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ABSTRACT: During the last two decades, the studies on ubiquitination in regulating transcription factor NF-κB activation have elucidated the expanding role of ubiquitination in modulating cellular events by non-proteolytic mechanisms, as well as by proteasomal degradation. The significance of ubiquitination has also been recognized in regulating gene transcription, epigenetic modifications, kinase activation, DNA repair and subcellular translocation. This progress has been translated into novel strategies for developing anti-cancer therapeutics, exemplified by the success of the first FDA-approved proteasome inhibitor drug Bortezomib. Here we discuss the current understanding of the ubiquitin-proteasome system and how it is involved in regulating NF-κB signaling pathways in response to a variety of stimuli. We also focus on the recent progress of anti-cancer drug development targeting various steps of ubiquitination process, and the potential of these drugs in cancer treatment as related to their impact on NF-κB activation.
Current pharmaceutical design 11/2012; · 4.41 Impact Factor
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ABSTRACT: UV-induced stress response involves expression change of a myriad of genes, which play critical roles in modulating cell cycle arrest, DNA repair, and cell survival. Alteration of microRNAs has been found in cells exposed to UV, yet their function in UV stress response remains elusive. Here, we show that UV radiation induces up-regulation of miR-125b, which negatively regulates p38α expression through targeting its 3'-UTR. Increase of miR-125b depends on UV-induced NF-κB activation, which enhances miR-125b gene transcription upon UV radiation. The DNA damage-responsive kinase ATM (ataxia telangiectasia mutated) is indispensable for UV-induced NF-κB activation, which may regulate p38α activation and IKKβ-dependent IκBα degradation in response to UV. Consequently, repression of p38α by miR-125b prohibits prolonged hyperactivation of p38α by UV radiation, which is required for protecting cells from UV-induced apoptosis. Altogether, our data support a critical role of NF-κB-dependent up-regulation of miR-125b, which forms a negative feedback loop to repress p38α activation and promote cell survival upon UV radiation.
Journal of Biological Chemistry 07/2012; 287(39):33036-47. · 4.77 Impact Factor
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ABSTRACT: NF-κB activation induced by genotoxic treatment in cancer cells has been associated with therapeutic resistance in multiple human malignancies. Therapeutic resistance also correlates with high metastatic potential in human cancers, including breast cancer. Whether genotoxic treatment-activated NF-κB also contributes to cancer metastasis following radiation and chemotherapy is unclear. Here, we show that chemotherapeutic drug-induced NF-κB activation promotes breast cancer cell migration and invasion. The increased metastatic potential is dependent on IL-6 induction mediated by genotoxic NF-κB activation. Moreover, genotoxic treatment also up-regulates oncogenic microRNA-21 (miR-21) expression through eliciting NF-κB recruitment to the miR-21 promoter region, where it cooperates with signal transducer and activator of transcription 3 (STAT3) to activate miR-21 transcription. DNA damage-induced histone H3 phosphorylation via activated MSK1 creates an open chromatin structure for NF-κB/STAT3-driven transactivation of miR-21. NF-κB-dependent IL-6 up-regulation is responsible for STAT3 activation and recruitment to the miR-21 promoter upon genotoxic stress. Induction of miR-21 may enable cancer cells to elude DNA damage-induced apoptosis and enhance the metastatic potential of breast cancer cells through repressing expression of PTEN and PDCD4. Our data support a critical role of DNA damage-induced NF-κB activation in promoting cancer metastasis following genotoxic treatment, and NF-κB-dependent miR-21 induction may contribute to both therapeutic resistance and metastasis in breast cancer.
Journal of Biological Chemistry 04/2012; 287(26):21783-95. · 4.77 Impact Factor
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ABSTRACT: DNA damage response upon UV radiation involves a complex network of cellular events required for maintaining the homeostasis and restoring genomic stability of the cells. As a new class of players involved in DNA damage response, the regulation and function of microRNAs in response to UV remain poorly understood. Here we show that UV radiation induces a significant increase of miR-22 expression, which appears to be dependent on the activation of DNA damage responding kinase ATM (ataxia telangiectasia mutated). Increased miR-22 expression may result from enhanced miR-22 maturation in cells exposed to UV. We further found that tumor suppressor gene phosphatase and tensin homolog (PTEN) expression was inversely correlated with miR-22 induction and UV-induced PTEN repression was attenuated by overexpression of a miR-22 inhibitor. Moreover, increased miR-22 expression significantly inhibited the activation of caspase signaling cascade, leading to enhanced cell survival upon UV radiation. Collectively, these results indicate that miR-22 is an important player in the cellular stress response upon UV radiation, which may promote cell survival via the repression of PTEN expression.
