Dap-Kinase Participates in TNF-α–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)-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.

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Prevention of TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00342.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. G479-G490 ◽

Cited By ~ 36

Author(s):

Sujoy Bhattacharya ◽

Ramesh M. Ray ◽

Leonard R. Johnson

Keyword(s):

Epithelial Cells ◽

Cytochrome C ◽

Critical Role ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Cytochrome C Release ◽

Erk Phosphorylation ◽

Tnf Α ◽

Induced Apoptosis ◽

Jnk Activation

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-α/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by α-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-α/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-α/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-α/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-α/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.

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Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7838-7848.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7838-7848 ◽

Cited By ~ 67

Author(s):

Nerina Gnesutta ◽

Audrey Minden

Keyword(s):

Cell Death ◽

Cell Survival ◽

Intracellular Signaling ◽

Death Receptor ◽

Caspase 8 ◽

Death Domain ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Different Types ◽

Promote Cell Survival

ABSTRACT Normal cell growth requires a precisely controlled balance between cell death and survival. This involves activation of different types of intracellular signaling cascades within the cell. While some types of signaling proteins regulate apoptosis, or programmed cell death, other proteins within the cell can promote survival. The serine/threonine kinase PAK4 can protect cells from apoptosis in response to several different types of stimuli. As is the case for other members of the p21-activated kinase (PAK) family, one way that PAK4 may promote cell survival is by phosphorylating and thereby inhibiting the proapoptotic protein Bad. This leads in turn to the inhibition of effector caspases such as caspase 3. Here we show that in response to cytokines which activate death domain-containing receptors, such as the tumor necrosis factor and Fas receptors, PAK4 can inhibit the death signal by a different mechanism. Under these conditions, PAK4 inhibits apoptosis early in the caspase cascade, antagonizing the activation of initiator caspase 8. This inhibition, which does not require PAK4's kinase activity, may involve inhibition of caspase 8 recruitment to the death domain receptors. This role in regulating initiator caspases is an entirely novel role for the PAK proteins and suggests a new mechanism by which these proteins promote cell survival.

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Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

Molecular and Cellular Biology ◽

10.1128/mcb.19.10.6500 ◽

1999 ◽

Vol 19 (10) ◽

pp. 6500-6508 ◽

Cited By ~ 39

Author(s):

Nanette J. Pazdernik ◽

David B. Donner ◽

Mark G. Goebl ◽

Maureen A. Harrington

Keyword(s):

Sequence Similarity ◽

Kinase Domain ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Death Domain ◽

Interacting Protein ◽

C Terminus ◽

Catalytically Active ◽

Induced Apoptosis

ABSTRACT The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-κB, events critical for proper immune system function. A screen for upstream activators of NF-κB identified a novel serine-threonine kinase capable of activating NF-κB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-κB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-κB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-κB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

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The Death Domain Kinase RIP Is Essential for TRAIL (Apo2L)-Induced Activation of IκB Kinase and c-Jun N-Terminal Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6638-6645.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6638-6645 ◽

Cited By ~ 165

Author(s):

Yong Lin ◽

Anne Devin ◽

Amy Cook ◽

Maccon M. Keane ◽

Michelle Kelliher ◽

...

Keyword(s):

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Death Domain ◽

Iκb Kinase ◽

Tnf Superfamily ◽

Key Factor ◽

Induced Apoptosis ◽

Jnk Activation ◽

Necrosis Factor

ABSTRACT Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-κB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IκB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559–671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.

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Death-Associated Protein Kinase-Related Protein 1, a Novel Serine/Threonine Kinase Involved in Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.1044-1054.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 1044-1054 ◽

Cited By ~ 97

Author(s):

Boaz Inbal ◽

Gidi Shani ◽

Ofer Cohen ◽

Joseph L. Kissil ◽

Adi Kimchi

Keyword(s):

Catalytic Domain ◽

Function Analysis ◽

Regulatory Region ◽

Dominant Negative ◽

Dependent Manner ◽

Related Protein ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Functional Connections ◽

