Article |
Address correspondence to Adi Kimchi, Dept. of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9342428. Fax: 972-8-9315938. E-mail: Adi.kimchi{at}weizmann.ac.il
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: DAP kinase; DRP-1; autophagy; membrane blebbing; programmed cell death
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study addresses the specific function of the death-associated protein kinase (DAPk)* family of genes, which have been shown previously to play a central role in various death signaling pathways. It focuses on two family members which display the highest homology, DAPk (Deiss et al., 1995) and its closest homologue DAPk-related protein kinase (DRP)-1 (Kawai et al., 1999; Inbal et al., 2000; for review see Kogel et al., 2001). DAPk and DRP-1 are both Ca2+-calmodulin (CaM)regulated Ser/Thr kinases whose death-promoting activity relies on the catalytic domain activity (Cohen et al., 1997, 1999; Kawai et al., 1999; Inbal et al., 2000). DAPk additionally contains ankyrin repeats and the death domain motif and is localized to actin microfilaments (Deiss et al., 1995; Feinstein et al., 1995; Cohen et al., 1997). DRP-1, which is found in soluble cytosolic fractions, lacks these motifs and instead contains a 40 amino acid COOH-terminal tail that is required for homodimerization (Inbal et al., 2000; Shani et al., 2001). Thus, the homology between the two proteins is restricted to the kinase and CaM-binding domains. Interestingly, both kinases undergo a similar inhibitory autophosphorylation on a single conserved Ser within the CaM-binding domain, which inhibits the catalytic activation and restrains their death-promoting functions (Shani et al., 2001; Shohat et al., 2001). Several studies demonstrated the ability of DAPk to induce cell death in various cell lines and furthermore established that suppression of DAPk function slows down cell death triggered by various stimuli (e.g., interferon [IFN]-, tumor necrosis factor [TNF]-
, Fas, detachment from extracellular matrix, TGF-ß, and C6-ceramide [Deiss et al., 1995; Cohen et al., 1997, 1999; Inbal et al., 1997; Jang et al., 2002; Pelled et al., 2002]). Other studies have linked the wide death-promoting effects of DAPk to its function as a tumor and a metastasis suppressor gene (Inbal et al., 1997; Kissil et al., 1997; Raveh et al., 2001). The latter paved the way to recent large scale cancer patient screens, which showed that DAPk expression is lost frequently in a wide range of tumor types (for review see Raveh and Kimchi, 2001). DRP-1 shares with DAPk some of the death-promoting functions, such as the participation in TNF-
cell death signaling (Inbal et al., 2000). The high degree of similarity in the catalytic and CaM regulatory domains of DAPk and DRP-1 suggests that they may share common substrates as evident by their joint ability to phosphorylate myosin light chain in in vitro kinase assays (Cohen et al., 1997; Kawai et al., 1999; Inbal et al., 2000). In addition, there is functional interaction between the two kinases, suggesting that they may be connected to similar subcellular outcomes (Inbal et al., 2000).
Here we found that DAPk and DRP-1 mediate the formation of autophagic vesicles that are characteristic of type II programmed cell death and also mediate a more prevalent event of cell death membrane blebbing. These two morphological events occur in a caspase-independent manner. DAPk family members are among the first identified death-promoting genes that regulate autophagy during type II cell death.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
We next wished to test whether DAPk family proteins are connected to the mitochondrial signaling pathway. Neither DAPk nor DRP-1 reduced the mitochondrial membrane potential (m), in contrast to transfections with Bax used as a positive control (Fig. 2 A) (Xiang et al., 1996). Furthermore, in contrast to Bax-transfected cells in which cytochrome C was released from the mitochondria to the cytoplasm (Fig. 2 B, 3 and 5, for spread and blebbed cell morphologies, respectively) DRP-1 and DAPk-transfected blebbed cells continued to display punctate mitochondrial staining (Fig. 2 B, 7 and 9), similar to the staining observed in control luciferase-transfected cells (Fig. 2 B, 1). Also, upon cotransfection experiments Bcl-2 or Bcl-XL failed to block the early effects induced by DAPk family proteins, such as membrane blebbing and partial detachment from the substrate, although they significantly inhibited the death effects of Bax (Fig. 2 C). These results rule out the direct involvement of mitochondrial events in this specific setting of DAPk family proteins' induced cell death.
