E3 ubiquitin ligase COP1 regulates the stability and functions of MTA1

Department of Biochemistry and Molecular Biology and Institute of Coregulator Biology, The George Washington University Medical Center, Washington, DC 20037, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2009; 106(41):17493-8. DOI: 10.1073/pnas.0908027106
Source: PubMed


Metastasis-associated protein 1 (MTA1), a component of the nucleosome remodeling and histone deacetylation (NuRD) complex, is widely upregulated in human cancers. However, the mechanism for regulating its protein stability remains unknown. Here we report that MTA1 is an ubiquitinated protein and targeted by the RING-finger E3 ubiquitin-protein ligase constitutive photomorphogenesis protein 1 (COP1) for degradation via the ubiquitin-proteasome pathway. Induced expression of wild-type COP1 but not its RING motif mutants promotes the ubiquitination and degradation of MTA1, indicating that the ligase activity is required for the COP1-mediated proteolysis of MTA1. Conversely, depletion of endogenous COP1 resulted in a marked decrease in MTA1 ubiquitination, accompanied by a pronounced accumulation of MTA1 protein. MTA1, in turn, destabilizes COP1 by promoting its autoubiquitination, thus creating a tight feedback loop that regulates both MTA1 and COP1 protein stability. Accordingly, disruption of the COP1-mediated proteolysis by ionizing radiation leads to MTA1 stabilization, accompanied by an increased coregulatory function of MTA1 on its target. Furthermore, we discovered that MTA1 is required for optimum DNA double-strand break repair after ionizing radiation. These findings provide novel insights into the regulation of MTA1 protein and reveal a novel function of MTA1 in DNA damage response.

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    • "RT-qPCR and Western blot analysis were performed following the protocol described previously [53], [54]. In brief, total RNA was isolated by using TRIzol reagent (Invitrogen), and 2 µg of total RNA was reverse-transcribed using the SuperScript III First-strand synthesis system for reverse transcriptase-PCR (Invitrogen). "
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    ABSTRACT: P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1 biology.
    PLoS ONE 08/2013; 8(8):e66585. DOI:10.1371/journal.pone.0066585 · 3.23 Impact Factor
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    • "In mammalian cells, COP1 regulates various cellular targets, including stress-responsive transcription factors, p53 tumor suppressor (Dornan et al., 2004a), c-JUN (Bianchi et al., 2003; Savio et al., 2008; Wertz et al., 2004), acetyl-coA carboxylase (Qi et al., 2006), TORC2 (Dentin et al., 2007), MVP (Yi et al., 2005) and nucleosome remodeling factor MTA1 (Li et al., 2009a), suggesting its versatile functions. Many COP1 associated proteins remain to be characterized. "
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    ABSTRACT: 14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop, which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediators such as Akt. In this study, we identify mammalian constitutive photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasomal degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 expression leads to stabilization of COP1 through reducing COP1 self-ubiquitination and decelerating COP1's turnover rate. We also show that CSN6-mediated 14-3-3σ ubiquitination is compromised when COP1 is knocked down. Thus, CSN6 mediates 14-3-3σ ubiquitination through enhancing COP1 stability. Subsequently, we show that CSN6 causes 14-3-3σ downregulation, thereby activating Akt and promoting cell survival. Also, CSN6 overexpression leads to increased cell growth, transformation and promotes tumorigenicity. Significantly, 14-3-3σ expression can correct the abnormalities mediated by CSN6 expression. These data suggest that the CSN6-COP1 axis is involved in 14-3-3σ degradation, and that deregulation of this axis will promote cell growth and tumorigenicity.
    Oncogene 05/2011; 30(48):4791-801. DOI:10.1038/onc.2011.192 · 8.46 Impact Factor
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    • "In mammalian cells, COP1 regulates various cellular functions, such as proliferation and survival, by facilitating the degradation of physiological substrates through ubiquitin-mediated protein degradation. The ubiquitinated targets of COP1 include stress-responsive transcription factors, p53 tumor suppressor [2], c-JUN [3-5], transducer of regulated CREB activity 2 (TORC2, a glucose metabolite regulator) [6], FOXO1 [7] and nucleosome remodeling factor MTA1 [8]. It has been shown that DNA damage leads to COP1 nuclear exclusion, [9,10] however, there is a knowledge gap about how DNA damage regulates COP1's translocation from the nucleus to the cytoplasm. "
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    ABSTRACT: Mammalian constitutive photomorphogenic 1 (COP1) is a p53 E3 ubiquitin ligase involved in regulating p53 protein level. In plants, the dynamic cytoplasm/nucleus distribution of COP1 is important for its function in terms of catalyzing the degradation of target proteins. In mammalian cells, the biological consequence of cytoplasmic distribution of COP1 is not well characterized. Here, we show that DNA damage leads to the redistribution of COP1 to the cytoplasm and that 14-3-3σ, a p53 target gene product, controls COP1 subcellular localization. Investigation of the underlying mechanism suggests that COP1 S387 phosphorylation is required for COP1 to bind 14-3-3σ. Significantly, upon DNA damage, 14-3-3σ binds to phosphorylated COP1 at S387, resulting in COP1's accumulation in the cytoplasm. Cytoplasmic COP1 localization leads to its enhanced ubiquitination. We also show that N-terminal 14-3-3σ interacts with COP1 and promotes COP1 nuclear export through its NES sequence. Further, we show that COP1 is important in causing p53 nuclear exclusion. Finally, we demonstrate that 14-3-3σ targets COP1 for nuclear export, thereby preventing COP1-mediated p53 nuclear export. Together, these results define a novel, detailed mechanism for the subcellular localization and regulation of COP1 after DNA damage and provide a mechanistic explanation for the notion that 14-3-3σ's impact on the inhibition of p53 E3 ligases is an important step for p53 stabilization after DNA damage.
    Molecular Cancer 09/2010; 9(1):243. DOI:10.1186/1476-4598-9-243 · 4.26 Impact Factor
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