Dap Kinase

ABSTRACT In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca2+/calmodulin (CaM)-regulated serine threonine kinase which shows high degree of homology to DAP kinase. The region of homology spans the catalytic domain and the CaM-regulatory region, whereas the remaining C-terminal part of the protein differs completely from DAP kinase and displays no homology to any known protein. The catalytic domain is also homologous to the recently identified ZIP kinase and to a lesser extent to the catalytic domains of DRAK1 and -2. Thus, DAP kinase DRP-1, ZIP kinase, and DRAK1/2 together form a novel subfamily of serine/threonine kinases. DRP-1 is localized to the cytoplasm, as shown by immunostaining and cellular fractionation assays. It binds to CaM, undergoes autophosphorylation, and phosphorylates an exogenous substrate, the myosin light chain, in a Ca2+/CaM-dependent manner. The truncated protein, deleted of the CaM-regulatory domain, was converted into a constitutively active kinase. Ectopically expressed DRP-1 induced apoptosis in various types of cells. Cell killing by DRP-1 was dependent on two features: the status of the catalytic activity, and the presence of the C-terminal 40 amino acids shown to be required for self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region could override the indispensable role of the C-terminal tail in apoptosis and generated a “superkiller” mutant. A dominant negative fragment of DAP kinase encompassing the death domain was found to block apoptosis induced by DRP-1. Conversely, a catalytically inactive mutant of DRP-1, which functioned in a dominant negative manner, was significantly less effective in blocking cell death induced by DAP kinase. Possible functional connections between DAP kinase and DRP-1 are discussed.

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Induced focal adhesion kinase expression suppresses apoptosis by activating NF-κB signaling in intestinal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00450.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. C1310-C1320 ◽

Cited By ~ 29

Author(s):

Huifang M. Zhang ◽

Kaspar M. Keledjian ◽

Jaladanki N. Rao ◽

Tongtong Zou ◽

Lan Liu ◽

...

Keyword(s):

Epithelial Cells ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Intestinal Epithelial Cells ◽

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Intestinal Epithelial ◽

Tnf Α ◽

Induced Apoptosis

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-κB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-κB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-α plus cycloheximide (TNF-α/CHX). Specific inhibition of NF-κB by the recombinant adenoviral vector containing the IκBα superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-κB activity and increased the sensitivity to TNF-α/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-κB activity and resulted in increased resistance to TNF-α/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-α/CHX-induced apoptosis, at least partially, through the activation of NF-κB signaling in IECs.

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Apoptotic Signal of Fas Is Not Mediated by Ceramide

Blood ◽

10.1182/blood.v91.8.2658.2658_2658_2663 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2658-2663 ◽

Cited By ~ 44

Author(s):

Shu-Ching Hsu ◽

Chia-Cheng Wu ◽

Tien-Yau Luh ◽

Chen-Kung Chou ◽

Shau-Hwa Han ◽

...

Keyword(s):

Cell Death ◽

Free Radical Scavenger ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Radical Scavenger ◽

Metal Chelator ◽

Secondary Messenger ◽

Induced Apoptosis ◽

Erk Kinase ◽

Apoptotic Signal

Ceramide has been suggested as the secondary messenger mediating the apoptotic signal for Fas engagement. By using different inhibitors, we demonstrated here that ceramide is unlikely a mediator of Fas-initiated apoptosis. First, cAMP prevented cell death induced by ceramide but not by Fas. Second, ceramide-triggered, but not Fas-triggered, apoptosis was antagonized by the free radical scavenger C60. Third, the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent Fas-induced apoptosis. Finally, activation of NF-κB inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the mediator for Fas-initiated apoptotic signal.

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Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6286 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6286-6296 ◽

Cited By ~ 257

Author(s):

Tadahiro Kitamura ◽

Yukari Kitamura ◽

Shoji Kuroda ◽

Yasuhisa Hino ◽

Miwa Ando ◽

...