To examine the morphological distinctions of DAPk family members' induced death in more detail, we analyzed 293 transfectants by transmission EM (TEM). p55/TNFR1-transfected cells showed membrane blebbing, mitochondrial darkening, chromatin clumping, and fragmentation (Fig. 3 A, 2a). At later stages, these cells were further condensed, engulfed by neighboring cells (Fig. 3 A, 2b), and completely phagocytosed (Fig. 3 A, 2c). In contrast, DRP-1 73 and DAPk
CaMtransfected 293 cells were neither condensed and fragmented nor phagocytosed consistent with the light microscopy observations. In both cases, the kinase-transfected cells exhibited marked membrane blebbing. Additionally and quite surprisingly, a relatively high number of autophagic vesicles appeared (Fig. 3 A, 3a, 3b, and 4). These reflected different stages of autophagosomal development (Dunn, 1994), such as double membrane structures engulfing cytoplasmic matter and organelles (among them mitochondria were clearly identified), formation of mature autophagic vesicles and autolysosomes, and finally the degradation of interautophagic content, leading to the formation of a residual body (marked by double arrows, single black arrows, and dashed arrows, respectively). These autophagic vesicles could be mainly observed in perinuclear areas and less frequently inside the blebbed areas themselves (Fig. 3 A, 3a and 3b). In p55/TNFR1 transfections, autophagic vesicles were rarely observed (Fig. 3 A, 2a). DRP-1
73transfected 293 cells also displayed packed, yet uncondensed, unfragmented chromatin, disappearance of nuclear membranes, and in a few cases mixing between the organelles and the nuclear material (Fig. 3 A, 3a and 3b). Similar results were observed in TEM studies of MCF-7 cells. Transfection of DRP-1
73 or DAPk
CaM in these cells also resulted in the formation of autophagic vesicles in the cytoplasm (Fig. 3 B, 3a, 3b, and 4), packed chromatin that was only minimally condensed and rarely fragmented, and the disappearance of nuclear membranes resulting in mixing between the organelles and the nuclear material (Fig. 3 B, 3a, 3b, and 4). In contrast, the induction of cell death in MCF-7 cells by p55/TNFR1 expression resulted in a round cell morphology, chromatin condensation, and fragmentation (Fig. 3 B, 2a, 2b, and 2c) and condensation (darkening) of mitochondria similar to what has been described under conditions of high oxidative phosphorylation (Hackenbrock et al., 1971). At later stages, there was a marked packing and condensation of cytoplasmic constituents, including hundreds of empty vesicles, none of which were autophagic in nature (Fig. 3 B, 2c).
DRP-1 73induced autophagy was quantified by scoring for the appearance of autophagic vesicles 4872 h after transfection using the specific lysosomal/autophagic vacuole marker monodansylcadaverin (MDC) (Biederbick et al., 1995; Niemann et al., 2000). DRP-1
73 expression statistically elevated MDC staining in both cell types (Fig. 4, A and B) in agreement with the TEM analysis of 293- and MCF-7transfected cells described above. Thus, DRP-1
73 induces autophagy-related elevation of MDC staining in 293 and MCF-7 cells in an analogous manner to the steroid withdrawal effect on MCF-7 cells (Bursch et al., 2000).
