RESUMEN
Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.
Asunto(s)
Endopeptidasas/química , Lisina/química , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/química , Ubiquitina/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Senescencia Celular/fisiología , Chaperonas de Histonas/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Senescencia Celular/genética , Cromatina/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Chaperonas de Histonas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Papiloma/patología , Neoplasias Cutáneas/patología , Tamoxifeno/farmacología , Factores de Transcripción/genéticaRESUMEN
Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acetilglucosamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Factor A de Crecimiento Endotelial Vascular/farmacología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Alanina/química , Alanina/metabolismo , Sustitución de Aminoácidos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glucosa/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Hexosaminas/biosíntesis , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleótidos/farmacología , Serina/química , Serina/metabolismoRESUMEN
Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2CRTC2HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMPPKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes.
Asunto(s)
Glucosa/metabolismo , Histona Desacetilasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Línea Celular , AMP Cíclico/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Células HEK293 , Histona Desacetilasas/genética , Humanos , Ratones , Ratones Noqueados , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies.
Asunto(s)
Macrófagos/inmunología , Mutación , ARN Mensajero/inmunología , Salmonelosis Animal/inmunología , Tristetraprolina/inmunología , Alanina/genética , Alanina/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Citocinas/antagonistas & inhibidores , Citocinas/genética , Citocinas/inmunología , Femenino , Expresión Génica , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosforilación , Cultivo Primario de Células , Estabilidad del ARN , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Salmonelosis Animal/genética , Salmonelosis Animal/patología , Salmonella typhimurium/inmunología , Serina/genética , Serina/metabolismo , Tristetraprolina/genéticaRESUMEN
PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3.
Asunto(s)
Proteínas 14-3-3/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas 14-3-3/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Quinasas Ciclina-Dependientes/genética , Ciclinas/química , Ciclinas/genética , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Alineación de SecuenciaRESUMEN
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.
Asunto(s)
Lisina/química , Poliubiquitina/química , Pliegue de Proteína , Ubiquitina-Proteína Ligasas/química , Animales , Humanos , Lisina/genética , Lisina/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.
Asunto(s)
Adenosina Desaminasa/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Edición de ARN , Receptores AMPA/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Fibroblastos/metabolismo , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN , UbiquitinaciónRESUMEN
BACKGROUND: Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. METHODS: Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. RESULTS: The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. CONCLUSIONS: For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells.
Asunto(s)
Carcinogénesis/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias/genética , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor , Quinasa 5 Dependiente de la Ciclina/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas del Tejido Nervioso/genética , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Empalme del ARN/genética , Serina/metabolismoRESUMEN
We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.
Asunto(s)
Expresión Génica , Glucosiltransferasas , Glucógeno Sintasa , Glicoproteínas , Complejos Multienzimáticos , Animales , Glucosiltransferasas/biosíntesis , Glucosiltransferasas/genética , Glucosiltransferasas/aislamiento & purificación , Glucógeno Sintasa/biosíntesis , Glucógeno Sintasa/genética , Glucógeno Sintasa/aislamiento & purificación , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Humanos , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , SpodopteraRESUMEN
Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)-ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]-NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Ebolavirus/fisiología , Quinasas Quinasa Quinasa PAM/biosíntesis , Subunidad p50 de NF-kappa B/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Regulación hacia Arriba/genética , Proteínas Reguladoras y Accesorias Virales/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Ebolavirus/genética , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/genética , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/genética , Subunidad p50 de NF-kappa B/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transfección , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
BACKGROUND: Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. FINDINGS: Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. CONCLUSIONS: The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.
