RESUMO
Because off-target effects hamper interpretation and validation of RNAi screen data, we developed a bioinformatics method, genome-wide enrichment of seed sequence matches (GESS), to identify candidate off-targeted transcripts in primary screening data. GESS analysis revealed a prominent off-targeted transcript in several screens, including MAD2 (MAD2L1) in a screen for genes required for the spindle assembly checkpoint. GESS analysis results can enhance the validation rate in RNAi screens.
Assuntos
Biologia Computacional/métodos , Interferência de RNA , Transcrição Gênica/genética , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Bases de Dados Genéticas , Biblioteca Gênica , Genoma/genética , Humanos , Proteínas Mad2 , Camundongos , Proteínas Repressoras/genética , Reprodutibilidade dos Testes , Fuso Acromático/metabolismoRESUMO
Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Quimioinformática/métodos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Furthermore, we demonstrate how this data can be incorporated into a drug discovery project aiming to develop therapeutics for schizophrenia using human stem cell-derived neurons. We identified both an on-target activation signature, induced by a set of chemically distinct positive allosteric modulators of the N-methyl-d-aspartate (NMDA) receptor, and independent off-target effects. Overall, the protocol and open-source analysis pipeline are a step toward industrializing RNA-seq for high-complexity transcriptomics studies performed at a saturating scale.
Assuntos
Descoberta de Drogas , Transcriptoma , Descoberta de Drogas/métodos , Humanos , RNA , Reprodutibilidade dos Testes , Análise de Sequência de RNA/métodosRESUMO
Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.
Assuntos
Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Sistemas CRISPR-Cas , Genes Reporter , Humanos , Regiões Promotoras Genéticas , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.
Assuntos
Sistema de Sinalização das MAP Quinases , Melanoma/genética , Melanoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Melanoma/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genéticaRESUMO
The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Pirimidinas/metabolismo , Regulação para Cima , Animais , Linhagem Celular , Cricetinae , Ativação Enzimática , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Monomethylated histone H4 lysine 20 (H4K20me1) is tightly regulated during the cell cycle. The H4K20me1 demethylase PHF8 transcriptionally regulates many cell cycle genes and is therefore predicted to play key roles in the cell cycle. Here, we show that PHF8 protein levels are the highest during G2 phase and mitosis, and we found PHF8 protein stability to be regulated by the ubiquitin-proteasome system. Purification of the PHF8 complex led to the identification of many subunits of the anaphase-promoting complex (APC) associated with PHF8. We showed that PHF8 interacts with the CDC20-containing APC (APC(cdc20)) primarily during mitosis. In addition, we defined a novel, KEN- and D-box-independent, LXPKXLF motif on PHF8 that is required for binding to CDC20. Through various in vivo and in vitro assays, we demonstrate that mutations of the LXPKXLF motif abrogate polyubiquitylation of PHF8 by the APC. APC substrates are typically cell cycle regulators, and consistent with this, the loss of PHF8 leads to prolonged G2 phase and defective mitosis. Furthermore, we provide evidence that PHF8 plays an important role in transcriptional activation of key G2/M genes during G2 phase. Taken together, these findings suggest that PHF8 is regulated by APC(cdc20) and plays an important role in the G2/M transition.
Assuntos
Proteínas Cdc20/metabolismo , Histona Desmetilases/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Sequência Conservada , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Histona Desmetilases/química , Histona Desmetilases/genética , Humanos , Células MCF-7 , Mitose , Mutação de Sentido Incorreto , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Pontos de Checagem da Fase S do Ciclo Celular , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , UbiquitinaçãoRESUMO
Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.
Assuntos
Dano ao DNA , Genoma Humano/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Recombinação Homóloga , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the 12 years since the process of RNA interference (RNAi) was first discovered, great progress has been made in understanding its mechanism and exploiting its ability to silence gene expression to study gene function at a genome-wide level. Its extensive use as a screening method has yielded many published lists of genes that play novel roles in higher eukaryotes. However, the usefulness of this information is potentially limited by the occurrence of unintended off-target effects. Here we review the potential causes of off-target effects and the impact of this phenomenon in interpreting the results of high-throughput RNAi screens. In addition to targeting the intended gene product, artificial short interfering RNAs (siRNAs) can produce off-target effects by down-regulating the expression of multiple mRNAs through microRNA-like targeting of the 3' untranslated region. We examine why this phenomenon can produce high hit rates in siRNA screens and why independent validation of screening results is critical for the approach to yield new biological insights.
