Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microinjection of the restriction endonuclease HaeIII, which causes DNA double-strand breaks with blunt ends, induces nuclear accumulation of p53 protein in normal and xeroderma pigmentosum (XP) primary fibroblasts. In contrast, this induction of p53 accumulation is not observed in ataxia telangiectasia (AT) fibroblasts. HaeIII-induced p53 protein in normal fibroblasts is phosphorylated at serine 15, as determined by immunostaining with an antibody specific for phosphorylated serine 15 of p53. This phosphorylation correlates well with p53 accumulation. Treatment with lactacystin (an inhibitor of the proteasome) or heat shock leads to similar levels of p53 accumulation in normal and AT fibroblasts, but the p53 protein lacks a phosphorylated serine 15. Following microinjection of HaeIII into lactacystin-treated normal fibroblasts, lactacystin-induced p53 protein is phosphorylated at serine 15 and stabilized even in the presence of cycloheximide. However, neither stabilization nor phosphorylation at serine 15 is observed in AT fibroblasts under the same conditions. These results indicate the significance of serine 15 phosphorylation for p53 stabilization after DNA double-strand breaks and an absolute requirement for ATM in this phosphorylation process.
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PMID:Requirement of ATM in phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks. 1008 48

Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a protein that targets p53 for degradation in the proteasome. Dissociation of p53 from Mdm2 could be caused by DNA damage-induced p53 posttranslational modifications. The ATM and ATR kinases, whose activation in response to ionizing radiation (IR) and UV light, respectively, is required for p53 stabilization, directly phosphorylate p53 on Ser-15. However, phosphorylation of Ser-15 is critical for the apoptotic activity of p53 and not for p53 stabilization. Thus, whether any p53 modifications, and which, underlie disruption of the p53-Mdm2 complex after DNA damage remains to be determined. We analyzed the IR- and UV light-induced stabilization of p53 proteins with substitutions of Ser known to be posttranslationally modified after DNA damage. Substitution of Ser-20 was sufficient to abrogate p53 stabilization in response to both IR and UV light. Furthermore, both IR and UV light induced phosphorylation of p53 on Ser-20, which involved the majority of nuclear p53 protein and weakened the interaction of p53 with Mdm2 in vitro. ATM and ATR cannot phosphorylate p53 on Ser-20. We therefore propose that ATM and ATR activate an, as yet unidentified, kinase that stabilizes p53 by phosphorylating it on Ser-20.
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PMID:Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. 1057 Jan 49

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.
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PMID:Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after gamma-irradiation by stabilization of the protein. 1077 Oct 89

The p53 tumour suppressor protein is a short-lived transcription factor that becomes stabilized in response to a wide range of cellular stresses. Ubiquitination and the targeting of p53 for degradation by the proteasome are mediated by Mdm2 (mouse double minute clone 2), a negative regulatory partner of p53. Previous studies have suggested that DNA-damage-induced phosphorylation of p53 at key N-terminal sites has a pivotal role in regulating the interaction with Mdm2 but the precise role of phosphorylation of serines 15 and 20 is still unclear. Here we show that replacement of serine 15 and a range of other key N-terminal phosphorylation sites with alanine, which cannot be phosphorylated, has little effect on the ubiquitination and degradation of full-length human p53. In contrast, replacement of serine 20 makes p53 highly sensitive to Mdm2-mediated turnover. These results define distinct roles for serines 15 and 20, two sites previously demonstrated to be dependent on phosphorylation through mechanisms mediated by DNA damage and ATM (ataxia telangiectasia mutated). We also show that the polyproline region of p53, a domain that has a key role in p53-induced apoptosis, exerts a critical influence over the Mdm2-mediated turnover of p53.
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PMID:Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover. 1158 95

