RESUMEN
Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.
RESUMEN
Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Pirazinas/farmacología , Tiofenos/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ácidos Borónicos/uso terapéutico , Bortezomib , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Dexametasona/farmacología , Dexametasona/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Quimioterapia Combinada , Humanos , Lenalidomida , Ratones , Ratones SCID , Datos de Secuencia Molecular , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazinas/uso terapéutico , Distribución Aleatoria , Talidomida/análogos & derivados , Talidomida/farmacología , Talidomida/uso terapéutico , Tiofenos/uso terapéutico , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Asunto(s)
Proteínas Musculares/metabolismo , Poliubiquitina/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica , Secuencias Repetidas en Tándem , Proteínas de Motivos Tripartitos , UbiquitinaciónRESUMEN
As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z'=0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
Asunto(s)
Endopeptidasas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Mediciones Luminiscentes , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Humanos , Proteína NEDD8 , Sensibilidad y Especificidad , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
Converting lead compounds into drug candidates is a crucial step in drug development, requiring early assessment of potency, selectivity, and off-target effects. We have utilized activity-based chemical proteomics to determine the potency and selectivity of deubiquitylating enzyme (DUB) inhibitors in cell culture models. Importantly, we characterized the small molecule PR-619 as a broad-range DUB inhibitor, and P22077 as a USP7 inhibitor with potential for further development as a chemotherapeutic agent in cancer therapy. A striking accumulation of polyubiquitylated proteins was observed after both selective and general inhibition of cellular DUB activity without direct impairment of proteasomal proteolysis. The repertoire of ubiquitylated substrates was analyzed by tandem mass spectrometry, identifying distinct subsets for general or specific inhibition of DUBs. This enabled identification of previously unknown functional links between USP7 and enzymes involved in DNA repair.
Asunto(s)
Aminopiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Proteómica , Tiocianatos/farmacología , Tiofenos/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Aminopiridinas/química , Anticuerpos/química , Anticuerpos/inmunología , Línea Celular , Cromatografía Líquida de Alta Presión , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Interferencia de ARN , Espectrometría de Masas en Tándem , Tiocianatos/química , Tiofenos/química , Ubiquitina Tiolesterasa/inmunología , Ubiquitina Tiolesterasa/metabolismo , Peptidasa Específica de Ubiquitina 7RESUMEN
This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism.
Asunto(s)
Isoenzimas/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Proteínas de Plantas/metabolismo , Ricinus/enzimología , Isoenzimas/genética , Fosfoenolpiruvato Carboxilasa/genética , Fosforilación , Proteínas de Plantas/genética , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Ricinus/genética , Ricinus/metabolismoRESUMEN
Wnt proteins control multiple cell behaviors during development and tissue homeostasis. However, pathological activation of Wnt signaling is the underlying cause of various human diseases. The ubiquitin-proteasome system plays important regulatory functions within the Wnt pathway by regulating the activity of several of its core components. Hence, multiple E3 ubiquitin ligases have been implicated in its regulation. Less is known, however, about the role of ubiquitin-specific proteases in Wnt signaling. Analysis of purified axin-containing protein complexes by liquid chromatography-tandem mass spectrometry revealed the presence of the ubiquitin protease USP34. Our results indicate that USP34 functions downstream of the ß-catenin destruction complex to control the stability of axin and opposes its tankyrase-dependent ubiquitination. Reflecting on the requirement for tight control of axin homeostasis during Wnt signaling, interfering with USP34 function by RNA interference leads to the degradation of axin and to the inhibition of ß-catenin-mediated transcription. Given the numerous human diseases exhibiting spurious Wnt pathway activation, the development of USP34 inhibitors may offer a novel therapeutic opportunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endopeptidasas/metabolismo , Proteínas Represoras/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina , Western Blotting , Diferenciación Celular , Cromatografía Liquida , Neoplasias Colorrectales , Endopeptidasas/genética , Células HEK293 , Homeostasis , Humanos , Células L , Ratones , Fosforilación , Reacción en Cadena de la Polimerasa , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/genética , Estabilidad Proteica , ARN Interferente Pequeño , Transducción de Señal , Espectrometría de Masas en Tándem , Tanquirasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Musculares/antagonistas & inhibidores , Atrofia Muscular/prevención & control , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Biocatálisis/efectos de los fármacos , Western Blotting , Línea Celular , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Glucocorticoides/farmacología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas , Especificidad por Sustrato , Proteínas de Motivos Tripartitos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.
