Interaction of a cyclin E fragment with Ku70 regulates Bax-mediated apoptosis.
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ABSTRACT:
SUBMITTER: Mazumder S
PROVIDER: S-EPMC1899959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA repair deficiency results in neurodegenerative disease and increased susceptibility to excitotoxic cell death, suggesting a critical but undefined role for DNA damage in neurodegeneration. We compared DNA damage, Ku70-Bax interaction, and Bax-dependent excitotoxic cell death in kainic acid-treated primary cortical neurons derived from both wild-type mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wild-type and Prkdc(-/-) neurons, kainic acid treatment resulted in rapid induction of DNA damage (53BP1 foci formation) followed by nuclear pyknosis. Bax deficiency, by either Bax shRNA-mediated knockdown or gene deletion, protected wild-type and heterozygous but not Prkdc(-/-) neurons from kainate-induced excitotoxicity. Cotransfection of DNA-PKcs with Bax shRNA restored Bax shRNA-mediated neuroprotection in Prkdc(-/-) neurons, suggesting that DNA-PKcs is required for kainate-induced activation of the pro-apoptotic Bax pathway. Immunoprecipitation studies revealed that the DNA-PKcs-nonphosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wild-type Ku70, suggesting that DNA-PKcs-mediated Ku70 phosphorylation causes release of Bax from Ku70. In support of this, kainic acid induced translocation of a Bax-EGFP fusion protein to the mitochondria in the presence of a cotransfected wild-type, but not mutant Ku70 (S6A/S51A) gene when examined at 4 and 8 h following kainate addition. We conclude that DNA-PKcs links DNA damage to Bax-dependent excitotoxic cell death, by phosphorylating Ku70 on serines 6 and/or 51, to initiate Bax translocation to the mitochondria and directly activate a pro-apoptotic Bax-dependent death cascade.
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Project description:The mammalian KU70 is a pleiotropic protein functioning in DNA repair and cytoplasmic suppression of apoptosis. We report a regulatory mechanism by which KU70's cytoplasmic function is enabled due to a methylation at K570 of KU70 by SET-domain-containing protein 4 (SETD4). While SETD4 silencing reduces the level of methylated KU70, over-expression of SETD4 enhances methylation of KU70. Mutations of Y272 and Y284 of SETD4 abrogate methylation of KU70. Although SETD4 is predominantly a nuclear protein, the methylated KU70 is enriched in the cytoplasm. SETD4 knockdown enhances staurosporine (STS)-induced apoptosis and cell killing. Over-expression of the wild-type (WT) SETD4, but not the SETD4-Y272/Y284F mutant, suppresses STS-induced apoptosis. The KU70-K570R (mouse Ku70-K568R) mutation dampens the anti-apoptosis activity of KU70. Our study identifies KU70 as a non-histone substrate of SETD4, discovers a post-translational modification of KU70, and uncovers a role for SETD4 and KU70-K570 methylation in the suppression of apoptosis.
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Project description:The severely debilitating disease Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the gene encoding laminin-alpha2. Bax-mediated muscle cell death is a significant contributor to the severe neuromuscular pathology seen in the Lama2-null mouse model of MDC1A. To extend our understanding of pathogenesis due to laminin-alpha2-deficiency, we have now analyzed molecular mechanisms of Bax regulation in normal and laminin-alpha2-deficient muscles and cells, including myogenic cells obtained from patients with a clinical diagnosis of MDC1A. In mouse myogenic cells, we found that, as in non-muscle cells, Bax co-immunoprecipitated with the multifunctional protein Ku70. In addition, cell permeable pentapeptides designed from Ku70, termed Bax-inhibiting peptides (BIPs), inhibited staurosporine-induced Bax translocation and cell death in mouse myogenic cells. We also found that acetylation of Ku70, which can inhibit binding to Bax and can be an indicator of increased susceptibility to cell death, was more abundant in Lama2-null than in normal mouse muscles. Furthermore, myotubes formed in culture from human laminin-alpha2-deficient patient myoblasts produced high levels of activated caspase-3 when grown on poly-L-lysine, but not when grown on a laminin-alpha2-containing substrate or when treated with BIPs. Finally, cytoplasmic Ku70 in human laminin-alpha2-deficient myotubes was both reduced in amount and more highly acetylated than in normal myotubes. Increased susceptibility to cell death thus appears to be an intrinsic property of human laminin-alpha2-deficient myotubes. These results identify Ku70 as a regulator of Bax-mediated pathogenesis and a therapeutic target in laminin-alpha2-deficiency.
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Project description:Intrinsic apoptosis requires mitochondrial outer membrane disruption triggered by recruitment, activation, and oligomerization of the Bcl-2 homology protein Bax. Following oxidative stress, we demonstrated that the transcriptional regulator cyclin C is released into the cytosol where it directs mitochondrial fragmentation and efficient apoptotic induction. This study reveals that cytoplasmic cyclin C is required for both normal Bax activation and its efficient mitochondrial localization. This activity appears direct as cyclin C co-immunoprecipitates with active Bax in stressed cells and binds recombinant Bax in vitro. In addition, stable cyclin C-Bax association requires the fission complex. Pharmacologically stimulating cyclin C nuclear release is sufficient for Bax association and their mitochondrial localization in the absence of any stress signals. However, these cells do not undergo cell death as Bax fails to oligomerize. These data support a model that cyclin C association defines an initial step in Bax mitochondrial recruitment and provides a physical connection between the fission and apoptotic factors. This strategy allows the cell to discriminate stress-induced fission able to recruit Bax from other types of mitochondrial divisions.
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Project description:The Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family, involved in inducing apoptosis at the mitochondrial level. Regulating the protein levels of Bax is essential to enhancing apoptosis. In the current study, we ascertained the presence of deubiquitinating enzymes (DUBs) associated with Bax by performing the yeast two-hybrid screening (Y2H). We determined that ubiquitin-specific protease 12 (USP12), one of the DUBs, is associated with Bax. The binding of USP12 to Bax shows the interaction as a DUB, which regulates ubiquitination on Bax. Taken together, we believe that USP12 regulates Bax by detaching ubiquitin on K63-linked chains, indicating that USP12 affects the cellular functions of Bax, but it is not related with proteasomal degradation. The half-life of the Bax protein was determined by performing the site-directed mutagenesis of putative ubiquitination sites on Bax (K128R, K189R, and K190R). Of these, Bax (K128R and K190R) showed less ubiquitination; therefore, we compared the half-life of Bax (WT) and Bax K mutant forms in vitro. Interestingly, Bax (K189R) showed a higher ubiquitination level and shorter half-life than Bax (WT), and the (K128R and K190R) mutant form has a longer half-life than Bax (WT).
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Project description:Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown.The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches.We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax.We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.
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