Asunto(s)
Cofilina 1/metabolismo , Cucurbitacinas/farmacología , Citoesqueleto de Actina/metabolismo , Cisteína/metabolismo , Humanos , Células MCF-7 , Unión ProteicaRESUMEN
Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)). As few molecular targets for PtdIns(3,4)P(2) have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P(2). A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P(2) selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates the presence of a pleckstrin homology domain whose lipid binding character remains to be established. IQ motif containing GAP1 lacks known lipid interacting components and a preliminary analysis here indicates that this may exemplify a novel class of atypical phosphoinositide (aPI) binding domain.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Línea Celular Tumoral , Estudios de Factibilidad , Humanos , Fragmentos de Péptidos/biosíntesis , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Resonancia por Plasmón de Superficie , Proteínas Activadoras de ras GTPasa/biosíntesisRESUMEN
Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.
Asunto(s)
Núcleo Celular/metabolismo , Neomicina/farmacología , Fosfatidilinositol 4,5-Difosfato/química , Proteómica/métodos , Secuencias de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Células Jurkat , Fosfatidilinositoles/química , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a 'methylation/acetylation switch'. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.
Asunto(s)
Proteína I de Unión a Poli(A)/química , Proteína I de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Animales , Arginina/metabolismo , Células Cultivadas , Células HeLa , Humanos , Cinética , Metilación , Ratones , Modelos Moleculares , Proteína I de Unión a Poli(A)/genética , Proteína Metiltransferasas/metabolismo , Proteína Metiltransferasas/fisiología , Procesamiento Proteico-Postraduccional/genética , Relación Estructura-Actividad , Distribución TisularRESUMEN
SIK2 (salt-inducible kinase 2) is a member of the AMPK (AMP-activated protein kinase) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP inducers or AICAR (5-amino-4-imidazolecarboxamide riboside), affected SIK2 activity towards peptide or protein substrates in vitro. However, stimulation with the cAMP-elevating agent forskolin and the ß-adrenergic receptor agonist CL 316,243 resulted in a PKA (protein kinase A)-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP induction, including Ser(358). Site-directed mutagenesis demonstrated that phosphorylation of Ser(358), but not the previously reported PKA site Ser(587), was required for 14-3-3 binding. Immunocytochemistry illustrated that the localization of exogenously expressed SIK2 in HEK (human embryonic kidney)-293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes, however, revealed a significant increase of wild-type, but not Ser358Ala, HA (haemagglutinin)-SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent relocalization in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.
Asunto(s)
Adipocitos/enzimología , AMP Cíclico/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Células 3T3 , Quinasas de la Proteína-Quinasa Activada por el AMP , Secuencia de Aminoácidos , Animales , AMP Cíclico/genética , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación/fisiología , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Serina/genéticaRESUMEN
Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP.
Asunto(s)
Calcio/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Transporte Biológico , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Fosforilación , Molécula de Interacción Estromal 1RESUMEN
PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteína Fosfatasa 1/química , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Biología Computacional/métodos , Bases de Datos de Proteínas , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Filogenia , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Estructuras de las Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/aislamiento & purificación , Proteína Fosfatasa 1/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
MOTIVATION: Complex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging. RESULTS: We have developed a knowledge base of criteria, which replicate expert assessment, allowing more than half of cases to be automatically validated and site assignments verified with a high degree of confidence. This was assessed by comparing automated spectral interpretation with careful manual examination of the assignments for 501 peptides above the 1% false discovery rate (FDR) threshold corresponding to 259 putative phosphorylation sites in 74 proteins of the Trypanosoma brucei proteome. Despite this stringent approach, we are able to validate 80 of the 91 phosphorylation sites (88%) positively identified by manual examination of the spectra used for the MASCOT searches with a FDR < 15%. CONCLUSIONS: High-throughput computational analysis can provide a viable second stage validation of primary mass spectrometry database search results. Such validation gives rapid access to a systems level overview of protein phosphorylation in the experiment under investigation. AVAILABILITY: A GPL licensed software implementation in Perl for analysis and spectrum annotation is available in the supplementary material and a web server can be assessed online at http://www.compbio.dundee.ac.uk/prophossi.
Asunto(s)
Fosfopéptidos/análisis , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem/métodos , Algoritmos , Automatización , Bases de Datos Factuales , Fosforilación , Programas Informáticos , Trypanosoma brucei brucei/metabolismoRESUMEN
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.