Asunto(s)
Endopeptidasas/metabolismo , Ésteres/metabolismo , Análisis por Matrices de Proteínas/métodos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Pruebas de Enzimas , Inhibidores Enzimáticos/farmacología , Enzimas Inmovilizadas/metabolismo , Humanos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Coloración y Etiquetado , Especificidad por Sustrato/efectos de los fármacosRESUMEN
The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.
Asunto(s)
Enzimas Reparadoras del ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Humanos , Luminiscencia , Proteínas del Tejido Nervioso/metabolismo , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His(6)-tagged small ubiquitin-like modifier (SUMO) to the amino terminus of the reporter enzyme phospholipase A(2) (PLA(2)). Following cleavage by a member of the sentrin specific proteases (SENPs), free PLA(2) is able to turn over its substrate, resulting in the release of a fluorescent product which is readily quantifiable using a fluorimeter or a fluorescence plate reader. The utility of this SUMO-CHOP-Reporter assay platform is demonstrated by its ability to determine K(m) values and to characterize inhibitors of SUMO proteases.
Asunto(s)
Técnicas Biosensibles/métodos , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Proteína SUMO-1/metabolismo , Catálisis , Técnicas de Laboratorio Clínico , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Humanos , Cinética , Péptido Hidrolasas/química , Fosfolipasas A2/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Sensibilidad y Especificidad , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factor de Transcripción CHOP/química , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/fisiologíaRESUMEN
Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme situated at the core of plant C-metabolism. Although its anaplerotic role and control by allosteric effectors, reversible phosphorylation, and oligomerization have been well documented in the endosperm of developing castor oil seeds (COS), relatively little is known about PEPC in germinating COS. The initial phase of COS germination was accompanied by elevated PEPC activity and accumulation of comparable amounts of pre-existing 107-kDa and inducible 110-kDa immunoreactive PEPC polypeptides (p107 and p110, respectively). A 440-kDa PEPC heterotetramer composed of an equivalent ratio of non-phosphorylated p110 and p107 subunits was purified from germinated COS. N-terminal microsequencing, mass spectrometry, and immunoblotting revealed that both subunits arose from the same gene (RcPpc3) that encodes the p107 subunit of a phosphorylated 410-kDa PEPC homotetramer in developing COS but that p110 is a monoubiquitinated form of p107. Tandem mass spectrometry sequencing of a diglycinated tryptic peptide identified Lys-628 as p110's monoubiquitination site. This residue is conserved in vascular plant PEPCs and is proximal to a PEP-binding/catalytic domain. Incubation with a human deubiquitinating enzyme (USP-2 core) converted the p110:p107 PEPC heterotetramer into a p107 homotetramer while significantly reducing the enzyme's K(m)(PEP) and sensitivity to allosteric activators (hexose-Ps, glycerol-3-P) and inhibitors (malate, aspartate). Monoubiquitination is a non-destructive and reversible post-translational modification involved in the control of diverse processes such as transcription, endocytosis, and signal transduction. The current study demonstrates that tissue-specific monoubiquitination of a metabolic enzyme can also occur and that this modification influences its kinetic and regulatory properties.
Asunto(s)
Aceite de Ricino/química , Regulación de la Expresión Génica , Fosfoenolpiruvato Carboxilasa/química , Ubiquitina/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Humanos , Cinética , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxilasa/metabolismo , Proteínas de Plantas/química , Estructura Terciaria de Proteína , Semillas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.
Asunto(s)
Endopeptidasas/metabolismo , Ubiquitina/metabolismo , Animales , Endopeptidasas/aislamiento & purificación , Ratones , Inhibidores de Proteasas/farmacologíaRESUMEN
The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.
Asunto(s)
Replicación del ADN/fisiología , Óvulo/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína SUMO-1/metabolismo , Xenopus laevis/metabolismo , Animales , Cromatina/metabolismo , Daño del ADN/fisiología , Replicación del ADN/genética , ADN de Cadena Simple/metabolismo , Lisina/metabolismo , Óvulo/enzimología , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Recombinantes/metabolismo , Fase S/genética , Factores de Tiempo , Xenopus laevis/genéticaRESUMEN
Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). In this study, we show that human I-2 directly and specifically stimulated recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity was not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A was unaffected by deletion of the I-2 N-terminal PP1 binding motif but was eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 was not additive and occurred without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase.