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Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

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Effect of mitochondria-localized active PKCδ on apoptosis. (A) Mitochondrial translocation of proteolyti-cally activated PKCδ. Mitochondrial fraction was prepared from cells exposed to 5.0 μM MG-132 for 90 or 120 min. Mitochondrial lysates were separated on SDS-PAGE and immunoblotted with PKCδ antibody, and the membrane was reprobed with COX IV to show equal protein loading. (B) Mitochondrial-localized active PKCδ activates caspase-3. After N27 cells were transfected with pCMV/myc/mito containing coding sequence for LacZ, PKCδ-RF or PKCδ-CF, double immunostaining was conducted using the mouse Myc tag primary antibody and rabbit active caspase-3 antibody. The Myc tag and active caspase-3 were visualized using confocal microscopy using Cy3 conjugated anti-mouse (red) and Alexa-488 conjugated anti-rabbit (green) secondary antibodies. (C) TUNEL staining in the transfected cells. After transfection for 24 hrs, cells were subjected to TUNEL staining (green) and immunostaining with Myc tag antibody (red). The images were analysed with fluorescence microscopy. The inserts show higher magnification pictures (600x).
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fig05: Effect of mitochondria-localized active PKCδ on apoptosis. (A) Mitochondrial translocation of proteolyti-cally activated PKCδ. Mitochondrial fraction was prepared from cells exposed to 5.0 μM MG-132 for 90 or 120 min. Mitochondrial lysates were separated on SDS-PAGE and immunoblotted with PKCδ antibody, and the membrane was reprobed with COX IV to show equal protein loading. (B) Mitochondrial-localized active PKCδ activates caspase-3. After N27 cells were transfected with pCMV/myc/mito containing coding sequence for LacZ, PKCδ-RF or PKCδ-CF, double immunostaining was conducted using the mouse Myc tag primary antibody and rabbit active caspase-3 antibody. The Myc tag and active caspase-3 were visualized using confocal microscopy using Cy3 conjugated anti-mouse (red) and Alexa-488 conjugated anti-rabbit (green) secondary antibodies. (C) TUNEL staining in the transfected cells. After transfection for 24 hrs, cells were subjected to TUNEL staining (green) and immunostaining with Myc tag antibody (red). The images were analysed with fluorescence microscopy. The inserts show higher magnification pictures (600x).

Mentions: Next, we examined whether activated PKCδ translocates to subcellular organelles to promote its proapoptotic function. As shown in Fig.5A, MG-132 treatment resulted in substantial and time-dependent accumulation of cleaved PKCδ in the mitochondrial fraction, and only a slight elevation of full-length PKCδ was observed. COX IV protein was used as marker of mitochondrial protein in the Western blot analysis (Fig.5A). Together, these results indicate proteasomal inhibition proteolytically activates PKCδ and causes translocation to mitochondria.


Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Sun F, Kanthasamy A, Song C, Yang Y, Anantharam V, Kanthasamy AG - J. Cell. Mol. Med. (2008)

Effect of mitochondria-localized active PKCδ on apoptosis. (A) Mitochondrial translocation of proteolyti-cally activated PKCδ. Mitochondrial fraction was prepared from cells exposed to 5.0 μM MG-132 for 90 or 120 min. Mitochondrial lysates were separated on SDS-PAGE and immunoblotted with PKCδ antibody, and the membrane was reprobed with COX IV to show equal protein loading. (B) Mitochondrial-localized active PKCδ activates caspase-3. After N27 cells were transfected with pCMV/myc/mito containing coding sequence for LacZ, PKCδ-RF or PKCδ-CF, double immunostaining was conducted using the mouse Myc tag primary antibody and rabbit active caspase-3 antibody. The Myc tag and active caspase-3 were visualized using confocal microscopy using Cy3 conjugated anti-mouse (red) and Alexa-488 conjugated anti-rabbit (green) secondary antibodies. (C) TUNEL staining in the transfected cells. After transfection for 24 hrs, cells were subjected to TUNEL staining (green) and immunostaining with Myc tag antibody (red). The images were analysed with fluorescence microscopy. The inserts show higher magnification pictures (600x).
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Related In: Results  -  Collection

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fig05: Effect of mitochondria-localized active PKCδ on apoptosis. (A) Mitochondrial translocation of proteolyti-cally activated PKCδ. Mitochondrial fraction was prepared from cells exposed to 5.0 μM MG-132 for 90 or 120 min. Mitochondrial lysates were separated on SDS-PAGE and immunoblotted with PKCδ antibody, and the membrane was reprobed with COX IV to show equal protein loading. (B) Mitochondrial-localized active PKCδ activates caspase-3. After N27 cells were transfected with pCMV/myc/mito containing coding sequence for LacZ, PKCδ-RF or PKCδ-CF, double immunostaining was conducted using the mouse Myc tag primary antibody and rabbit active caspase-3 antibody. The Myc tag and active caspase-3 were visualized using confocal microscopy using Cy3 conjugated anti-mouse (red) and Alexa-488 conjugated anti-rabbit (green) secondary antibodies. (C) TUNEL staining in the transfected cells. After transfection for 24 hrs, cells were subjected to TUNEL staining (green) and immunostaining with Myc tag antibody (red). The images were analysed with fluorescence microscopy. The inserts show higher magnification pictures (600x).
Mentions: Next, we examined whether activated PKCδ translocates to subcellular organelles to promote its proapoptotic function. As shown in Fig.5A, MG-132 treatment resulted in substantial and time-dependent accumulation of cleaved PKCδ in the mitochondrial fraction, and only a slight elevation of full-length PKCδ was observed. COX IV protein was used as marker of mitochondrial protein in the Western blot analysis (Fig.5A). Together, these results indicate proteasomal inhibition proteolytically activates PKCδ and causes translocation to mitochondria.

Bottom Line: PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity.Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis.Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.

Show MeSH
Related in: MedlinePlus