RESUMEN
Nuclear localization of the metabolic enzyme PKM2 is widely observed in various cancer types. We identify nuclear PKM2 as a non-canonical RNA-binding protein (RBP) that specifically interacts with folded RNA G-quadruplex (rG4) structures in precursor mRNAs (pre-mRNAs). PKM2 occupancy at rG4s prevents the binding of repressive RBPs, such as HNRNPF, and promotes the expression of rG4-containing pre-mRNAs (the "rG4ome"). We observe an upregulation of the rG4ome during epithelial-to-mesenchymal transition and a negative correlation of rG4 abundance with patient survival in different cancer types. By preventing the nuclear accumulation of PKM2, we could repress the rG4ome in triple-negative breast cancer cells and reduce migration and invasion of cancer cells in vitro and in xenograft mouse models. Our data suggest that the balance of folded and unfolded rG4s controlled by RBPs impacts gene expression during tumor progression.
RESUMEN
Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.
Asunto(s)
Endorribonucleasas/metabolismo , Presión Osmótica , ARN Polimerasa II/metabolismo , ARN/biosíntesis , Estrés Salino , Transcripción Genética , Activación Transcripcional , Regulación hacia Abajo , Endorribonucleasas/genética , Células HEK293 , Humanos , ARN/genética , ARN Polimerasa II/genética , Factores de TiempoRESUMEN
Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.
Asunto(s)
Fosfopéptidos , Proteoma , Humanos , Proteoma/metabolismo , Fosfopéptidos/metabolismo , Fosfoserina/metabolismo , Proteómica , Espectrometría de Masas , FosforilaciónRESUMEN
Gene expression noise (heterogeneity) leads to phenotypic diversity among isogenic individual cells. Our current understanding of gene expression noise is mostly limited to transcription, as separating translational noise from transcriptional noise has been challenging. It also remains unclear how translational heterogeneity originates. Using a transcription-normalized reporter system, we discovered that stop codon readthrough is heterogeneous among single cells, and individual cells with higher UGA readthrough grow faster from stationary phase. Our work also revealed that individual cells with lower protein synthesis levels exhibited higher UGA readthrough, which was confirmed with ribosome-targeting antibiotics (e.g., chloramphenicol). Further experiments and mathematical modeling suggest that varied competition between ternary complexes and release factors perturbs the UGA readthrough level. Our results indicate that fluctuations in the concentrations of translational components lead to UGA readthrough heterogeneity among single cells, which enhances phenotypic diversity of the genetically identical population and facilitates its adaptation to changing environments.
Asunto(s)
Codón de Terminación , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporteros , Microscopía Fluorescente , Transferasas del Grupo 1-Carbono , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Aptitud Genética , Genotipo , Cinética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Modelos Genéticos , Fenotipo , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transcripción Genética , Proteína Fluorescente RojaRESUMEN
Over the past two decades, synthetic biological systems have revolutionized the study of cellular physiology. The ability to site-specifically incorporate biologically relevant non-standard amino acids using orthogonal translation systems (OTSs) has proven particularly useful, providing unparalleled access to cellular mechanisms modulated by post-translational modifications, such as protein phosphorylation. However, despite significant advances in OTS design and function, the systems-level biology of OTS development and utilization remains underexplored. In this study, we employ a phosphoserine OTS (pSerOTS) as a model to systematically investigate global interactions between OTS components and the cellular environment, aiming to improve OTS performance. Based on this analysis, we design OTS variants to enhance orthogonality by minimizing host process interactions and reducing stress response activation. Our findings advance understanding of system-wide OTS:host interactions, enabling informed design practices that circumvent deleterious interactions with host physiology while improving OTS performance and stability. Furthermore, our study emphasizes the importance of establishing a pipeline for systematically profiling OTS:host interactions to enhance orthogonality and mitigate mechanisms underlying OTS-mediated host toxicity.
Asunto(s)
Aminoácidos , Procesamiento Proteico-Postraduccional , Aminoácidos/metabolismo , Fosforilación , AminasRESUMEN
Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.
Asunto(s)
Simportadores/química , Simportadores/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Alineación de Secuencia , Cotransportadores de K ClRESUMEN
Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway.
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Mitocondrias/metabolismo , Mitofagia , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular , Células HeLa , Humanos , Proteínas de Transporte de Membrana , Proteínas Nucleares/metabolismo , Fosforilación , Proteómica/métodos , Proteína Sequestosoma-1 , Factor de Transcripción TFIIIA/metabolismoRESUMEN
Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.
Asunto(s)
Codón de Terminación , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Concentración de Iones de Hidrógeno , MutaciónRESUMEN
Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Quinasas p21 Activadas/metabolismo , Catálisis , Línea Celular , Humanos , Mutagénesis/genética , Mutagénesis/fisiología , Fosforilación/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Quinasas p21 Activadas/genéticaRESUMEN
ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.
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Alanina-ARNt Ligasa/metabolismo , Aminoácidos Diaminos/metabolismo , Aminoacilación de ARN de Transferencia , Alanina/química , Alanina/metabolismo , Aminoácidos Diaminos/química , Cromatografía Liquida , Toxinas de Cianobacterias , Expresión Génica , Humanos , Cinética , Espectrometría de Masas , Serina/química , Serina/metabolismo , Serina-ARNt Ligasa/metabolismoRESUMEN
Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing â¼10(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.
