RESUMEN
The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai, which uses ß-glucosidase (akuBGL) to digest the laminarin in algae into glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect akuBGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo akuBGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP-TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. akuBGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits akuBGL by occupying the substrate-binding pocket, and EHEP protects akuBGL against this inhibition by binding with phlorotannin to free the akuBGL pocket.
Asunto(s)
Phaeophyceae , Proteínas , Animales , Proteínas/metabolismo , Phaeophyceae/metabolismo , Aplysia , Glucosa/metabolismo , Dominio CatalíticoRESUMEN
Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.
Asunto(s)
Arabidopsis , Brassica , Fosfolipasa D , Raphanus , Fosfolipasa D/genética , Fosfolipasa D/química , Raphanus/metabolismo , Fosfolipasas/metabolismo , Esfingolípidos/metabolismo , Brassica/genética , Brassica/química , Arabidopsis/genética , Arabidopsis/metabolismoRESUMEN
Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Serratia marcescens/metabolismo , Anticuerpos de Dominio Único/biosíntesis , Secuencia de Aminoácidos , Animales , Antígenos/metabolismo , Camelus , Medios de Cultivo , Proteínas Fluorescentes Verdes/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificaciónRESUMEN
Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.
Asunto(s)
Receptores del Factor Natriurético Atrial/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Células HEK293 , Células HeLa , Humanos , Ligandos , Proteínas Musculares/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Factores de Transcripción/farmacologíaRESUMEN
Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Citrus/química , Neoplasias Cutáneas/tratamiento farmacológico , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Frutas/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patologíaRESUMEN
Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects ß-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48â Å using wavelengths of 1.0 and 2.1â Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.
Asunto(s)
Aplysia/química , Cristalización/métodos , Proteínas/química , Animales , Aplysia/enzimología , Aplysia/genética , Aplysia/metabolismo , Cristalografía por Rayos X , Hidrólisis , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica , Dominios Proteicos/genética , Proteínas/aislamiento & purificaciónRESUMEN
Although we recently reported that sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis.
Asunto(s)
Apoptosis/efectos de los fármacos , Citrus/química , Flavonoides/farmacología , Glicósidos/farmacología , Queratinocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/metabolismo , HumanosRESUMEN
Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5â¯kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5â¯kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.
Asunto(s)
Aplysia/enzimología , Sistema Digestivo/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Animales , Aplysia/genética , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Serina Endopeptidasas/genética , Especificidad por SustratoRESUMEN
Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.
Asunto(s)
Aplysia/enzimología , Celulasa/química , Celulosa/química , Digestión/genética , Animales , Aplysia/genética , Dominio Catalítico/genética , Celobiosa/química , Celulasa/genética , Celulosa/análogos & derivados , Celulosa/genética , Celulosa/metabolismo , Digestión/fisiología , Glucosa/química , Glucosa/metabolismo , Hidrólisis , Cinética , Oligosacáridos/química , Oligosacáridos/metabolismo , Unión Proteica , Dominios Proteicos/genética , Tetrosas/química , Tetrosas/metabolismoRESUMEN
A variety of polyphenols have been isolated from plants, and their biological activities have been examined. Sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone) is a polymethoxyflavone that is isolated from the peel of Citrus sudachi. Although we previously reported that sudachitin possesses an anti-inflammatory activity, its other biological activities are not yet understood. In this study, we report a novel biological activity of sudachitin, which selectively induced apoptosis in human keratinocyte HaCaT cells. Another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), promoted autophagy but not apoptosis in HaCaT cells. On the other hand, 3'-demethoxysudachitin (5,7,4'-trihydroxy-6,8-dimethoxyflavone) failed to induce apoptosis and autophagy. These results show that three polymethoxyflavones have different effects on apoptosis and autophagy in HaCaT cells. Understanding the structure and biological activity of polymethoxyflavones may lead to the discovery of potential candidates for cancer drug development without significant toxic side effects. Abbreviations: ROS: reactive oxygen species; DMSO: dimethyl sulfoxide; MTT: 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; MAPK: mitogen-activated protein kinase.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Citrus/química , Flavonas/farmacología , Flavonoides/farmacología , Glicósidos/farmacología , Queratinocitos/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Flavonas/química , Flavonoides/química , Glicósidos/química , Humanos , Queratinocitos/citología , Relación Estructura-ActividadRESUMEN
Death associated protein kinase (DAPK)-related apoptosis-inducing protein kinase (DRAK)-1 is a positive apoptosis regulator. However, the molecular mechanisms underlying the DRAK1-mediated apoptotic pathway remain unclear. In this study, we demonstrated the intracellular localization and binding partners of DRAK1. In human osteosarcoma cell line U2OS cells, DRAK1 was mainly localized in the nucleus and translocated outside the nucleus through Ser395 phosphorylation by protein kinase C. In the nucleus, DRAK1 associated with tumor suppressor p53 and positively regulated p53 transcriptional activity in response to DNA-damaging agent cisplatin. On the other hand, DRAK1 interacted with the mitochondrial inner-membrane protein, adenine nucleotide translocase (ANT)-2, an anti-apoptotic oncoprotein, outside the nucleus. These findings suggest that DRAK1 translocates in response to stimuli and induces apoptosis through its interaction with specific binding partners, p53 and/or ANT2.
