RESUMEN
Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.
Asunto(s)
Arabidopsis , Calcio , Homeostasis , Inmunidad de la Planta , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Citosol/metabolismo , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Antiportadores/metabolismoRESUMEN
Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.
Asunto(s)
Oryza , Inmunidad de la Planta , Proteínas de Plantas , Ubiquitina , Animales , Quitina/metabolismo , Homeostasis , Ligandos , Oryza/enzimología , Oryza/inmunología , Oryza/metabolismo , Oryza/microbiología , Fosforilación , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Fosfoserina/metabolismo , Secuencia ConservadaRESUMEN
Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.
Asunto(s)
Fosfoproteínas , Proteínas Serina-Treonina Quinasas , Proteoma , Serina , Treonina , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Especificidad por Sustrato , Treonina/metabolismo , Proteoma/química , Proteoma/metabolismo , Conjuntos de Datos como Asunto , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Línea Celular , Fosfoserina/metabolismo , Fosfotreonina/metabolismoRESUMEN
Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for full-blown metastasis is largely unknown. Here, we show that AMPK (AMP-activated protein kinase), activated in mouse metastasis models, drives pyruvate dehydrogenase complex (PDHc) activation to maintain TCA cycle (tricarboxylic acid cycle) and promotes cancer metastasis by adapting cancer cells to metabolic and oxidative stresses. This AMPK-PDHc axis is activated in advanced breast cancer and predicts poor metastasis-free survival. Mechanistically, AMPK localizes in the mitochondrial matrix and phosphorylates the catalytic alpha subunit of PDHc (PDHA) on two residues S295 and S314, which activates the enzymatic activity of PDHc and alleviates an inhibitory phosphorylation by PDHKs, respectively. Importantly, these phosphorylation events mediate PDHc function in cancer metastasis. Our study reveals that AMPK-mediated PDHA phosphorylation drives PDHc activation and TCA cycle to empower cancer cells adaptation to metastatic microenvironments for metastasis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Ciclo del Ácido Cítrico , Complejo Piruvato Deshidrogenasa/metabolismo , Animales , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular , Activación Enzimática , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Fosforilación , Fosfoserina/metabolismo , Transducción de Señal , Estrés Fisiológico , Análisis de SupervivenciaRESUMEN
Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.
Asunto(s)
Proteína BRCA1/deficiencia , Proteína BRCA2/deficiencia , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN , Exodesoxirribonucleasas/metabolismo , Histonas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisina/metabolismo , Modelos Biológicos , Mutación/genética , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica/efectos de los fármacos , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/genéticaRESUMEN
In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
Asunto(s)
Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separasa/química , Separasa/metabolismo , Secuencias de Aminoácidos , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/ultraestructura , Quinasas CDC2-CDC28/química , Quinasas CDC2-CDC28/metabolismo , Quinasas CDC2-CDC28/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Microscopía por Crioelectrón , Ciclina B1/ultraestructura , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Fosfoserina/metabolismo , Unión Proteica , Dominios Proteicos , Securina/ultraestructura , Separasa/antagonistas & inhibidores , Separasa/ultraestructura , Especificidad por SustratoRESUMEN
Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting-feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes.
Asunto(s)
Colesterol/biosíntesis , Ingestión de Alimentos/fisiología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Línea Celular , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Hígado/metabolismo , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Metabolismo/genética , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fosfoserina/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/deficiencia , Ubiquitinación , Aumento de PesoRESUMEN
Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change-domain closure-that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly; the data suggest that the kinase Ypk2 directly phosphorylates Ser176. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.
Asunto(s)
Arginina , Fosfoserina , Biosíntesis de Proteínas , Proteínas Quinasas , Proteínas Ribosómicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/química , Arginina/metabolismo , Arginina/química , Fosfoserina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Fosforilación , Evolución MolecularRESUMEN
The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.
Asunto(s)
Proteínas 14-3-3 , 3',5'-AMP Cíclico Fosfodiesterasas , Proteínas Quinasas Dependientes de AMP Cíclico , Sistema de Señalización de MAP Quinasas , Humanos , Proteínas 14-3-3/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Fosfoserina/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismoRESUMEN
Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.
Asunto(s)
Segregación Cromosómica , Cinetocoros , Proteínas Serina-Treonina Quinasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Transducción de Señal , Anafase , Anticuerpos Fosfo-Específicos/inmunología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Mitosis , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/inmunología , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismoRESUMEN
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
Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(ßTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.
