RESUMEN
The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Solanum tuberosum/metabolismo , Sacarosa/metabolismo , Calcio/metabolismo , Solanum tuberosum/enzimología , Solanum tuberosum/crecimiento & desarrolloRESUMEN
We isolated a full-length cDNA clone (StCDPK1) encoding a calcium-dependent protein kinase (CDPK) by screening a stolon tip cDNA library from potato plants (Solanum tuberosum L.). The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family except in the N-terminal region. As described for other CDPKs, StCDPK1 has a putative N-terminal myristoylation sequence. A coupled transcription/translation system was used to demonstrate that this post-translational modification occurs in vitro. The behaviour of the myristoylated form of StCDPK1 during its purification on a phenyl-Sepharose column mimics that of the endogenous potato enzyme suggesting that this modification occurs in vivo. In addition, a possible palmitoylation site is present in StCDPK1. Southern blot analysis suggests that more than one CDPK isoform is present in potato plants. Northern blot analysis of steady-state mRNA levels for StCDPK1 in different tissues of potato plants shows that the transcript is differentially expressed in tuberizing stolons. The transcript appears in the early steps of tuber formation before the induction of other genes, such as Pin2 and patatin. This result parallels previous data on CDPK activity in potato plants which was highest at the beginning of tuberization. Our results suggest that StCDPK1 is developmentally regulated. The early and transient expression of this CDPK isoform in the tuberization process suggests that this kinase could trigger a cascade of phosphorylation events involved in tuber induction.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Plantas , Proteínas Quinasas/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Northern Blotting , Southern Blotting , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Ácido Mirístico/metabolismo , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Solanum tuberosum/enzimología , Solanum tuberosum/crecimiento & desarrolloRESUMEN
Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.
Asunto(s)
Proteínas Protozoarias , Proteínas Ribosómicas/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Microscopía Electrónica , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/química , Ribosomas/química , Ribosomas/metabolismoRESUMEN
Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.
Asunto(s)
Ciclinas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2 , Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/aislamiento & purificación , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/enzimología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Histonas/metabolismo , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Cinetina , Mitocondrias/enzimología , Datos de Secuencia Molecular , Piperidinas/farmacología , Pruebas de Precipitina , Proteínas Quinasas/aislamiento & purificación , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Purinas/farmacología , Proteína de Retinoblastoma/metabolismo , Alineación de Secuencia , Trypanosoma cruzi/metabolismoRESUMEN
Protein kinase C (PKC) has been implicated in the control of proliferation and differentiation of many cell types. There is evidence indicating that it plays a role in signal transduction mechanisms related to myogenesis, but little is known about the individual functions of PKC isoforms in muscle cell development. Data obtained in previous studies using cultured chick embryo skeletal muscle cells suggested that PKC alpha is linked to the regulation of myoblast proliferation. However, this causal relationship could not be definitively established as no experiments based on selective inhibition of this isoform were carried out. In the present work, specific inhibition of the expression of PKC alpha in cultured myoblasts by using antisense oligonucleotide technology resulted in a significant decrease of culture cell density and DNA synthesis, clearly showing that this isoenzyme is involved in signalling pathways which promote muscle cell proliferation.
Asunto(s)
Isoenzimas/genética , Isoenzimas/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/enzimología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Animales , Muerte Celular/genética , División Celular/genética , Células Cultivadas , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Músculo Esquelético/citología , Músculo Esquelético/embriología , Oligonucleótidos Antisentido/farmacología , Fosfatidiletanolaminas , Proteína Quinasa C-alfa , Transducción de Señal/fisiologíaRESUMEN
Regulation of muscle cell Ca(2+) metabolism by 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] is mediated by the classic nuclear mechanism and a fast, nongenomic mode of action that activates signal transduction pathways. The role of individual protein kinase C (PKC) isoforms in the regulation of intracellular Ca(2+) levels ([Ca(2+)](i)) by the hormone was investigated in cultured proliferating (myoblasts) and differentiated (myotubes) chick skeletal muscle cells. 1,25(OH)(2)D(3) (10(-9) M) induced a rapid (30- to 60-s) and sustained (>5-min) increase in [Ca(2+)](i) which was markedly higher in myotubes than in myoblasts. The effect was suppressed by the PKC inhibitor calphostin C. In differentiated cells, PKC activity increased in the particulate fraction and decreased in cytosol to a greater extent than in proliferating cells after 5-min treatment with 1,25(OH)(2)D(3). By Western blot analysis, these changes were correlated to translocation of the PKC alpha isoform from cytosol to the particulate fraction, which was more pronounced in myotubes than in myoblasts. Specific inhibition of PKC alpha activity using antibodies against this isoform decreased the 1, 25(OH)(2)D(3)-induced [Ca(2+)](i) sustained response associated with Ca(2+) influx through voltage-dependent calcium channels. Neomycin, a phospholipase C (PLC) inhibitor, blocked its effects on [Ca(2+)](i), PKC activity, and translocation of PKC alpha. Exposure of myotubes to 1,2-dioleyl-rac-glycerol (1,2-diolein), also increased [Ca(2+)](i), PKC activity, and the amount of PKC alpha associated with the particulate fraction. Changes in [Ca(2+)](i) induced by diolein were inhibited by calphostin C and nifedipine. The results indicate that PKC alpha activation via PLC-catalyzed phosphoinositide hydrolysis is part of the mechanism by which 1, 25(OH)(2)D(3) regulates muscle intracellular Ca(2+) through modulation of the Ca(2+) influx pathway of the Ca(2+) response to the sterol.
