p38 kinase mediates nitric oxide-induced apoptosis of chondrocytes through the inhibition of protein kinase C ζ by blocking autophosphorylationKim, J-S; Park, Z-Y; Yoo, Y-J; Yu, S-S; Chun, J-S
doi: 10.1038/sj.cdd.4401511pmid: 15665819
This study investigated the molecular mechanisms underlying inhibition of protein kinase C (PKC) ζ by p38 kinase during nitric oxide (NO)-induced apoptosis of chondrocytes. Coimmunoprecipitation experiments showed that activation of p38 kinase following addition of an NO donor resulted in a physical association between PKCζ and p38 kinase. Direct interaction of p38 kinase with PKCζ was confirmed in vitro using p38 kinase and PKCζ recombinant proteins. p38 kinase interacts with the regulatory domain of PKCζ and its association blocked PKCζ autophosphorylation. Micro LC–MS/MS analysis using recombinant proteins indicated that the interaction of p38 kinase with PKCζ blocked autophosphorylation of PKCζ on Thr-560, which is required for PKCζ activation. Collectively, our results demonstrate a novel mechanism of PKCζ regulation: following activation by the production of NO, p38 kinase binds directly to the PKCζ regulatory domain, preventing PKCζ autophosphorylation on Thr-560, thereby inhibiting PKCζ activation.
Akt inhibition upregulates FasL, downregulates c-FLIPs and induces caspase-8-dependent cell death in Jurkat T lymphocytesUriarte, S M; Joshi-Barve, S; Song, Z; Sahoo, R; Gobejishvili, L; Jala, V R; Haribabu, B; McClain, C; Barve, S
doi: 10.1038/sj.cdd.4401549pmid: 15665818
In T lymphocytes, the role of Akt in regulating Fas/Fas ligand (FasL)-mediated apoptotic signaling and death is not clearly understood. In this study, we observed that inhibition of Akt causes enhanced expression of FasL mRNA and protein and increased death-inducing signaling complex (DISC) formation with Fas-associated death domain (FADD) and procaspase-8 recruitment. Also, caspase-8 was activated at the DISC with accompanying decrease in c-FLIPs expression. FasL neutralizing antibody significantly decreased apoptotic death in the Akt-inhibited T cells. Additionally, Akt inhibition-induced Fas signaling was observed to link to the mitochondrial pathway via Bid cleavage. Further, inhibition of caspase-8 activity effectively blocked the loss of mitochondrial membrane potential and DNA fragmentation, suggesting that DISC formation and subsequent caspase-8 activation are critical initiating events in Akt inhibition-induced apoptotic death in T lymphocytes. These data demonstrate yet another important survival function governed by Akt kinase in T lymphocytes, which involves the regulation of FasL expression and consequent apoptotic signaling.
Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytesCohen, L Y; Bourbonnière, M; Sabbagh, L; Bouchard, A; Chew, T; Jeannequin, P; Lazure, C; Sékaly, R-P
doi: 10.1038/sj.cdd.4401568pmid: 15650752
Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-κB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.
The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosisPrecht, T A; Phelps, R A; Linseman, D A; Butts, B D; Le, S S; Laessig, T A; Bouchard, R J; Heidenreich, K A
doi: 10.1038/sj.cdd.4401552pmid: 15637643
Cerebellar granule neurons (CGNs) require depolarization for their survival in culture. When deprived of this stimulus, CGNs die via an intrinsic apoptotic cascade involving Bim induction, Bax translocation, cytochrome c release, and caspase-9 and -3 activation. Opening of the mitochondrial permeability transition pore (mPTP) is an early event during intrinsic apoptosis; however, the precise role of mPTP opening in neuronal apoptosis is presently unclear. Here, we show that mPTP opening acts as an initiating event to stimulate Bax translocation to mitochondria. A C-terminal (α9 helix) GFP-Bax point mutant (T182A) that constitutively localizes to mitochondria circumvents the requirement for mPTP opening and is entirely sufficient to induce CGN apoptosis. Collectively, these data indicate that the major role of mPTP opening in CGN apoptosis is to trigger Bax translocation to mitochondria, ultimately leading to cytochrome c release and caspase activation.
Mechanism of mitochondrial stress-induced resistance to apoptosis in mitochondrial DNA-depleted C2C12 myocytesBiswas, G; Anandatheerthavarada, H K; Avadhani, N G
doi: 10.1038/sj.cdd.4401553pmid: 15650755
In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3–4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-XL, and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.
Motoneuronal death during spinal cord development is mediated by oxidative stressSánchez-Carbente, M R; Castro-Obregón, S; Covarrubias, L; Narváez, V
doi: 10.1038/sj.cdd.4401560pmid: 15637642
The involvement of reactive oxygen species (ROS) in neuronal death has been determined in culture, and in association with several neurodegenerative disorders. We examined whether ROS participate in the cell death observed during spinal cord development. We found that the general pattern of high ROS levels, gene expression for some antioxidant enzymes, and motoneuron death correlated positively along spinal cord development. ROS were reduced in spinal cords cultured in the presence of a synthetic superoxide dismutase and catalase mimetic, with a concomitant reduction in cell death and an increase in the number of motoneurons. The number of motoneurons was higher in spinal cords treated with the antioxidant than in those treated with caspase inhibitors. In general, the increase in motoneuron survival did not correlate with the reduction in cells undergoing DNA degradation in the motoneuronal region. These results suggest that ROS are signaling molecules controlling caspase-dependent and caspase-independent programmed motoneuron death, and support the hypothesis that this mechanism is abnormally turned on in some neurodegenerative disorders and aging.
Apoptosis of non-small-cell lung cancer cell lines after paclitaxel treatment involves the BH3-only proapoptotic protein BimLi, R; Moudgil, T; Ross, H J; Hu, H -M
doi: 10.1038/sj.cdd.4401554pmid: 15711598
A significant variation in susceptibility to paclitaxel-mediated killing was observed among a panel of short-term cultured non-small-cell lung cancer (NSCLC) cell lines. Susceptibility to killing by paclitaxel correlated with expression of the BH3-only protein, Bim, but not with other members of Bcl-2 family. NSCLC cell lines with the highest level of Bim expression are most susceptible to apoptosis induction after paclitaxel treatment. Forced expression of Bim increased paclitaxel-mediated killing of cells expressing an undetectable level of Bim. Conversely, knock down of Bim, but not Bcl-2 expression, decreased the susceptibility of tumor cells to paclitaxel-mediated killing. Similar observations were made using a panel of breast and prostate cancer cell lines. Paclitaxel impairs microtubule function, causes G2/M cell cycle blockade, mitochondria damage, and p53-independent apoptosis. These results established Bim as a critical molecular link between the microtubule poison, paclitaxel, and apoptosis.