Eukaryotic cells respond to DNA damage and stalled replication forks by activating protein kinase-mediated signaling pathways that promote cell cycle arrest and DNA repair. A central target of the cell cycle arrest program is the Cdc25A protein phosphatase. Cdc25A is required for S-phase entry and dephosphorylates tyrosine-15 phosphorylated Cdk1 (Cdc2) and Cdk2, positive regulators of cell division. Cdc25A is unstable during S-phase and is degraded through the ubiquitin-proteasome pathway, but its turnover is enhanced in response to DNA damage. Although basal and DNA-damage-induced turnover depends on the ATM-Chk2 and ATR-Chk1 pathways, how these kinases engage the ubiquitin ligase machinery is unknown. Here, we demonstrate a requirement for SCFbeta-TRCP in Cdc25A turnover during an unperturbed cell cycle and in response to DNA damage. Depletion of beta-TRCP stabilizes Cdc25A, leading to hyperactive Cdk2 activity. SCFbeta-TRCP promotes Chk1-dependent Cdc25A ubiquitination in vitro, and this involves serine 76, a known Chk1 phosphorylation site. However, recognition of Cdc25A by beta-TRCP occurs via a noncanonical phosphodegron in Cdc25A containing phosphoserine 79 and phosphoserine 82, sites that are not targeted by Chk1. These data indicate that Cdc25A turnover is more complex than previously appreciated and suggest roles for an additional kinase(s) in Chk1-dependent Cdc25A turnover.
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PMID:SCFbeta-TRCP links Chk1 signaling to degradation of the Cdc25A protein phosphatase. 1468 Dec 6

Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin-proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells.
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PMID:Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells. 1569 85

Camptothecin (CPT) is a potent inhibitor of DNA topoisomerase I with a wide spectrum of anti-tumor activity. Relatively little information is available regarding the relation of known topoisomerase-mediated DNA damage with other intracellular pathways. To gain an insight into the intracellular molecular mechanisms of Topoisomerase I inhibitor camptothecin-mediated DNA damage leading to cell death, we used a high-density cDNA microarray to assess sensitive early gene expression profiles in SGC7901 (gastric cancer), Hela (cervical adenocarcinoma), K562 (chronic myelogenous leukemia) and HL60 (promyelocytic leukemia) tumor cells stimulated with camptothecin for 1 h at the concentrations of GI50 (50 % growth inhibition after 24 h of treatment). Analysis of the differentially expressed genes obtained 29 response genes common to all four cell lines. Moreover, these cell lines also shared the direction of regulation. Most of these common response genes were functionally related to cell proliferation or apoptosis, and some of them were involved in ATM (ataxia-telangiectasia mutated) and ATR (ATM-and Rad3 related) checkpoint pathways, JNK (c-Jun N-terminal kinase) pathway, the survival phosphatidylinositol (PI) 3 kinase-Akt-dependent pathway, mitochondrial cell death pathway, endoplasmic reticulum (ER)-related cell death pathway, and to ubiquitin/proteasome dependent protein degradation pathway. The data provides evidence for a linkage between topoisomerase-mediated DNA damage and intracellular signaling events, which may facilitate our understanding of the camptothecin mediated molecular mechanisms of action.
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PMID:Analysis of common gene expression patterns in four human tumor cell lines exposed to camptothecin using cDNA microarray: identification of topoisomerase-mediated DNA damage response pathways. 1636 68

In response to DNA breaks, human cells delay their progression through the G1, S, and G2 phases of the cell cycle. This response requires the coordinated effort of the ATM-CHK2-p53 and ATR-CHK1 DNA damage-sensing pathways and DNA repair (eg, DNA-PK and RAD51 complexes). The turnover of many of these DNA damage-associated proteins is controlled by the 26S proteasome. In this article, we review molecular strategies that target each of these pathways using silencing RNA (siRNA), antisense, or small-molecule inhibition. Although these agents can radiosensitize tumor cells, little data are available regarding potential effects on normal tissues to determine the potential therapeutic ratio of these strategies after fractionated radiotherapy. Clinical trials using such agents will require novel correlative science endpoints to track DNA repair and cell-cycle arrest and will need careful assessment of normal tissue toxicity and stability.
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PMID:Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. 1637 7

Mammalian DNA polymerase (Pol) delta is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol delta in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol delta by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol delta function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR(-/-) cells but not in ATM(-/-) cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol delta in both DNA replication and DNA repair.
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PMID:A novel DNA damage response: rapid degradation of the p12 subunit of dna polymerase delta. 1731 65

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.
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PMID:A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints. 1747 28


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