Effects of DAPk and DRP-1 on membrane blebbing and the formation of autophagic vesicles are caspase independent
To test whether caspases, in general, mediate the two major effects of DAPk and DRP-1 on membrane blebbing and autophagy, we performed transfections with DAPk CaM and DRP-1
73 in 293 and MCF-7 cells in the presence or absence of high doses of the pan-caspase inhibitors, BD-fmk and z-VAD-fmk (100 µM). As a positive control, we used p55/TNFR1 transfections, which are extremely sensitive to these pan-caspase inhibitors due to the rate-limiting effects of caspases both in the death-inducing signalling complex at the cell surface level and in further downstream events. Both caspase inhibitors blocked almost completely all phenotypic changes induced by p55/TNFR1 transfections in 293 and MCF-7 cells (Fig. 5 A and B, respectively), resulting in normal spread cell morphology (Fig. 5 D, 1). In contrast, these caspase inhibitors failed to alter the pattern of morphological changes induced by DAPk family members (Fig. 5, A and B). Similar results were obtained in cotransfection experiments using the cowpox caspase inhibitor crmA (Fig. 5 C). The lack of protection by caspase inhibitors was observed more closely in TEM studies of DAPk
CaM and DRP-1
73transfected 293 cells exposed to 100 µM BD-fmk. As seen in Fig. 5 D, 2a and 2b, both membrane blebbing and the formation of autophagic vesicles at the different maturation stages were not affected at all by the addition of BD-fmk. In contrast, the p55/TNFR1 transfectants were fully protected and looked normal in the presence of BD-fmk with intact nuclei and cytoplasm and no signs of membrane blebbing (Fig. 5 D, 1). Western blot analysis of poly (ADP-ribose) polymerase (PARP), caspase-8, and caspase-3 cleavage in HeLa cells at 24 h after transfection demonstrated that these proteins were clearly processed in cells transfected with p55/TNFR1 but not in cells transfected with DRP-1
73 or DAPk
CaM (Fig. 5 E). Together, these results demonstrate that caspases do not act downstream of DAPk family proteins in the pathway, which is responsible for membrane blebbing and autophagy.
|
|
|
|
|
DRP-1 is localized to the inner part of autophagic and autolysosomic vesicles
To check the intracellular localization of DAPk and DRP-1 in 293 cells, immunogold labeling was used to detect HA epitopetagged wild-type DAPk and DRP-1. In both TEM sections, a remarkable induction of autophagic vesicles was observed, similar to the previous results shown in Fig. 3. The immunogold staining of DAPk was mostly cytoplasmic without yielding a definitive pattern (unpublished data). In contrast, examination of DRP-1 transfectants, especially those which expressed moderate levels of the protein, clearly revealed that DRP-1 was specifically stained inside the lumen of autophagic/autolysosomal structures (Fig. 10, AD). This staining was seen in autophagic vesicles harboring a double membrane and containing cytoplasmic matter (Fig. 10, A and B), in autophagic vesicles containing mitochondria and vacuoles (Fig. 10 C), and inside normal (Fig. 10 D) or large sized (unpublished data) autolysosomal structures. The gold particles did not overlap with the degraded organelles or lysosomes (Fig. 10, C and D). DRP-1 is therefore localized specifically inside autophagic/autolysosomal structures, implying a direct role in this process.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work is part of our attempts to dissect at the subcellular level the different functional "arms" emanating from these death kinases. In a previous work, we found that in primary embryonic fibroblasts DAPk activates a strong and rapid cascade of caspase-dependent events, including DNA and cytoplasmic fragmentation (Raveh et al., 2001). This requires p53, which DAPk stabilizes and further activates in a p19ARF-dependent mechanism. As a consequence, the p53 pathway becomes the predominant functional arm in this system, and DAPk-induced cell death can be rescued by caspase inhibitors. Only in p53-deficient mouse embryonic fibroblasts in which the p53-mediated caspase-dependent mechanisms were not activated were alternate functional arms of DAPk apparent (Raveh et al., 2001). Here, the use of epithelial cell lines, which lack functional p53, enabled us to analyze the caspase-independent functions of DAPk family members, which include membrane blebbing and the formation of autophagic vesicles. It should be noted that in these same cells, extremely high expression levels of DAPk are capable of inducing caspase-dependent events, which are suppressed by crmA and Bcl-2 (Cohen et al., 1999). Likewise, DAPk mediates a mitochondrial-based apoptotic pathway in response to TGFß in a p53-deficient hepatoma cell line (Jang et al., 2002). Therefore, although DAPk in certain cell settings can be linked to caspase-dependent type I apoptotic cell death, it possesses an independent death function that can only be assessed in the absence of caspase activation as was seen in the experimental conditions presented here. These subcellular changes are sufficient to induce an irreversible commitment to cell death, since it was found that the number of colonies which appeared on plates after two weeks of culturing of kinase-transfected cells was reduced by several logs (unpublished data).