Biochemical and Biophysical Research Communications 12/2011; 417(1):546-51. · 2.48 Impact Factor
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ABSTRACT: The transcription factor nuclear factor κB (NF-κB) regulates various cellular processes such as inflammation and apoptosis. The NF-κB essential modulator (NEMO/IKKγ) is indispensable for NF-κB activation by diverse stimuli including genotoxic stress. Here, we show that NEMO linear ubiquitination on K285/309 is critical for genotoxic NF-κB activation. The E3 ligase linear ubiquitin chain assembly complex (LUBAC) facilitates NEMO linear ubiquitination upon genotoxic stress. Inhibiting LUBAC function interrupts the genotoxic NF-κB signalling cascade by attenuating the activation of IKK and TAK1 in response to DNA damage. We further show that the linear ubiquitination of NEMO is a cytoplasmic event, potentially downstream of NEMO nuclear exportation. Moreover, ELKS ubiquitination appears to facilitate linear ubiquitination of NEMO through stabilizing NEMO:LUBAC association upon DNA damage. Deubiquitinating enzyme CYLD inhibits NEMO linear ubiquitination, possibly by disassembling both K63-linked and linear polyubiquitin. We also found that abrogating linear ubiquitination of NEMO significantly increased genotoxin-induced apoptosis, resulting in enhanced sensitivity to chemodrug in cancer cells. Therefore, LUBAC-dependent NEMO linear ubiquitination is critical for genotoxic NF-κB activation and protects cells from DNA damage-induced apoptosis.
The EMBO Journal 08/2011; 30(18):3741-53. · 9.20 Impact Factor
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ABSTRACT: Activation of the transcription factor NF-κB by multiple genotoxic stimuli modulates cancer cell survival. This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic IκB kinase (IKK); however, the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKKγ/NEMO and ELKS (a protein rich in glutamate, leucine, lysine, and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM- and NEMO-dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-κB activation following genotoxic stimuli.
Molecular cell 10/2010; 40(1):75-86. · 14.61 Impact Factor
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ABSTRACT: TDP-43 (43-kDa TAR DNA-binding domain protein) is a major constituent of ubiquitin-positive cytoplasmic aggregates present in neurons of patients with fronto-temporal lobular dementia and amyotrophic lateral sclerosis (ALS). The pathologic significance of TDP-43 aggregation is not known; however, dominant mutations in TDP-43 cause a subset of ALS cases, suggesting that misfolding and/or altered trafficking of TDP-43 is relevant to the disease process. Here, we show that the presenilin-binding protein ubiquilin 1 (UBQLN) plays a role in TDP-43 aggregation. TDP-43 interacted with UBQLN both in yeast and in vitro, and the carboxyl-terminal ubiquitin-associated domain of UBQLN was both necessary and sufficient for binding to polyubiquitylated forms of TDP-43. Overexpression of UBQLN recruited TDP-43 to detergent-resistant cytoplasmic aggregates that colocalized with the autophagosomal marker, LC3. UBQLN-dependent aggregation required the UBQLN UBA domain, was mediated by non-overlapping regions of TDP-43, and was abrogated by a mutation in UBQLN previously linked to Alzheimer disease. Four ALS-associated alleles of TDP-43 also coaggregated with UBQLN, and the extent of aggregation correlated with in vitro UBQLN binding affinity. Our findings suggest that UBQLN is a polyubiquitin-TDP-43 cochaperone that mediates the autophagosomal delivery and/or proteasome targeting of TDP-43 aggregates.