Asunto(s)
Calcitriol/farmacología , Calcio/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular , División Celular , Células Cultivadas , Embrión de Pollo , Diglicéridos/farmacología , Inhibidores Enzimáticos/farmacología , Líquido Intracelular/metabolismo , Isoenzimas/metabolismo , Músculo Esquelético/citología , Proteína Quinasa C/antagonistas & inhibidores , Fracciones Subcelulares/enzimologíaRESUMEN
Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.
Asunto(s)
Proteína Quinasa C/metabolismo , Trypanosoma cruzi/enzimología , Animales , Western Blotting , Bovinos , Cromatografía , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Durapatita , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ésteres del Forbol/metabolismo , Fosforilación , Proteína Quinasa C/inmunología , Proteína Quinasa C/aislamiento & purificación , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
A Trypanosoma cruzi gene, PKB, coding for a putative protein kinase was cloned and sequenced. Analysis of the sequence showed that the encoded protein (called PKB) corresponds to a relatively novel subgroup of Ser/Thr protein kinases denominated protein kinases B (PKB), related to A and C protein kinases (RAC), or protein kinases of the transforming retrovirus AKT8 (Akt) in which the catalytic domains show similarity to corresponding domains of protein kinases A and protein kinases C. Unlike mammalian enzymes belonging to the same subgroup, PKB did not have a pleckstrin (PH)-homologous domain. PKB was expressed in Escherichia coli and the recombinant protein was found to be a Thr-specific protein kinase that required Mn2+ for activity and used ATP as phosphate donor (Km = 1.8 microM). Classical protein kinase A and protein kinase C modulators and inhibitors were found to have only marginal or no effect on PKB activity. Antisera raised against the recombinant protein recognized PKB in Western blotting analysis of cell extracts as a membrane bound protein. Evidence was obtained suggesting the presence of a Cys-linked acyl anchor. Northern and Western blotting analysis showed that PKB was constitutively expressed in the lag, exponential and stationary phases of T. cruzi epimastigote growth, as well as in the amastigote and metacyclic trypomastigote stages of differentiation. This is the first description of the existence of a protein kinase B in trypanosomatid protozoa.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Western Blotting , Clonación Molecular , ADN Protozoario , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Fosforilación , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Temperatura , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Changes in morphology and DNA synthesis in cultured myoblasts in response to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] have previously suggested that the vitamin D hormone may affect muscle cell proliferation and differentiation. However, this interpretation was not substantiated by measurement of specific biochemical markers of myogenesis. To study the effect of 1,25(OH)2D3 on muscle development, chicken embryo myoblasts were cultured for 1-6 days in the presence or absence of 1,25(OH)2D3 (10(-9) M). The hormone increased DNA synthesis and decreased creatine kinase activity, indicating stimulation of cell proliferation and inhibition of myogenesis, in undifferentiated myoblasts (1 day of culture). At longer culture intervals, when myoblasts elongate and fuse to form differentiated myotubes, 1,25(OH)2D3 promoted myogenesis, as indicated by an inhibition of DNA synthesis and an increase in specific muscle differentiation markers as creatine kinase activity and myosin expression. The role of protein kinase C (PKC) in mediating the effects of hormone and the likely PKC isoform involved were also investigated. Increased PKC activity was observed during 1,25(OH)2D3 stimulation of myoblast proliferation whereas inhibition of PKC activity accompanied the effects of the hormone on myoblast differentiation. The specific PKC inhibitor calphostin suppressed hormone potentiation of DNA synthesis in proliferating myoblasts. 1,25(OH)2D3-dependent changes in the expression of PKC isoforms alpha, beta, delta, epsilon and zeta during myogenesis were investigated by Western blot analysis. The early stimulation of myoblast proliferation by the hormone mainly correlated to increased PKC alpha expression whereas decreased PKC alpha levels were observed during the subsequent activation of myoblast differentiation. These results support that 1,25(OH)2D3 has a function in embryonic muscle growth and maturation, and PKC alpha may participate in the signal transduction pathway which mediates the response to the hormone.