We show here for the first time that IFN- induces autophagic cell death in HeLa cells. Autophagy in HeLa cells and that induced by antiestrogen or amino acid starvation in MCF7 cells occurs independently of caspases, similar to newly isolated NGF-deprived superior cervical ganglia neurons and
-irradiated MCF-7 cells (Xue et al., 1999; Paglin et al., 2001). This may dictate the slow progression of autophagic death in which the cells eventually die by the loss of essential organelles, such as mitochondria (Lemasters et al., 1998; Xue et al., 2001). Whereas rapid apoptosis ensures the immediate and quick elimination of hazardous cells, the slower nature of autophagic cell death may help to shape the tissue more accurately, enable enough time for a decision between cell survival and cell death in cases where minimal damage takes place, or simply allow cell death in cases where heterophagy is not available. Autophagic cell death probably represents a more ancient-based type of cell death (Klionsky and Emr, 2000), since many of the autophagy-associated genes are evolutionary conserved between yeast and mammalian organisms. Genes like DAPk or DRP-1, which do not exist in yeast, were probably added in evolution to provide a link between the basic evolutionary conserved autophagic machinery and programmed cell death. Another link between mammalian death-associated protein and the basic autophagy is provided by beclin 1, a tumor suppressor protein that interacts with Bcl-2 and is structurally similar to the yeast autophagic gene Apg6/Vps30p (Liang et al., 1998, 1999).
DRP-1 protein was localized to the lumen of autophagosomes and autolysosomes by immunogold staining. Since staining was not observed within the lysosomal compartment of the autolysosomes or within the engulfed organelles, we can conclude that DRP-1 is a resident protein of the autophagosome itself and was not nonspecifically delivered by lysosomes or the engulfed organelle. Of note, DRP-1 does not contain the peptide recognition signal KFERQ, which is used in the carrier-mediated proteolysis pathway (Dunn, 1994). Other mammalian proteins that localize to autophagosomes are involved in the structural stages of autophagic vesicle formation. These include the complex of mammalian Apg5p-Apg12p required for elongation of autophagic isolation membranes (Mizushima et al., 2001) and the mammalian homologue of yeast Apg8p, LC3, whose processed form is later targeted to autophagosome membranes (Kabeya et al., 2000). DRP-1's localization is consistent with it having a direct role in autophagosome formation, perhaps through phosphorylation of one or more of these autophagy-related proteins. In contrast, DAPk does not localize to the autophagosomes but rather is associated with actin microfilaments. DAPk may still influence the autophagic process through its association with the cytoskeletal structures that are involved in autophagosome formation (Dunn, 1994; Bursch et al., 2000). Thus, although both DAPk and DRP-1 are mediators of autophagy, the different intracellular localization of the two kinases suggests that they are not completely redundant in their mode of action.