Journal of Biological Chemistry 01/2009; 284(12):8083-92. · 4.77 Impact Factor
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ABSTRACT: The functionally related ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases are critical regulators of DNA damage responses in mammalian cells. ATM and ATR share highly overlapping substrate specificities and show a strong preference for the phosphorylation of Ser or Thr residues followed by Gln. In this report we used a polyreactive phosphospecific antibody (alpha-pDSQ) that recognizes a subset of phosphorylated Asp-Ser-Gln sequences to purify candidate ATM/ATR substrates. This led to the identification of phosphorylation sites in the carboxyl terminus of the minichromosome maintenance protein 3 (MCM3), a component of the hexameric MCM DNA helicase. We show that the alpha-DSQ antibody recognizes tandem DSQ phosphorylation sites (Ser-725 and Ser-732) in the carboxyl terminus of murine MCM3 (mMCM3) and that ATM phosphorylates both sites in vitro. ATM phosphorylated the carboxyl termini of mMCM3 and human MCM3 in vivo and the phosphorylated form of MCM3 retained association with the canonical MCM complex. Although DNA damage did not affect steady-state levels of chromatin-bound MCM3, the ATM-phosphorylated form of MCM3 was preferentially localized to the soluble, nucleoplasmic fraction. This finding suggests that the carboxyl terminus of chromatin-loaded MCM3 may be sequestered from ATM-dependent checkpoint signals. Finally, we show that ATM and ATR jointly contribute to UV light-induced MCM3 phosphorylation, but that ATM is the predominant UV-activated MCM3 kinase in vivo. The carboxyl-terminal ATM phosphorylation sites are conserved in vertebrate MCM3 orthologs suggesting that this motif may serve important regulatory functions in response to DNA damage. Our findings also suggest that DSQ motifs are common phosphoacceptor motifs for ATM family kinases.
Journal of Biological Chemistry 04/2007; 282(12):9236-43. · 4.77 Impact Factor
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ABSTRACT: The transcription factor NF-kappaB modulates apoptotic responses induced by genotoxic stress. We show that NF-kappaB essential modulator (NEMO), the regulatory subunit of IkappaB kinase (IKK) (which phosphorylates the NF-kappaB inhibitor IkappaB), associates with activated ataxia telangiectasia mutated (ATM) after the induction of DNA double-strand breaks. ATM phosphorylates serine-85 of NEMO to promote its ubiquitin-dependent nuclear export. ATM is also exported in a NEMO-dependent manner to the cytoplasm, where it associates with and causes the activation of IKK in a manner dependent on another IKK regulator, a protein rich in glutamate, leucine, lysine, and serine (ELKS). Thus, regulated nuclear shuttling of NEMO links two signaling kinases, ATM and IKK, to activate NF-kappaB by genotoxic signals.
Science 03/2006; 311(5764):1141-6. · 31.20 Impact Factor
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ABSTRACT: The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.
Journal of Biological Chemistry 01/2006; 280(48):40195-200. · 4.77 Impact Factor
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ABSTRACT: Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.
Journal of Biological Chemistry 09/2004; 279(32):34010-4. · 4.77 Impact Factor
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ABSTRACT: Ataxia-telangiectasia (A-T) is a syndrome of cancer susceptibility, immune dysfunction, and neurodegeneration that is caused by mutations in the A-T-mutated (ATM) gene. ATM has been implicated as a critical regulator of cellular responses to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although defective cell cycle-checkpoint regulation and associated genomic instability presumably contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. In addition, although ATM is required for the induction of the p53 transcriptional program in response to DNA damage, the identities of the relevant transcription factors that mediate ATM-dependent changes in gene expression remain largely undetermined. In this article, we describe a signal transduction pathway linking ATM directly to the Ca(2+)/cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylated CREB in vitro and in vivo in response to ionizing radiation (IR) and H(2)O(2) on a stress-inducible domain. IR-induced phosphorylation of CREB correlated with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional coactivator, CREB-binding protein (CBP). A CREB mutant containing Ala substitutions at ATM phosphorylation sites displayed enhanced transactivation potential, resistance to inhibition by IR, and increased binding to CBP. We propose that ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREB-dependent gene expression and that dysregulation of the ATM-CREB pathway may contribute to neurodegeneration in A-T.
Proceedings of the National Academy of Sciences 05/2004; 101(16):5898-903. · 9.68 Impact Factor
