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Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity.
McDonald KR, Guise AJ, Pourbozorgi-Langroudi P, Cristea IM, Zakian VA, Capra JA, Sabouri N
(2016) PLoS Genet 12: e1006238
MeSH Terms: Binding Sites, DNA Helicases, DNA Replication, DNA-Directed DNA Polymerase, Genomic Instability, Multienzyme Complexes, Protein Binding, S Phase, Schizosaccharomyces, Schizosaccharomyces pombe Proteins
Show Abstract · Added April 18, 2017
Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.
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10 MeSH Terms
Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.
Mazalouskas MD, Godoy-Ruiz R, Weber DJ, Zimmer DB, Honkanen RE, Wadzinski BE
(2014) J Biol Chem 289: 4219-32
MeSH Terms: Amino Acid Substitution, Animals, Cattle, Cell Line, Extracellular Signal-Regulated MAP Kinases, Glycoproteins, Humans, Multienzyme Complexes, Mutation, Missense, Phosphorylation, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-raf, Proto-Oncogene Proteins p21(ras), Rats, rac1 GTP-Binding Protein, ras Proteins
Show Abstract · Added March 26, 2014
Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.
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16 MeSH Terms
BRAF fusions define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition.
Hutchinson KE, Lipson D, Stephens PJ, Otto G, Lehmann BD, Lyle PL, Vnencak-Jones CL, Ross JS, Pietenpol JA, Sosman JA, Puzanov I, Miller VA, Pao W
(2013) Clin Cancer Res 19: 6696-702
MeSH Terms: Adult, Cell Line, Tumor, Female, High-Throughput Nucleotide Sequencing, Humans, MAP Kinase Signaling System, Male, Melanoma, Mitogen-Activated Protein Kinase Kinases, Multienzyme Complexes, Mutation, Oncogene Proteins, Fusion, Protein Kinase Inhibitors, Proto-Oncogene Proteins B-raf, Skin Neoplasms, Sulfate Adenylyltransferase
Show Abstract · Added March 10, 2014
PURPOSE - Recurrent "driver" mutations at specific loci in BRAF, NRAS, KIT, GNAQ, and GNA11 define clinically relevant molecular subsets of melanoma, but more than 30% are "pan-negative" for these recurrent mutations. We sought to identify additional potential drivers in "pan-negative" melanoma.
EXPERIMENTAL DESIGN - Using a targeted next-generation sequencing (NGS) assay (FoundationOne™) and targeted RNA sequencing, we identified a novel PAPSS1-BRAF fusion in a "pan-negative" melanoma. We then analyzed NGS data from 51 additional melanomas genotyped by FoundationOne™, as well as melanoma RNA, whole-genome and whole-exome sequencing data in The Cancer Genome Atlas (TCGA), to determine the potential frequency of BRAF fusions in melanoma. We characterized the signaling properties of confirmed molecular alterations by ectopic expression of engineered cDNAs in 293H cells.
RESULTS - Activation of the mitogen-activated protein kinase (MAPK) pathway in cells by ectopic expression of PAPSS1-BRAF was abrogated by mitogen-activated protein kinase kinase (MEK) inhibition but not by BRAF inhibition. NGS data analysis of 51 additional melanomas revealed a second BRAF fusion (TRIM24-BRAF) in a "pan-negative" sample; MAPK signaling induced by TRIM24-BRAF was also MEK inhibitor sensitive. Through mining TCGA skin cutaneous melanoma dataset, we further identified two potential BRAF fusions in another 49 "pan-negative" cases.
CONCLUSIONS - BRAF fusions define a new molecular subset of melanoma, potentially comprising 4% to 8% of "pan-negative" cases. Their presence may explain an unexpected clinical response to MEK inhibitor therapy or assist in selecting patients for MEK-directed therapy.
©2013 AACR.
1 Communities
4 Members
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16 MeSH Terms
The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development.
Venkateswaran A, Sekhar KR, Levic DS, Melville DB, Clark TA, Rybski WM, Walsh AJ, Skala MC, Crooks PA, Knapik EW, Freeman ML
(2014) Cancer Res 74: 38-43
MeSH Terms: Animals, Animals, Genetically Modified, Endothelium, Vascular, Multienzyme Complexes, NADH, NADPH Oxidoreductases, Neovascularization, Physiologic, Zebrafish
Show Abstract · Added March 7, 2014
ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)-mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1's oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization.