Although DAPk and DRP-1 induce caspase-independent type II cell death, their functions are recruited by classical type I stimuli, such as TNF, to mediate membrane blebbing, a phenomenon common to both death processes. It has been proposed that during cell death myosin-mediated contraction of the cortical actin ring, occurring simultaneously with proteolytic degradation of cytoskeletal proteins that connect actin filaments to the cell membrane, gives rise to bleb extrusion in areas of structural weakness (Mills et al., 1998). Both kinases and a third family member, ZIP-kinase, are capable of phosphorylating the myosin regulatory light chain in vitro and in vivo on Ser19, a site responsible for activation of nonmuscle myosin (Cohen et al., 1997; Jin et al., 2001; Murata-Hori et al., 2001; Shani et al., 2001; unpublished data). Additional kinases that phosphorylate myosin light chain, such as the Rho-associated coiled-coilcontaining protein kinase (ROCK), have been shown to mediate apoptotic membrane blebbing (Mills et al., 1998; Coleman et al., 2001; Sebbagh et al., 2001). Like DAPk and DRP-1, ROCK, once activated, was capable of inducing membrane blebbing in a caspase-independent manner. It will be important to precisely dissect the role of each of these kinases to determine whether there is cross talk between these pathways or whether each is a specific mediator of particular death signals. DAPk and DRP-1 are activated by the increase in intracellular Ca2+ (Cohen et al., 1997) and by means of dephosphorylation of autoinhibitory sites (Shani et al., 2001; Shohat et al., 2001), whereas ROCK activation during apoptosis requires caspase cleavage (Coleman et al., 2001; Sebbagh et al., 2001). Thus, DAPk protein members and ROCK could be differentially regulated in programmed cell death, depending on the nature of the trigger. For example, DAPk rather than ROCK may mediate the caspase-independent blebbing observed in Myc-induced death of Rat-1 cells (McCarthy et al., 1997).
Together, these results imply a major role of DAPk proteins in membrane blebbing and in the less studied process of autophagic (type II) cell death. Elucidating the exact molecular events taking place during this type of cell death, its cellular consequences, and relation to the apoptotic (type I) cell death are of primary importance for both scientific and clinical purposes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Western blot analysis
Detection of proteins was performed by standard Western blot analysis using anti-HA antibodies (clone 16B12; Babco), anti-FLAG antibodies (M2; Sigma-Aldrich), anti-PARP antibodies (clone C-210; Biomol), anticaspase-8 antibodies (clone 1C12; Cell Signaling), anticaspase-3 antibodies (H-277; Santa Cruz Biotechnology), or antiß-tubulin antibodies (clone TUB 2.1; Sigma-Aldrich).
Immunocytochemistry
Cells grown on coverslips were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton in PBS for 10 min, and blocked with 10% normal goat serum in PBS for 30 min. Mouse monoclonal anticytochrome C antibodies were applied for 60 min (clone 6H2.B4; BD PharMingen) followed by Cy3-conjugated goat antimouse secondary antibodies (Jackson ImmunoResearch Laboratories) for 30 min. The coverslips were mounted in Mowiol and examined under transmitted light fluorescence microscope (Axioscope 2; ZEISS) with an excitation filter of 550570 nm.
Loss of mitochondrial membrane potential
Cells transfected with the various death-inducing constructs were incubated 24 h after transfection with tetramethylrhodamine methyl ester (TMRM; 0.2 µM) (Molecular Probes) for 30 min at 37°C and then subjected to flow cytometry analysis using an argon-ion filter for TMRM detection (excitation wavelength at 514 nm, emission at 585 ± 21 nm). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (10 µM; Sigma-Aldrich) was added 3 h before TMRM staining and was used as a positive control for mitochondrial depolarization.
Induction of type II cell death in MCF-7 breast carcinoma and HeLa cell lines
1 x 105 MCF-7 cells were seeded per 3-cm well and subjected to steroid depletion in the presence of 10-6 M tamoxifen (Sigma-Aldrich) for 35 d as described (Bursch et al., 1996). For amino acid starvation, cells were grown in Earle's balanced salt solution medium (GIBCO BRL) lacking amino acids and serum for 56 h with hourly replacement of the medium to discard secreted amino acids. HeLa cells (1 x 105/3-cm well) were treated with IFN- (1,000 µ/ml; Pepro Tech) for 23 d. Where indicated, MCF-7 cells were transfected with 1.5 µg dominant negative kinase DRP-1 K42A and 0.5 µg pEGFP-NI. 24 h later, cells were steroid depleted and left for another 2448 h or AA-deprived for 56 h. Staining of autophagic vacuoles with 0.05 mM monodansylcadaverin (MDC; Sigma-Aldrich) was as described previously (Biederbick et al., 1995) and observed under inverted fluorescent microscope (IX50; Olympus) with an excitation filter range of 330385 nm and a barrier filter of 420 nm.