Asunto(s)
Fosfoserina/química , Fosfotreonina/química , Estructura Terciaria de Proteína , Proteínas/química , Ciclo Celular/genética , Daño del ADN , Reparación del ADN , Humanos , Modelos Moleculares , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Unión Proteica , Proteínas/genética , Proteínas/metabolismoRESUMEN
The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicación del ADN , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína BRCA1/genética , Línea Celular Tumoral , Replicación del ADN/genética , Inestabilidad Genómica/genética , Humanos , Isomerismo , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Recombinasa Rad51/metabolismoRESUMEN
The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3ß) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Cardiopatías/prevención & control , Cardiopatías/fisiopatología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/química , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Animales , Autofagia , Células Cultivadas , Progresión de la Enfermedad , Activación Enzimática , Everolimus/farmacología , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Cardiopatías/genética , Cardiopatías/patología , Humanos , Hipertrofia/tratamiento farmacológico , Hipertrofia/patología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Mutación , Miocitos Cardíacos/patología , Fosforilación , Fosfoserina/metabolismo , Presión , Ratas , Ratas Wistar , Serina/genética , Serina/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genéticaRESUMEN
The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.
Asunto(s)
Diferenciación Celular , Células Epidérmicas/citología , Epidermis/fisiología , Factores Reguladores del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Anomalías Múltiples/genética , Animales , Labio Leporino/genética , Fisura del Paladar/genética , Quistes/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Epidérmicas/metabolismo , Epidermis/embriología , Anomalías del Ojo/genética , Femenino , Dedos/anomalías , Regulación de la Expresión Génica , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Rodilla/anomalías , Articulación de la Rodilla/anomalías , Labio/anomalías , Metabolismo de los Lípidos/genética , Deformidades Congénitas de las Extremidades Inferiores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Sindactilia/genética , Anomalías Urogenitales/genéticaRESUMEN
The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Fosfoserina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/biosíntesis , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Fibroblastos , Genoma/genética , Células HEK293 , Humanos , Ratones , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S , Complejo Shelterina , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed â¼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.
Asunto(s)
Aterosclerosis , Inflamación , Quinasas Asociadas a Receptores de Interleucina-1 , Interleucina-1beta , Músculo Liso Vascular , Miocitos del Músculo Liso , Fosfoserina , Ubiquitina Tiolesterasa , Animales , Humanos , Ratones , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Hiperplasia/metabolismo , Hiperplasia/patología , Inflamación/metabolismo , Inflamación/patología , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fosforilación , Fosfoserina/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , FN-kappa B/metabolismo , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Interleucina-1beta/metabolismo , UbiquitinaciónRESUMEN
Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Dióxido de Carbono/farmacología , Luz , Fosfotransferasas/metabolismo , Estomas de Plantas/fisiología , Estomas de Plantas/efectos de la radiación , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/química , Modelos Biológicos , Mutación/genética , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Fosfoserina/metabolismo , Fosfotransferasas/química , Fototropinas/metabolismo , Estomas de Plantas/efectos de los fármacos , Plantas Modificadas Genéticamente , Dominios Proteicos , ATPasas de Translocación de Protón/metabolismoRESUMEN
Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.
Asunto(s)
Adenilato Quinasa/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , ADN/química , ADN/metabolismo , Metilación de ADN , Diabetes Mellitus/genética , Dioxigenasas , Estabilidad de Enzimas , Epigénesis Genética , Hemoglobina Glucada/análisis , Humanos , Hiperglucemia/metabolismo , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fosforilación , Fosfoserina/metabolismo , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alterations in both cell metabolism and transcriptional programs are hallmarks of cancer that sustain rapid proliferation and metastasis 1 . However, the mechanisms that control the interaction between metabolic reprogramming and transcriptional regulation remain unclear. Here we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We used a kinome-wide RNA interference-based screening method to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent stimulator of glycolysis 2 , is found to be a robust stimulator of SRC-3 that coregulates oestrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes the SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway and enables purine synthesis by transcriptionally upregulating the expression of the enzyme transketolase. In addition, the two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH), which are involved in purine metabolism, were identified as SRC-3 targets that may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation of SRC-3 at Ser857 increases its interaction with the transcription factor ATF4 by stabilizing the recruitment of SRC-3 and ATF4 to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumour growth in mice and prevents metastasis to the lung from an orthotopic setting, as does Ser857Ala-mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in oestrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.