1 Communities
3 Members
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7 MeSH Terms
Autotaxin as a novel serum marker of liver fibrosis.
Nakagawa H, Ikeda H, Nakamura K, Ohkawa R, Masuzaki R, Tateishi R, Yoshida H, Watanabe N, Tejima K, Kume Y, Iwai T, Suzuki A, Tomiya T, Inoue Y, Nishikawa T, Ohtomo N, Tanoue Y, Omata M, Igarashi K, Aoki J, Koike K, Yatomi Y
(2011) Clin Chim Acta 412: 1201-6
MeSH Terms: Aged, Area Under Curve, Biomarkers, Female, Hepatitis C, Chronic, Humans, Liver, Liver Cirrhosis, Male, Middle Aged, Multienzyme Complexes, Phosphodiesterase I, Phosphoric Diester Hydrolases, Pyrophosphatases, ROC Curve, Retrospective Studies
Show Abstract · Added May 2, 2014
BACKGROUND - The clinical significance of autotaxin (ATX), a key enzyme for the production of the bioactive lysophospholipid lysophosphatidic acid remains unknown. Serum ATX enzymatic activity reportedly increases in parallel with liver fibrosis and exhibits a gender difference.
METHODS - Serum ATX antigen level, measured easier than the activity, was evaluated as a marker of liver fibrosis in 2 cohorts of chronic liver disease caused by hepatitis C virus.
RESULTS - In the first cohort, serum ATX level correlated significantly with liver fibrosis stage and was the best parameter for prediction of cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.756 in male and 0.760 in female, when compared with serum hyaluronic acid and aminotransferase-to-platelet ratio index, an established marker of liver fibrosis. In another cohort, serum ATX level correlated significantly with liver stiffness, a novel reliable marker of liver fibrosis, being the second-best parameter in male (AUROC, 0.799) and in female (AUROC, 0.876) for prediction of significant fibrosis, and the best parameter in male (AUROC, 0.863) and the third-best parameter in female (AUROC, 0.872) for prediction of cirrhosis, both of which were judged by liver stiffness.
CONCLUSIONS - Serum ATX level may be a novel marker of liver fibrosis.
Copyright © 2011 Elsevier B.V. All rights reserved.
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16 MeSH Terms
Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid.
Altman MK, Gopal V, Jia W, Yu S, Hall H, Mills GB, McGinnis AC, Bartlett MG, Jiang G, Madan D, Prestwich GD, Xu Y, Davies MA, Murph MM
(2010) Mol Cancer 9: 140
MeSH Terms: Animals, Antineoplastic Agents, Cell Line, Tumor, Cell Survival, Gene Expression, Humans, Melanoma, Experimental, Mice, Multienzyme Complexes, Phosphatidic Acids, Phosphodiesterase I, Phosphoric Diester Hydrolases, Pyrophosphatases, RNA, Small Interfering, Receptors, Lysophosphatidic Acid, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Xenograft Model Antitumor Assays
Show Abstract · Added September 20, 2016
BACKGROUND - Although the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective.
RESULTS - Herein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor.
CONCLUSIONS - Thus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.
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18 MeSH Terms
Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system.
Friemann R, Lee K, Brown EN, Gibson DT, Eklund H, Ramaswamy S
(2009) Acta Crystallogr D Biol Crystallogr 65: 24-33
MeSH Terms: Catalytic Domain, Crystallization, Crystallography, X-Ray, Electron Transport Complex III, Flavin-Adenine Dinucleotide, Iron, Mixed Function Oxygenases, Models, Chemical, Multienzyme Complexes, NAD, Protein Binding, Pseudomonas putida, Substrate Specificity, Toluene
Show Abstract · Added August 31, 2017
Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
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14 MeSH Terms
How does arrestin assemble MAPKs into a signaling complex?
Song X, Coffa S, Fu H, Gurevich VV
(2009) J Biol Chem 284: 685-95
MeSH Terms: Animals, Arrestins, COS Cells, Cattle, Cercopithecus aethiops, Humans, MAP Kinase Signaling System, Mitogen-Activated Protein Kinase Kinases, Multienzyme Complexes, Protein Binding, Protein Structure, Tertiary
Show Abstract · Added December 10, 2013
Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule.