TEM and immunogold staining
293 cells grown in 35-mm dishes were fixed for 60 min in Karnovsky's fixative (3% paraformaldehyde, 2% glutaraldehyde, 5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.4, containing 0.1 M sucrose) and postfixed with 1% OsO4, 0.5% potassium dichromate, and 0.5% potassium hexacyanoferrate in 0.1 M cacodylate buffer. The cells were stained en bloc with 2% aqueous uranyl acetate followed by ethanol dehydration. The dishes were embedded in Epon 812 (Tuosimis). Frontal sections were cut, stained with 2% uranyl acetate in 50% ethanol and lead citrate, and examined using a Philips CM-12 transmission electron microscope at an accelerating voltage of 100 KV. For immunogold detection of DRP-1 and DAPk, the transfected 293 cells grown in 90-mm Falcon dishes were fixed in situ for 1 h at 24°C in a freshly prepared solution of Karnovsky's fixative. Scraped cell pellets were then incubated in 10% gelatin at 37°C for 30 min and centrifuged at 37°C. Excess gelatin was removed, and pellets were postfixed in Karnovsky's fixative overnight at 4°C. The pellets were cut into small pieces and cryoprotected by overnight infiltration with 2.3 M sucrose in 0.1 M cacodylate buffer. Samples were quick frozen in liquid nitrogen, and ultrathin frozen sections (75 nm) were cut at 110°C on Reichert Ultracat-S ultramicrotome. The sections were transferred to formvar-coated 200 mesh nickel grids and blocked with conditional medium as described (Tokuyasu, 1980). Sections were incubated with rabbit anti-HA antibodies (1:100200; Santa Cruz Biotechnology) for 2 h. After extensive washing in PBS-0.1% glycine, the primary antibodies were detected with goat antirabbit 10 nm colloidal gold conjugate (1:20; Zymed Laboratories). The grids were then washed in PBS-glycine, stained with neutral uranyl acetate oxalate for 5 min, and then stained with 2% uranyl acetate in H2O for 10 min. They were then embedded in 2% methyl cellulose/uranyl acetate as described (Tokuyasu, 1980).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by a grant from Deutsch-Israelische Projektkooperation. A. Kimchi is the incumbent of Helena Rubinstein Chair of Cancer Research.
Submitted: 26 September 2001
Revised: 19 March 2002
Accepted: 25 March 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biederbick, A., H.F. Kern, and H.P. Elsasser. 1995. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol. 66:314.[Medline]
Bursch, W., A. Ellinger, H. Kienzl, L. Torok, S. Pandey, M. Sikorska, R. Walker, and R.S. Hermann. 1996. Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis. 17:15951607.[Abstract]
Bursch, W., K. Hochegger, L. Torok, B. Marian, A. Ellinger, and R.S. Hermann. 2000. Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. J. Cell Sci. 113:11891198.
Christensen, S.T., J. Chemnitz, E.M. Straarup, K. Kristiansen, D.N. Wheatley, and L. Rasmussen. 1998. Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration. Cell Biol. Int. 22:591598.[CrossRef][Medline]
Cohen, O., E. Feinstein, and A. Kimchi. 1997. DAPk is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 16:9981008.
Cohen, O., B. Inbal, J.L. Kissil, T. Raveh, H. Berissi, T. Spivak-Kroizaman, E. Feinstein, and A. Kimchi. 1999. DAPk participates in TNF- and Fas-induced apoptosis and its function requires the death domain. J. Cell Biol. 146:141148.
Deiss, L.P., E. Feinstein, H. Berissi, O. Cohen, and A. Kimchi. 1995. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev. 9:1530.[Abstract]
Feinstein, E., A. Kimchi, D. Wallach, M. Boldin, and E. Varfolomeev. 1995. The death domain: a module shared by proteins with diverse cellular functions. Trends Biochem. Sci. 20:342344.[CrossRef][Medline]
Hackenbrock, C.R., T.G. Rehn, E.C. Weinbach, and J.J. Lemasters. 1971. Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell. J. Cell Biol. 51:123137.
Inbal, B., G. Shani, O. Cohen, J.L. Kissil, and A. Kimchi. 2000. Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis. Mol. Cell. Biol. 20:10441054.
