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Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H(2)O(2)) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H(2)O(2) treatment. Caspase 3 was activated rapidly, reaching a maximum in 12 +/- 3 min, while the average duration of caspase 9 activation was 21 +/- 3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H(2)O(2) treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H(2)O(2)-induced apoptosis. Our results suggest that, during H(2)O(2)-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation. Copyright (C) 2011 S. Karger AG, Basel

Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H(2)O(2)) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H(2)O(2) treatment. Caspase 3 was activated rapidly, reaching a maximum in 12 +/- 3 min, while the average duration of caspase 9 activation was 21 +/- 3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H(2)O(2) treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H(2)O(2)-induced apoptosis. Our results suggest that, during H(2)O(2)-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation. Copyright (C) 2011 S. Karger AG, Basel

Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H(2)O(2)) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H(2)O(2) treatment. Caspase 3 was activated rapidly, reaching a maximum in 12 +/- 3 min, while the average duration of caspase 9 activation was 21 +/- 3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H(2)O(2) treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H(2)O(2)-induced apoptosis. Our results suggest that, during H(2)O(2)-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation. Copyright (C) 2011 S. Karger AG, Basel

Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK] inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL. (C) 2010 Elsevier Inc. All rights reserved.

Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK] inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL. (C) 2010 Elsevier Inc. All rights reserved.

Bax, a member of Bcl-2 family, plays an essential role in apoptotic pathways induced by a number of apoptotic stimulus. In a search for new potential binding partners of Bax, we identified the receptor for activated C-kinase 1 (RACK1) by a yeast two-hybrid assay. We demonstrated that RACK1 interacts with Bax through its BH3 domain both in vitro and in vivo. Using immunostaining and immunoprecipitation experiments, we found that RACK1 colocalizes with Bax oligomers and promotes Bax oligomerization both in vitro and in vivo. Furthermore, we observed that RACK1 also interacts with Bcl-XL, an anti-apoptotic protein associated with Bax. Interestingly, the Bcl-XL/Bax interaction is decreased when RACK1 is overexpressed, but is increased when RACK1 is depleted, suggesting RACK1 disrupts the association of Bax and Bcl-XL. In addition, we found that overexpression of RACK1 promotes UV-induced apoptosis, while knocking down RACK] inhibits the effects. Together, these results indicate that RACK1 promotes apoptosis by promoting Bax oligomerization and dissociating the complex of Bax and Bcl-XL. (C) 2010 Elsevier Inc. All rights reserved.