Browsing by Author "Kornbluth, Sally"

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    A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.
    (Sci Signal, 2013-05-07) Kurokawa, Manabu; Kim, Jiyeon; Geradts, Joseph; Matsuura, Kenkyo; Liu, Liu; Ran, Xu; Xia, Wenle; Ribar, Thomas J; Henao, Ricardo; Dewhirst, Mark W; Kim, Wun-Jae; Lucas, Joseph E; Wang, Shaomeng; Spector, Neil L; Kornbluth, Sally
    In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.
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    Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.
    (J Cell Sci, 2008-11-01) Wu, Judy Qiju; Kornbluth, Sally
    Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.
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    Apoptosis in Drosophila: neither fish nor fowl (nor man, nor worm).
    (J Cell Sci, 2005-05-01) Kornbluth, Sally; White, Kristin
    Studies in a wide variety of organisms have produced a general model for the induction of apoptosis in which multiple signaling pathways lead ultimately to activation of the caspase family of proteases. Once activated, these enzymes cleave key cellular substrates to promote the orderly dismantling of dying cells. A broad similarity exists in the cell death pathways operating in different organisms and there is a clear evolutionary conservation of apoptotic regulators such as caspases, Bcl-2 family members, inhibitor of apoptosis (IAP) proteins, IAP antagonists and caspase activators. Despite this, studies in Caenorhabditis elegans, Drosophila and vertebrates have revealed some apparent differences both in the way apoptosis is regulated and in the way individual molecules contribute to the propagation of the death signal. For example, whereas cytochrome c released from mitochondria clearly promotes caspase activation in vertebrates, there is no documented role for cytochrome c in C. elegans apoptosis and its role in Drosophila is highly controversial. In addition, the apoptotic potency of IAP antagonists appears to be greater in Drosophila than in vertebrates, indicating that IAPs may be of different relative importance in different organisms. Thus, although Drosophila, worms and humans share a host of apoptotic regulators, the way in which they function may not be identical.
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    Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.
    (Proc Natl Acad Sci U S A, 2007-10-16) Wu, Judy Qiju; Hansen, David V; Guo, Yanxiang; Wang, Michael Zhuo; Tang, Wanli; Freel, Christopher D; Tung, Jeffrey J; Jackson, Peter K; Kornbluth, Sally
    Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase.
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    Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.
    (Proc Natl Acad Sci U S A, 2007-12-26) Johnson, Carrie E; Huang, Yolanda Y; Parrish, Amanda B; Smith, Michelle I; Vaughn, Allyson E; Zhang, Qian; Wright, Kevin M; Van Dyke, Terry; Wechsler-Reya, Robert J; Kornbluth, Sally; Deshmukh, Mohanish
    Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.
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    Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.
    (Mol Biol Cell, 2010-08-01) Tang, Wanli; Wu, Judy Qiju; Chen, Chen; Yang, Chih-Sheng; Guo, Jessie Yanxiang; Freel, Christopher D; Kornbluth, Sally
    Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.
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    Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells.
    (Proc Natl Acad Sci U S A, 2013-02-05) Kurokawa, Manabu; Ito, Takahiro; Yang, Chih-Sheng; Zhao, Chen; Macintyre, Andrew N; Rizzieri, David A; Rathmell, Jeffrey C; Deininger, Michael W; Reya, Tannishtha; Kornbluth, Sally
    Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations.
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    HLA-B-associated transcript 3 (Bat3)/Scythe is essential for p300-mediated acetylation of p53.
    (Genes Dev, 2007-04-01) Sasaki, Toru; Gan, Eugene C; Wakeham, Andrew; Kornbluth, Sally; Mak, Tak W; Okada, Hitoshi
    In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21. Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53-p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53.
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    Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.
    (J Cell Biol, 2005-04-11) Casaletto, Jessica B; Nutt, Leta K; Wu, Qiju; Moore, Jonathan D; Etkin, Laurence D; Jackson, Peter K; Hunt, Tim; Kornbluth, Sally
    Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.
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    Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication.
    (Genes Dev, 2007-09-15) Zhu, Wenge; Ukomadu, Chinweike; Jha, Sudhakar; Senga, Takeshi; Dhar, Suman K; Wohlschlegel, James A; Nutt, Leta K; Kornbluth, Sally; Dutta, Anindya
    The MCM2-7 helicase complex is loaded on DNA replication origins during the G1 phase of the cell cycle to license the origins for replication in S phase. How the initiator primase-polymerase complex, DNA polymerase alpha (pol alpha), is brought to the origins is still unclear. We show that And-1/Ctf4 (Chromosome transmission fidelity 4) interacts with Mcm10, which associates with MCM2-7, and with the p180 subunit of DNA pol alpha. And-1 is essential for DNA synthesis and the stability of p180 in mammalian cells. In Xenopus egg extracts And-1 is loaded on the chromatin after Mcm10, concurrently with DNA pol alpha, and is required for efficient DNA synthesis. Mcm10 is required for chromatin loading of And-1 and an antibody that disrupts the Mcm10-And-1 interaction interferes with the loading of And-1 and of pol alpha, inhibiting DNA synthesis. And-1/Ctf4 is therefore a new replication initiation factor that brings together the MCM2-7 helicase and the DNA pol alpha-primase complex, analogous to the linker between helicase and primase or helicase and polymerase that is seen in the bacterial replication machinery. The discovery also adds to the connection between replication initiation and sister chromatid cohesion.
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    Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11.
    (Proc Natl Acad Sci U S A, 2013-12-17) Chen, Chen; Zhang, Liguo; Huang, Nai-Jia; Huang, Bofu; Kornbluth, Sally
    Ataxia telangiectasia mutant (ATM) is an S/T-Q-directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.
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    The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.
    (J Cell Biol, 2012-04-30) Huang, Nai-Jia; Zhang, Liguo; Tang, Wanli; Chen, Chen; Yang, Chih-Sheng; Kornbluth, Sally
    Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.
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    Ubiquitylation of p53 by the APC/C inhibitor Trim39.
    (Proc Natl Acad Sci U S A, 2012-12-18) Zhang, Liguo; Huang, Nai-Jia; Chen, Chen; Tang, Wanli; Kornbluth, Sally
    Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.