Jia, L., R.R. Dourmashkin, P.D. Allen, A.B. Gray, A.C. Newland, and S.M. Kelsey. 1997. Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells. Br. J. Haematol. 98:673685.[Medline]
Jin, Y., E.K. Blue, S. Dixon, L. Hou, R.B. Wysolmerski, and P.J. Gallagher. 2001. Identification of a new form of death associated protein kinase that promotes cell survival. J. Biol Chem. 276:3966739678.
Kabeya, Y., N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, and T. Yoshimori. 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19:57205728.
Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26:239257.[Medline]
Klionsky, D.J., and S.D. Emr. 2000. Autophagy as a regulated pathway of cellular degradation. Science. 290:17171721.
Lemasters, J.J., A.L. Nieminen, T. Qian, L.C. Trost, S.P. Elmore, Y. Nishimura, R.A. Crowe, W.E. Cascio, C.A. Bradham, D.A. Brenner, and B. Herman. 1998. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim. Biophys. Acta. 1366:177196.[Medline]
Liang, X.H., L. Kleeman, H.H. Jiang, G. Gordon, J.E. Goldman, G. Berry, B. Herman, and B. Levine. 1998. Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein. J. Virol. 72:85868596.
McCarthy, N.J., M.K. Whyte, C.S. Gilbert, and G.I. Evan. 1997. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136:215227.
Mills, J.C., N.L. Stone, J. Erhardt, and R.N. Pittman. 1998. Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J. Cell Biol. 140:627636.
Mizushima, N., A. Yamamoto, M. Hatano, Y. Kobayashi, Y. Kabeya, K. Suzuki, T. Tokuhisa, Y. Ohsumi, and T. Yoshimori. 2001. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152:657668.
Niemann, A., A. Takatsuki, and H.P. Elsasser. 2000. The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe. J. Histochem. Cytochem. 48:251258.
Paglin, S., T. Hollister, T. Delohery, N. Hackett, M. McMahill, E. Sphicas, D. Domingo, and J. Yahalom. 2001. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 61:439444.
Pelled, D., T. Raveh, C. Riebeling, M. Fridkin, H. Berissi, A.H. Futerman, and A. Kimchi. 2002. Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons. J. Biol. Chem. 277:19571961.
Raveh, T., H. Berissi, M. Eisenstein, T. Spivak, and A. Kimchi. 2000. 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. Proc. Natl. Acad. Sci. USA. 97:15721577.
Sebbagh, M., C. Renvoize, J. Hamelin, N. Riche, J. Bertoglio, and J. Breard. 2001. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell Biol. 3:346352.[CrossRef][Medline]
Seglen, P.O., and P.B. Gordon. 1982. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad. Sci. USA. 79:18891892.[Abstract]
Shani, G., S. Henis-Korenblit, G. Jona, O. Gileadi, M. Eisenstein, T. Ziv, A. Admon, and A. Kimchi. 2001. Autophosphorylation restrains the apoptotic activity of DRP-1 kinase by controlling dimerization and calmodulin binding. EMBO J. 20:10991113.
Shohat, G., T. Spivak, S. Bialik, O. Cohen, G. Shani, M. Eisenstein, and A. Kimchi. 2001. A novel mechanism of negative autophosphorylation restrains the pro-apoptotic function of DAPk J. Biol. Chem. 276: 4745047467.
Wyllie, A.H., J.F. Kerr, and A.R. Currie. 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68:251306.[Medline]
Xiang, J., D.T. Chao, and S.J. Korsmeyer. 1996. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA. 93:1455914563.
Xue, L., G.C. Fletcher, and A.M. Tolkovsky. 2001. Mitochondria are selectively eliminated from eukaryotic cells after blockade of caspases during apoptosis. Curr. Biol. 11:361365.[CrossRef][Medline]
Zakeri, Z., W. Bursch, M. Tenniswood, and R. Lockshin. 1995. Cell death: programmed, apoptosis, necrosis, or other? Cell Death Diff. 2:8796.
Related Article