DAP kinase and DAP-3: novel positive mediators of apoptosis.
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Apoptosis↗
Biomedical subjects
A Kimchi
Publications and source records attributed to A Kimchi.
Death associated proteins (DAPs): from gene identification to the analysis of 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 approaches are being used to identify and study genes which function as positive mediators of apoptosis. Here, we address a novel approach of gene cloning aimed at isolating intracellular death promoting genes by utilizing a functional screen. This method, called TKO, was based on transfection of cells with an anti-sense cDNA library, followed by the selection of transfectants which survived in the continuous presence of a killing cytokine-interferon-gamma. 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 death process. The five novel apoptotic genes (named DAP genes for: Death Associated Proteins) code for proteins which display a diverse spectrum of biochemical activities. The list comprises a novel type of calcium/calmodulin-regulated kinase which carries ankyrin repeats and a death domain (DAP-kinase), a nucleotide-binding protein (DAP-3), a small proline-rich cytoplasmic protein (DAP-1), and a novel homolog of the eIF4G translation initiation factor (DAP-5). Extensive studies proved that these genes are critical for mediating 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 from extracellular matrix. Moreover, one of these genes, DAP-kinase, was recently found to display strong tumor suppressive activities, coupling the control 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 unbiased manner. As a consequence, novel targets and unpredicted mechanisms emerged. A few examples illustrating this important point will be discussed. One relates to the calcium/calmodulin-dependent DAP-kinase, which is localized 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 stress fibers--a typical hallmark of apoptosis. Another important breakthrough step in our understanding of apoptotic processes relates to the identification and analysis of the DAP-5 gene. The structure/ function features of this novel translation regulator resemble the proteolytically cleaved eIF4G which appears in cells upon infection with some RNA viruses and which directs cap-independent translation. Thus, the rescue of DAP-5 highlighted the importance of regulation of protein translation in certain apoptotic systems. Finally, the isolation of cathespin D by our method suggests that lysosomal proteases are recruited during apoptosis, in addition to the well known caspase family of proteases, and that a unique pattern of regulation affecting the processing of this protease takes place. The major challenge now is to analyse how these diverse DAP gene activities constitute biochemical pathway(s) leading to programmed cell death, and what is their functional position with respect to other known positive mediators and suppressors of apoptosis such as the Bcl2 and caspase family members.
Animals↗
DAP genes: novel apoptotic genes isolated by a functional approach to gene cloning.
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Adaptor Proteins, Signal Transducing↗
Death-associated proteins: from gene identification to the analysis of their apoptotic and tumour suppressive functions.
Aberrations of apoptosis are implicated in many diseases, including cancer, autoimmune disease, cardiovascular disease and neurodegeneration. The cell's apoptotic machinery is, therefore, an important potential target for the development of new therapies. Our laboratory has used a strategy called technical knockout (TKO) to identify novel genes involved in apoptosis. TKO is based on random inactivation of gene expression with antisense cDNA libraries, followed by selection of those cells that survive in the continuous presence of an apoptotic stimulus. Using this approach, we have isolated five novel genes, including a serine/threonine kinase, a nucleotide-binding protein and a homologue of the p220 translation initiation factor. Expression of one of these genes (DAP kinase) is lost in some cancers, and this loss appears to increase the metastatic potential of some tumours.
Animals↗
DAP kinase links the control of apoptosis to metastasis.
DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the 'death' domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-gamma, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign 'microenvironment' Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.
Animals↗
DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: possible implications for role as tumor suppressor gene.
DAP-kinase is a novel calmodulin dependent serine/threonine kinase that carries ankyrin repeats and the death domain. It was recently isolated, by a functional selection approach of gene cloning, as a positive mediator of programmed cell death. In this study the expression of DAP-kinase was examined in the cell lines derived from various human neoplasms. DAP-kinase mRNA and protein expression were below the limit of detection in eight out of ten neoplastic derived B-cell lines. In six out of 14 examined bladder carcinoma, in three out of five renal cell carcinoma, and in four out of ten tested breast carcinoma cell lines, the DAP-kinase protein levels were below detection limits or lower than 1% compared to the positive cell lines. Interestingly, DAP-kinase expression could be restored in some of the negative bladder carcinoma and B-cell lines by treatment of cells with 5'-azadeoxycytidine that causes DNA demethylation. The high frequency of loss of DAP-kinase expression in human tumor cell lines, and the occasional involvement of methylation in this process raise the possibility that this novel mediator of cell death may function as a tumor suppressor gene.
Apoptosis Regulatory Proteins↗
DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.
DAP-kinase was initially identified as a gene whose anti-sense-mediated reduced expression protected HeLa cells from interferon-gamma-induced programmed cell death. It was cloned in our laboratory by a functional gene selection approach. According to its amino acid sequence, this 160 kDa protein was predicted to be a novel type of calmodulin-regulated serine/threonine kinase which carries ankyrin repeats and the death domain. In this work we have shown that the kinase was autophosphorylated and capable of phosphorylating an exogenous substrate in a Ca2+/calmodulin-dependent manner. We proved that calmodulin binds directly to the recombinant kinase, and generated a constitutively active kinase mutant by the deletion of the calmodulin-regulatory domain. By immunostaining and biochemical fractionations we demonstrated that the kinase is localized to the cytoskeleton, in association with the microfilament system, and mapped a region within the protein which is responsible for binding to the cytoskeleton. Several assays attributed a cell death function to the gene. Ectopic expression of wild-type DAP-kinase induced the death of target cells, and the killing property depended strictly on the status of the intrinsic kinase activity. Conversely, a catalytically inactive mutant that carried a lysine to alanine substitution within the kinase domain, displayed dominant-negative features and protected cells from interferon-gamma-induced cell death. DAP-kinase is therefore a novel cytoskeletal-associated cell death serine/threonine kinase whose activation by Ca2+/calmodulin may be linked to the biochemical mechanism underlying the cytoskeletal alterations that occur during cell death.
Actin Cytoskeleton↗
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.
Amino Acid Sequence↗
Assignment of death associated protein 3 (DAP3) to human chromosome 1q21 by in situ hybridization.
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Cell Line↗
Double-stranded RNA-dependent protein kinase mediates c-Myc suppression induced by type I interferons.
The antiproliferative functions of interferons result from specific effects that these cytokines exert on several cell cycle-controlling genes. The possible coupling between the interferon-responsive genes that are directly transactivated by the interferon signaling and the genes that constitute the basic machinery of the cell cycle is not clear yet. We report in this work that interferon-induced double-stranded RNA-activated kinase (PKR) is one of the specific mediators of the antiproliferative effects of the cytokine. Transfections of M1 myeloid leukemia cells with two catalytically inactive mutant forms of PKR abrogated the ability of interferon to suppress c-Myc without interfering with the pRB/cyclin D responses. As a consequence, these genetically manipulated cells displayed a small but significant reduction in their growth sensitivity to interferons, a phenotype that characterizes a single pathway disruption. Transfection of the parental M1 cells with the functional wild-type human PKR restricted their proliferation in the absence of interferons. This PKR-mediated growth inhibition could be efficiently rescued by the ectopic expression of deregulated c-myc. Taken together these results prove the existence of direct or indirect links between PKR and c-Myc suppression, thereby placing this gene along one of the complementary growth suppressive pathways that are triggered by interferons.
Animals↗
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.
Apoptosis↗
Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible G0-like arrest.
Alpha interferon is a potent growth inhibitor of Daudi Burkitt's lymphoma cells. We show here that alpha-interferon signaling interacted simultaneously with several components of the basic cell cycle machinery, causing cells to enter into a state that had many features characteristic of the G0 state. Within a few hours after alpha-interferon treatment, cyclin D3 mRNA and protein levels dropped to undetectable levels and, in parallel, the activities of cyclin A- and cyclin E-associated kinases were significantly reduced. The latter resulted from the rapid alpha-interferon-mediated elimination of cdc25A, a phosphatase that is required for antagonism of negative tyrosine phosphorylation of cdk2 in cyclin-cdk complexes. This regulation represents a novel mechanism through which an external inhibitory cytokine interacts with the cell cycle machinery. At later time points after alpha-interferon treatment, the levels of the 55-kDa slowly migrating hyperphosphorylated form of cyclin E and of cyclin A were also reduced. The antiproliferative effects were reversible, and cultures from which alpha interferon was removed reentered S phase after a lag that typically corresponded to approximately two doubling times. During this lag period, the expression of cyclin D3 and cyclin A, as well as of the cdc25A phosphatase, continued to be switched off, in spite of the removal of alpha interferon from the cell surface. In contrast, c-myc, which represents another downstream target gene that is subjected to negative regulation by alpha interferon, was relieved from suppression much earlier, concomitant with the decay in early signaling of the cytokine. The delayed pattern of cyclin reexpression provides evidence that alpha-interferon signaling imposes a G0-like state on this system.
Burkitt Lymphoma↗
Isolation of DAP3, a novel mediator of interferon-gamma-induced cell death.
Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.
Amino Acid Sequence↗
Assignment of DAP1 and DAPK--genes that positively mediate programmed cell death triggered by IFN-gamma--to chromosome regions 5p12.2 and 9q34.1, respectively.
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Animals↗
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.
Amino Acid Sequence↗
The death domain: a module shared by proteins with diverse cellular functions.
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Amino Acid Sequence↗
p53 undergoes epitopic changes in vitro by sodium-vanadate.
p53 has at least two conformations that differ in their immunoreactivity. They consist of the functional tumor suppressor form, characteristic of the wild type p53 and the mutant form, generated by changes in the primary amino acid sequence of the protein. It has been previously shown that the wild type p53 protein also acquires the mutant conformation upon certain changes in growth conditions. Here we report that similar epitopic changes can be induced in crude cell lysate by addition of vanadate anions at 1 mM final concentration. A panel of anti p53 antibodies was used to discriminate between the different immunoreactive forms of wild type p53 in SV80 fibroblasts. It was found that addition of sodium vanadate to the lysis buffer converted part of p53 molecules into a mutant conformation that is recognized by the PAb 240 monoclonal antibodies. The effect of vanadate on p53 conformation was prominent even if it was added to the cell lysates after 15 min of pre-incubation at 37 degrees C. This further excluded its possible role as phosphatase inhibitor in the system and suggested a direct interaction with the p53 protein itself. Based on these data we recommend to avoid using sodium vanadate as a phosphatase inhibitor in experiments where in vivo conformational changes of wild type p53 are studied.
Antibodies, Monoclonal↗
Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.
Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.
Cyclins↗