Assuntos
Técnicas Genéticas/normas , MicroRNAs/genética , Interferência de RNA , RNA de Cadeia Dupla/química , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Regulação para Baixo , Expressão Gênica , Genômica , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Regiões não Traduzidas , Estudos de Validação como AssuntoRESUMO
BACKGROUND: Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments. METHODOLOGY/PRINCIPAL FINDINGS: Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment. CONCLUSIONS/SIGNIFICANCE: This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division.
Assuntos
Interfase/fisiologia , Microscopia de Fluorescência/métodos , Mitose/fisiologia , Imagem com Lapso de Tempo/métodos , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Interfase/genética , Mitose/genéticaRESUMO
The flux through the de novo pyrimidine biosynthetic pathway is controlled by the multifunctional protein CAD, which catalyzes the first three steps. The cell cycle dependent regulation of pyrimidine biosynthesis is a consequence of sequential phosphorylation of CAD Thr456 and Ser1406 by the MAP kinase and PKA cascades, respectively. Coordinated regulation of the pathway requires precise timing of the two phosphorylation events. These studies show that phosphorylation of purified CAD by PKA antagonizes MAP kinase phosphorylation, and vice versa. Similar results were observed in vivo. Forskolin activation of PKA in BHK-21 cells resulted in a 8.5 fold increase in Ser1406 phosphorylation and severely curtailed the MAP kinase mediated phosphorylation of CAD Thr456. Moreover, the relative activity of MAP kinase and PKA was found to determine the extent of Thr456 phosphorylation. Transfectants expressing elevated levels of MAP kinase resulted in a 11-fold increase in Thr456 phosphorylation, whereas transfectants that overexpress PKA reduced Thr456 phosphorylation 5-fold. While phosphorylation of one site by one kinase may induce conformational changes that interfere with phosphorylation by the other, the observation that both MAP kinase and PKA form stable complexes with CAD suggest that the mutual antagonism is the result of steric interference by the bound kinases. The reciprocal antagonism of CAD phosphorylation by MAP kinase and PKA provides an elegant mechanism to coordinate the cell cycle-dependent regulation of pyrimidine biosynthesis ensuring that signals for up- and down-regulation of the pathway do not conflict.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Di-Hidro-Orotase/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Linhagem Celular , Colforsina/metabolismo , Cricetinae , Cricetulus , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Subunidades Proteicas/metabolismoRESUMO
CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Núcleo Celular/enzimologia , Di-Hidro-Orotase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Aspartato Carbamoiltransferase/genética , Neoplasias da Mama , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Divisão Celular/fisiologia , Fracionamento Celular , Linhagem Celular Tumoral , Cricetinae , Citoplasma/enzimologia , Di-Hidro-Orotase/genética , Di-Hidro-Orotato Desidrogenase , Fator de Crescimento Epidérmico/farmacologia , Imunofluorescência , Humanos , Rim/citologia , Microscopia Confocal , Complexos Multienzimáticos/metabolismo , Mutagênese Sítio-Dirigida , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosforilação , Pirimidinas/metabolismo , Treonina/genéticaRESUMO
The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.
Assuntos
Neoplasias da Mama/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Pirimidinas/biossíntese , Regulação Alostérica , Aspartato Carbamoiltransferase/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Transfecção , Células Tumorais Cultivadas , Uridina Trifosfato/farmacologiaRESUMO
CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Complexos Multienzimáticos/metabolismo , Pirimidinas/biossíntese , Sequência de Aminoácidos , Animais , Aspartato Carbamoiltransferase/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Linhagem Celular , Cricetinae , Humanos , Cinética , Mamíferos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Fragmentos de Peptídeos/química , Fosforilação , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Pirimidinas/biossíntese , Regulação Alostérica , Animais , Carbamoil-Fosfato Sintase (Amônia)/genética , Divisão Celular , Linhagem Celular , Cricetinae , Rim , Cinética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforribosil Pirofosfato/farmacologia , Fosforilação , Proteínas Recombinantes/metabolismo , Transfecção , Uridina Trifosfato/farmacologiaRESUMO
De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G(1), the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G(1). Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.