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11 MeSH Terms
Thyroid hormone effects on LKB1, MO25, phospho-AMPK, phospho-CREB, and PGC-1alpha in rat muscle.
Branvold DJ, Allred DR, Beckstead DJ, Kim HJ, Fillmore N, Condon BM, Brown JD, Sudweeks SN, Thomson DM, Winder WW
(2008) J Appl Physiol (1985) 105: 1218-27
MeSH Terms: AMP-Activated Protein Kinases, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Animals, Antithyroid Agents, Blotting, Western, Cyclic AMP Response Element-Binding Protein, Disease Models, Animal, Electric Stimulation, Hyperthyroidism, Hypothyroidism, Male, Mitochondrial Proteins, Multienzyme Complexes, Muscle, Skeletal, Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha, Phosphoprotein Phosphatases, Phosphorylation, Promoter Regions, Genetic, Propylthiouracil, Protein Phosphatase 2C, Protein-Serine-Threonine Kinases, RNA, Messenger, RNA-Binding Proteins, Rats, Rats, Sprague-Dawley, Signal Transduction, Thyroxine, Transcription Factors, Triiodothyronine
Show Abstract · Added October 23, 2017
Expression of all of the isoforms of the subunits of AMP-activated protein kinase (AMPK) and AMPK activity is increased in skeletal muscle of hyperthyroid rats. Activity of AMPK in skeletal muscle is regulated principally by the upstream kinase, LKB1. This experiment was designed to determine whether the increase in AMPK activity is accompanied by increased expression of the LKB1, along with binding partner proteins. LKB1, MO25, and downstream targets were determined in muscle extracts in control rats, in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 4 wk, and in rats given 0.01% propylthiouracil (PTU; an inhibitor of thyroid hormone synthesis) in drinking water for 4 wk (hypothyroid group). LKB1 and MO25 increased in the soleus of thyroid hormone-treated rats vs. the controls. In other muscle types, LKB1 responses were variable, but MO25 increased in all. In soleus, MO25 mRNA increased with thyroid hormone treatment, and STRAD mRNA increased with PTU treatment. Phospho-AMPK and phospho-ACC were elevated in soleus and gastrocnemius of hyperthyroid rats. Thyroid hormone treatment also increased the amount of phospho-cAMP response element binding protein (CREB) in the soleus, heart, and red quadriceps. Four proteins having CREB response elements (CRE) in promoter regions of their genes (peroxisome proliferator-activated receptor-gamma coactivator-1alpha, uncoupling protein 3, cytochrome c, and hexokinase II) were all increased in soleus in response to thyroid hormones. These data provide evidence that thyroid hormones increase soleus muscle LKB1 and MO25 content with subsequent activation of AMPK, phosphorylation of CREB, and expression of mitochondrial protein genes having CRE in their promoters.
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30 MeSH Terms
AMP-activated protein kinase phosphorylates transcription factors of the CREB family.
Thomson DM, Herway ST, Fillmore N, Kim H, Brown JD, Barrow JR, Winder WW
(2008) J Appl Physiol (1985) 104: 429-38
MeSH Terms: AMP-Activated Protein Kinases, Acetyl-CoA Carboxylase, Activating Transcription Factor 1, Aminoimidazole Carboxamide, Animals, Cell Line, Cyclic AMP Response Element Modulator, Cyclic AMP Response Element-Binding Protein, Cyclic AMP-Dependent Protein Kinases, Enzyme Activation, Enzyme Induction, Genes, Reporter, Hexokinase, Humans, Liver, Luciferases, Male, Mice, Mice, Knockout, Multienzyme Complexes, Muscle, Skeletal, Phosphorylation, Promoter Regions, Genetic, Protein Kinase Inhibitors, Protein-Serine-Threonine Kinases, Pyrazoles, Pyrimidines, Rats, Rats, Wistar, Recombinant Proteins, Ribonucleotides, Signal Transduction, Time Factors, Transfection
Show Abstract · Added October 23, 2017
AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle-specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein that is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cAMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cAMP-response element (CRE) binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of HEK-293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately threefold over a 24-h time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a CRE in their promoters.
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34 MeSH Terms