Asunto(s)
Translocador 2 del Nucleótido Adenina/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fracciones Subcelulares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas , Distribución TisularRESUMEN
Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca2+-dependent K+ channels. TBHQ elevated intracellular Ca2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents.
Asunto(s)
Aditivos Alimentarios/efectos adversos , Hidroquinonas/efectos adversos , Linfocitos/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Canales Iónicos/metabolismo , Linfocitos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Línea Celular , Línea Celular Tumoral , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilación/fisiología , Proteínas Tirosina Quinasas/metabolismo , Seudópodos/metabolismo , Transducción de Señal/fisiología , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
To develop new whitening agents from natural products, we screened 80 compounds derived from crude drugs in Kampo medicine in a melanin synthesis inhibition assay using murine B16 melanoma cells. The screen revealed that treatment with alisol B, a triterpene from Alismatis rhizoma, significantly decreased both melanin content and cellular tyrosinase activity in B16 cells. However, alisol B did not directly inhibit mushroom tyrosinase activity in vitro. Therefore, we investigated the mechanism underlying the inhibitory effect of alisol B on melanogenesis. Alisol B suppressed mRNA induction of tyrosinase and its transcription factor, microphthalmia-associated transcription factor (MITF). Furthermore, alisol B reduced the phosphorylation of CREB and maintained the activation of ERK1/2. These results suggest that the reduction in melanin production by alisol B is due to the downregulation of MITF through the suppression of CREB and activation of ERK and that alisol B may be useful as a new whitening agent.
Asunto(s)
Alisma/química , Colestenonas/farmacología , Melaninas/biosíntesis , Melanoma Experimental/metabolismo , Preparaciones para Aclaramiento de la Piel/farmacología , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Melaninas/antagonistas & inhibidores , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Fosforilación/efectos de los fármacos , Rizoma/química , Transducción de Señal/efectos de los fármacosRESUMEN
The digestive fluid of the sea hare Aplysia kurodai can liberate approximately 2.5 mg of glucose from 10 mg of dried Eisenia bicyclis powder. Although laminaran, a major storage polysaccharide in E. bicyclis, is easily digested to glucose by the synergistic action of the 110 and 210 kDa A. kurodai ß-glucosidases (BGLs), glucose is not liberated from E. bicyclis by direct incubation with these BGLs. To clarify this discrepancy, we searched for an Eisenia hydrolysis enhancing protein (EHEP) in the digestive fluid of A. kurodai. A novel 25 kDa protein that enhances E. bicyclis saccharification by ß-glucosidases was purified to a homogeneous state from the digestive fluid of A. kurodai, and its cDNA was cloned from total cDNAs reverse-transcribed from hepatopancreas total RNA. The E. bicyclis extract strongly inhibited BGLs, suggesting some compound within this brown alga functioned as a feeding deterrent. However, when E. bicyclis was incubated with BGLs in the presence of EHEP, glucose production was markedly increased. As E. bicyclis is rich in phlorotannin, which are only found in brown algae, our study suggested that these compounds are the main BGL inhibitors in E. bicyclis extract. EHEP protects BGLs from phlorotannin inhibition by binding to phlorotannins and forming an insoluble complex with phloroglucinol and phlorotannins. These findings indicated that EHEP plays a key role in the saccharification of brown seaweeds containing phlorotannins in the digestive fluid of A. kurodai. This is the first report of EHEP as a phlorotannin-binding protein that protects BGLs from inhibition.
Asunto(s)
Aplysia/genética , Digestión/genética , Glucosa/metabolismo , Proteínas/genética , Taninos/metabolismo , Animales , Aplysia/metabolismo , Celulasas/genética , Celulasas/metabolismo , Clonación Molecular , ADN Complementario , Glucanos/metabolismo , Hidrólisis , Phaeophyceae/química , Phaeophyceae/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Alineación de Secuencia , Análisis de Secuencia de Proteína , Taninos/química , Taninos/genéticaRESUMEN
Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.
Asunto(s)
Condrosarcoma/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Condrocitos/metabolismo , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Sistema de Señalización de MAP Quinasas , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/genética , Ratas , Serina/química , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo II/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , GMP Cíclico/metabolismo , Regulación Alostérica , Animales , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule ß-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/ß-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with ß-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to ß-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.
Asunto(s)
Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Nocodazol/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Sustitución de Aminoácidos , Núcleo Celular/metabolismo , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación , Estructura Terciaria de Proteína , ARN Helicasas/metabolismo , Interferencia de ARN , Subunidades Ribosómicas Grandes de Eucariotas/metabolismoRESUMEN
Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.