Asunto(s)
Aminoácidos/síntesis química , Aminoácidos/farmacología , Contención de Riesgos Biológicos/métodos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Viabilidad Microbiana/efectos de los fármacos , Biología Sintética/métodos , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Dominio Catalítico/genética , Codón/genética , Medios de Cultivo/química , Medios de Cultivo/farmacología , Ambiente , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Transferencia de Gen Horizontal/genética , Genes Esenciales/genética , Código Genético/genética , Ingeniería Genética/métodos , Genoma Bacteriano/genética , Viabilidad Microbiana/genética , Datos de Secuencia Molecular , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Organismos Modificados Genéticamente/metabolismo , Factores de Terminación de Péptidos/genética , Fenilalanina/química , Fenilalanina/metabolismo , Multimerización de Proteína/genética , ARN de Transferencia/genéticaRESUMEN
Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Animales , Animales Modificados Genéticamente , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Fosforilación , Proteína Quinasa C-epsilon/genética , Proteómica/métodos , ARN Interferente Pequeño/metabolismo , Ratas , Receptor de Insulina/metabolismo , Proteína S6 Ribosómica/metabolismo , Transducción de Señal/fisiologíaRESUMEN
AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK's ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Membrana Celular/enzimología , Uniones Estrechas/enzimología , Animales , Membrana Celular/química , Perros , Células de Riñón Canino Madin Darby , Ratones , Fosforilación/fisiología , Proteína Quinasa C/metabolismo , Uniones Estrechas/química , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.
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Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas de Escherichia coli/biosíntesis , Escherichia coli/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Kanamicina/farmacología , Espectrometría de Masas , Viabilidad Microbiana/efectos de los fármacos , Mutación , Estrés Oxidativo/genética , Peroxirredoxinas/biosíntesis , Peroxirredoxinas/genética , Pliegue de Proteína , Proteómica/métodos , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Estreptomicina/farmacología , Factores de TiempoRESUMEN
With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.
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Angiotensina II/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Animales , Volumen Sanguíneo , Células COS , Chlorocebus aethiops , Electrólitos/metabolismo , Furosemida/farmacología , Células HEK293 , Humanos , Túbulos Renales Distales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/metabolismo , Espironolactona/farmacología , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity. Phenylalanyl-tRNA synthetase (PheRS) maintains specificity via an editing pathway that targets non-cognate Tyr-tRNAPhe. While the primary role of aaRS editing is to prevent misaminoacylation, we demonstrate editing of misaminoacylated tRNA is also required for detection of amino acid starvation by Gcn2p. Ablation of PheRS editing caused accumulation of Tyr-tRNAPhe (5%), but not deacylated tRNAPhe during amino acid starvation, limiting Gcn2p kinase activity and suppressing Gcn4p-dependent gene expression. While the PheRS-editing ablated strain grew 50% slower and displayed a 27-fold increase in the rate of mistranslation of Phe codons as Tyr compared to wild type, the increase in mistranslation was insufficient to activate an unfolded protein stress response. These findings show that during amino acid starvation a primary role of aaRS quality control is to help the cell mount an effective stress response, independent of the role of editing in maintaining translational accuracy.
Asunto(s)
Fenilalanina-ARNt Ligasa/metabolismo , Edición de ARN , ARN de Transferencia de Fenilalanina/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoacilación de ARN de Transferencia , Respuesta de Proteína Desplegada , Aminoácidos/metabolismo , Fenilalanina/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Estrés Fisiológico , Tirosina/metabolismoRESUMEN
The human proteome encodes >500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase-substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library-kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of >100000 serine-oriented human peptides expressed heterologously in Escherichia coli, we perform in vitro kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKCß mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future.
Asunto(s)
Biblioteca de Péptidos , Proteínas Quinasas/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Cromatografía Liquida , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr(1092), Tyr(1094), and Tyr(1143), and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr(1092) or Tyr(1143) to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4(Y1092/1094F). Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser(1196), an effect that was abrogated in the double mutant. WNK4(Y1143F) inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4(Y1143F) could not be reversed by SGK1. The failure to reverse WNK4(Y1143F)-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4-PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4(Y1143F)-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr(1092) and Tyr(1143) plays an important role in modulating the inhibitory effect of WNK4 on ROMK.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Sitios de Unión , Cromatografía Liquida , Electrofisiología , Células HEK293 , Humanos , Hiperpotasemia/metabolismo , Hipopotasemia/metabolismo , Ratones , Mutación , Nefronas/metabolismo , Fosforilación , Ratas , Espectrometría de Masas en Tándem , Titanio/química , Tirosina/químicaRESUMEN
The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB(S65A)-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB(S65A)-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.
Asunto(s)
Mitocondrias/metabolismo , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Células HeLa , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Mitofagia , Modelos Biológicos , Fosforilación , Proteínas Quinasas/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Recent advances in mass spectrometry-based proteomics have revealed translation of previously nonannotated microproteins from thousands of small open reading frames (smORFs) in prokaryotic and eukaryotic genomes. Facile methods to determine cellular functions of these newly discovered microproteins are now needed. Here, we couple semiquantitative comparative proteomics with whole-genome database searching to identify two nonannotated, homologous cold shock-regulated microproteins in Escherichia coli K12 substr. MG1655, as well as two additional constitutively expressed microproteins. We apply molecular genetic approaches to confirm expression of these cold shock proteins (YmcF and YnfQ) at reduced temperatures and identify the noncanonical ATT start codons that initiate their translation. These proteins are conserved in related Gram-negative bacteria and are predicted to be structured, which, in combination with their cold shock upregulation, suggests that they are likely to have biological roles in the cell. These results reveal that previously unknown factors are involved in the response of E. coli to lowered temperatures and suggest that further nonannotated, stress-regulated E. coli microproteins may remain to be found. More broadly, comparative proteomics may enable discovery of regulated, and therefore potentially functional, products of smORF translation across many different organisms and conditions.