Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.
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Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks. (+info)
Activation of caspases and p53 by bovine herpesvirus 1 infection results in programmed cell death and efficient virus release.
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Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. Bovine herpesvirus 1 (BHV-1) induces PCD in peripheral blood mononuclear cells at the G0/G1 phase of the cell cycle (E. Hanon, S. Hoornaert, F. Dequiedt, A. Vanderplasschen, J. Lyaku, L. Willems, and P.-P. Pastoret, Virology 232:351-358, 1997). However, penetration of virus particles is not required for PCD (E. Hanon, G. Meyer, A. Vanderplasschen, C. Dessy-Doize, E. Thiry, and P. P. Pastoret, J. Virol. 72:7638-7641, 1998). The mechanism by which BHV-1 induces PCD in peripheral blood mononuclear cells is not understood, nor is it clear whether nonlymphoid cells undergo PCD following infection. This study demonstrates that infection of bovine kidney (MDBK) cells with BHV-1 leads to PCD, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, DNA laddering, and chromatin condensation. p53 appears to be important in this process, because p53 levels and promoter activity increased after infection. Expression of proteins that are stimulated by p53 (p21(Waf1) and Bax) is also activated after infection. Cleavage of Bcl-xL, a protein that inhibits PCD, occurred after infection, suggesting that caspases (interleukin-1beta-converting enzyme-like proteases) were activated. Other caspase substrates [poly(ADP-ribose) polymerase and actin] are also cleaved during the late stages of infection. Inhibition of caspase activity delayed cytotoxic activity and virus release but increased the overall virus yield. Taken together, these results indicate that nonlymphoid cells undergo PCD near the end of productive infection and further suggest that caspases enhance virus release. (+info)
Caspase-8 is required for cell death induced by expanded polyglutamine repeats.
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We show here that caspase-8 is required for the death of primary rat neurons induced by an expanded polyglutamine repeat (Q79). Expression of Q79 recruited and activated caspase-8. Inhibition of caspase-8 blocked polyglutamine-induced cell death. Coexpression of Q79 with the caspase inhibitor CrmA, a dominant-negative mutant of FADD (FADD DN), Bcl-2, or Bcl-xL, but not an N-terminally tagged Bcl-xL, prevented the recruitment of caspase-8 and inhibited polyglutamine-induced cell death. Furthermore, Western blot analysis revealed the presence of activated caspase-8 in the insoluble fraction of affected brain regions from Huntington's disease (HD) patients but not in those from neurologically unremarkable controls, suggesting the relocation and activation of caspase-8 during the pathogenesis of HD. These results suggest an essential role of caspase-8 in HD-related neural degenerative diseases. (+info)
Caspase-mediated degradation of T-cell receptor zeta-chain.
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We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein. (+info)
Antiapoptotic signaling in LNCaP prostate cancer cells: a survival signaling pathway independent of phosphatidylinositol 3'-kinase and Akt/protein kinase B.
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Constitutive activation of the phosphatidylinositol 3'-kinase (PI3 kinase)-Akt/protein kinase B (PKB) "survival signaling" pathway is a likely mechanism by which many cancers become refractory to cytotoxic therapy. In LNCaP prostate cancer cells, the PTEN phosphoinositide phosphatase is inactivated, leading to constitutive activation of Akt/PKB and resistance to apoptosis. However, apoptosis and inactivation of Akt/PKB can be induced in these cells by treatment with PI3 kinase inhibitors. Surprisingly, androgen, epidermal growth factor, or serum can protect these cells from apoptosis, even in the presence of PI3 kinase inhibitors and without activation of Akt/PKB, indicating the activity of a novel, Akt/PKB-independent survival pathway. This pathway blocks apoptosis at a level prior to caspase 3 activation and release of cytochrome c from mitochondria. (+info)
Tumor necrosis factor-alpha sensitizes prostate cancer cells to gamma-irradiation-induced apoptosis.
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LNCaP prostate cancer cells are highly resistant to induction of programmed cell death by y-irradiation and somewhat sensitive to the death-inducing effects of tumor necrosis factor (TNF)-alpha. Simultaneous exposure of LNCaP cells to TNF-alpha and 8 Gy of irradiation was synergistic and resulted in a 3-fold increase of apoptotic cells within 72 h compared to TNF-alpha alone. It appeared that TNF-alpha sensitized the cells to irradiation because, when cells were irradiated 24 h after exposure to TNF-alpha, increased cell death was observed. In contrast, irradiation delivered 24 h prior to TNF-alpha exposure did not result in more cell death than after TNF-alpha alone. TNF-alpha induced expression of its own mRNA, but TNF-alpha mRNA induction was neither induced nor enhanced by irradiation. Activation of the transcription factor nuclear factor kappaB can be induced by TNF-alpha and has a modulating antiapoptotic effect. But enhancement of TNF-alpha-induced cell death by irradiation did not result from altered activation of nuclear factor kappaB. TNF-alpha treatment of LNCaP cells resulted in partial activation of caspase-8 and -6 but not caspase-3. There was only minimal poly(ADP-ribose) polymerase cleavage seen in LNCaP cells after exposure to both TNF-alpha and irradiation at 72 h, a time when 60% of the cells were apoptotic. Experiments with peptide inhibitors of cysteine and serine proteases suggested that caspases were the predominant mediators of apoptosis induced by TNF-alpha alone but that serine proteases contributed significantly to cell death induced by TNF-alpha plus irradiation. TNF-alpha increased production of ceramide in LNCaP cells 48 h after exposure. Although irradiation alone had no effect on ceramide production in LNCaP cells, TNF-alpha plus irradiation induced significantly more ceramide than TNF-alpha alone. Ceramide production did not occur immediately after exposure to TNF-alpha, but rather was delayed such that ceramide levels were increased only 24 h after exposure to apoptotic stimuli. Moreover, non-toxic levels of exogenous C2-ceramide sensitized LNCaP cells to irradiation similarly to TNF-alpha, suggesting that one mechanism by which LNCaP cells were sensitized to irradiation was by increased intracellular ceramide. Hence, ceramide generation is a critical component in radiation-induced apoptosis in human prostate cancer cells. Inhibition of ceramide generation may provide a selective advantage in the development of radioresistance in prostate cancer. (+info)
The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.
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Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling. (+info)
DRONC, an ecdysone-inducible Drosophila caspase.
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Caspases play an essential role in the execution of programmed cell death in metazoans. Although 14 caspases are known in mammals, only a few have been described in other organisms. Here we describe the identification and characterization of a Drosophila caspase, DRONC, that contains an amino terminal caspase recruitment domain. Ectopic expression of DRONC in cultured cells resulted in apoptosis, which was inhibited by the caspase inhibitors p35 and MIHA. DRONC exhibited a substrate specificity similar to mammalian caspase-2. DRONC is ubiquitously expressed in Drosophila embryos during early stages of development. In late third instar larvae, dronc mRNA is dramatically up-regulated in salivary glands and midgut before histolysis of these tissues. Exposure of salivary glands and midgut isolated from second instar larvae to ecdysone resulted in a massive increase in dronc mRNA levels. These results suggest that DRONC is an effector of steroid-mediated apoptosis during insect metamorphosis. (+info)