Keyword(s):

Cyclic Nucleotide ◽

Second Messengers ◽

Dominant Negative ◽

Cyclic Nucleotide Phosphodiesterase ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Serine Threonine Kinase ◽

Intact Cells ◽

Threonine Kinase

ABSTRACT Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

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Effects of apigenin, lycopene and astaxanthin on 7β-hydroxycholesterol-induced apoptosis and Akt phosphorylation in U937 cells

British Journal Of Nutrition ◽

10.1017/s0007114507898643 ◽

2008 ◽

Vol 100 (2) ◽

pp. 287-296 ◽

Cited By ~ 20

Author(s):

Sinéad Lordan ◽

Cora O'Neill ◽

Nora M. O'Brien

Keyword(s):

Cell Death ◽

Cell Viability ◽

U937 Cells ◽

Caspase Activation ◽

Serine Threonine Kinase ◽

Akt Phosphorylation ◽

Akt Activation ◽

Threonine Kinase ◽

Low Concentrations ◽

Induced Apoptosis

Oxysterols arise from the enzymic or non-enzymic oxidation of cholesterol and have been shown to be cytotoxic to certain cell lines. In particular, apoptosis induced by the oxysterol 7β-hydroxycholesterol (7β-OH) has been associated with the generation of oxidative stress, cytochrome c release and caspase activation. Due to the fundamental importance of apoptosis in pathological processes, the identification of substances capable of modulating this form of cell death is now actively researched. The objective of the present study was to investigate if apigenin, lycopene and astaxanthin could inhibit 7β-OH-induced apoptosis in U937 cells. Pretreatment with 0·1 μm-astaxanthin protected against apoptosis, while lycopene did not oppose the adverse effects of 7β-OH. At low concentrations, apigenin did not protect against oxysterol-induced apoptosis; however, at higher concentrations it intensified cell death. Additionally, we investigated the effect of 7β-OH, apigenin and astaxanthin on the activation of the serine threonine kinase Akt (phosphorylated Akt:Akt ratio) to determine whether the effect on cell viability and growth was linked to the Akt signalling pathway. Akt activation was decreased in the oxysterol-treated cells compared with control cells; however, this did not attain significance. Interestingly, activation of Akt was significantly reduced compared with control cells following incubation with apigenin and astaxanthin both in the absence and in the presence of 7β-OH. Our data suggest that apigenin, lycopene and astaxanthin failed to protect against 7β-OH-induced apoptosis, and the decrease in cell viability and the increase in apoptotic nuclei induced by the antioxidants appear to be associated with down regulation of Akt activity.

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Cell Death Signaling Pathway Induced by Cholix Toxin, a Cytotoxin and eEF2 ADP-Ribosyltransferase Produced by Vibrio cholerae

Toxins ◽

10.3390/toxins13010012 ◽

2020 ◽

Vol 13 (1) ◽

pp. 12

Author(s):

Kohei Ogura ◽

Kinnosuke Yahiro ◽

Joel Moss

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Vibrio Cholerae ◽

Elongation Factor ◽

Host Cells ◽

Mitogen Activated Protein Kinase ◽

Pseudomonas Exotoxin A ◽

Tnf Α ◽

Induced Apoptosis ◽

Adp Ribosyltransferase

Pathogenic microorganisms produce various virulence factors, e.g., enzymes, cytotoxins, effectors, which trigger development of pathologies in infectious diseases. Cholera toxin (CT) produced by O1 and O139 serotypes of Vibrio cholerae (V. cholerae) is a major cytotoxin causing severe diarrhea. Cholix cytotoxin (Cholix) was identified as a novel eukaryotic elongation factor 2 (eEF2) adenosine-diphosphate (ADP)-ribosyltransferase produced mainly in non-O1/non-O139 V. cholerae. The function and role of Cholix in infectious disease caused by V. cholerae remain unknown. The crystal structure of Cholix is similar to Pseudomonas exotoxin A (PEA) which is composed of an N-terminal receptor-recognition domain and a C-terminal ADP-ribosyltransferase domain. The endocytosed Cholix catalyzes ADP-ribosylation of eEF2 in host cells and inhibits protein synthesis, resulting in cell death. In a mouse model, Cholix caused lethality with severe liver damage. In this review, we describe the mechanism underlying Cholix-induced cytotoxicity. Cholix-induced apoptosis was regulated by mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, which dramatically enhanced tumor necrosis factor-α (TNF-α) production in human liver, as well as the amount of epithelial-like HepG2 cancer cells. In contrast, Cholix induced apoptosis in hepatocytes through a mitochondrial-dependent pathway, which was not stimulated by TNF-α. These findings suggest that sensitivity to Cholix depends on the target cell. A substantial amount of information on PEA is provided in order to compare/contrast this well-characterized mono-ADP-ribosyltransferase (mART) with Cholix.

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