by factor i, but certain cistrons are stimulated at the expense of others. Interfering factors such as factor i could be important in controlling translation in E. coli.