Asunto(s)
Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Isoenzimas/metabolismo , Músculo Esquelético/citología , Proteína Quinasa C/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Creatina Quinasa/metabolismo , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Músculo Esquelético/embriología , Naftalenos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Factores de TiempoRESUMEN
There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms alpha, beta, delta, epsilon, and zeta during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferation stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC alpha in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down-regulated PKC alpha, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC beta, delta, and epsilon was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts.
Asunto(s)
Isoenzimas/metabolismo , Músculo Esquelético/citología , Proteína Quinasa C/metabolismo , Animales , Diferenciación Celular , División Celular , Embrión de Pollo , Replicación del ADN/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Naftalenos/farmacología , Proteína Quinasa C-alfa , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The presence of Ca2+/calmodulin (Ca2+/CaM)-dependent protein kinase (TcCaM K) and some stage-specific substrates that appeared during morphogenesis of the parasite Trypanosoma cruzi were identified. Western blot analysis using a polyclonal antibody against rat brain CaM K type 11 recognized the same subunit composition (52, 59/62 kDa) observed for the mammalian enzyme, as well as the previously characterized TcCaM K found in epimastigote forms. Differential protein phosphorylation profiles were observed after enzyme activation in the stages of T. cruzi. Co-immunoprecipitation of stage-specific substrates with the TcCaM K suggested that the enzyme might be involved in the phosphorylation of a different set of proteins through the life cycle. Three phosphoproteins, pp105 and pp87 from epimastigotes and pp23 from trypomastigotes were identified as potential substrates for TcCaM K. The characterization of these endogenous stage markers might be a useful tool to understand the developmental cycles of these pathogenic protozoa.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Animales , Chlorocebus aethiops , Péptidos y Proteínas de Señalización Intercelular , Péptidos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Ratas , Especificidad por Sustrato , Células VeroRESUMEN
Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732-735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359-1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.
Asunto(s)
Adenosina Trifosfato/farmacología , Bradiquinina/farmacología , GMP Cíclico/metabolismo , Endotelio Vascular/metabolismo , Proteína Quinasa C/metabolismo , Animales , Aorta Torácica , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Feto , Cinética , NG-Nitroarginina Metil Éster/farmacología , Naftalenos/farmacología , Óxido Nítrico/farmacología , Ésteres del Forbol/farmacología , Polietilenglicoles/farmacología , Estaurosporina/farmacología , Porcinos , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Vasodilatadores/farmacologíaRESUMEN
Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.
Asunto(s)
Ciclinas/metabolismo , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Células COS , Clonación Molecular , Genes Protozoarios , Prueba de Complementación Genética , Histonas/metabolismo , Humanos , Técnicas de Sonda Molecular , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Retinoblastoma/metabolismo , Alineación de Secuencia , Transfección , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote-infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock-infected cytosols (heat-killed trypomastigotes were used in the parasite-macrophage interaction assay). GTP gamma S addition potentiates the fusogenic activity driven by trypomastigote-infected macrophage cytosol-mediated assays, unlike the biphasic fusogenic effect obtained with GTP gamma S treatment of macrophage cytosol controls. Calcium-stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP-regulated factors, and not calcium-regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote-infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment-fusion of the host lysosomes upon trypomastigote infection and further survival of the parasite within its host.
Asunto(s)
Citosol/fisiología , Endosomas/fisiología , Macrófagos/parasitología , Fusión de Membrana/fisiología , Trypanosoma cruzi/fisiología , Animales , Calcio/fisiología , Citosol/parasitología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/fisiología , Macrófagos/ultraestructura , RatonesRESUMEN
Trypanosoma cruzi, the etiological agent of Chagas disease, is an adequate model for studies on the evolution of signal transduction pathways. These pathways involve molecular entities such as membrane receptors, transduction G proteins, protein kinases and second messengers (Ca(2+), cyclic AMP, cyclic GMP, nitric oxide). In this article, Mirtha M. Flawiá, María T. Téllez-Iñón and Héctor N. Torres describe the studies performed on T. cruzi transduction pathways and their role in the control of metacyclogenesis and cell motility.
RESUMEN
In vitro culture was used to study morphogenetic aspects of the tuberization process under controlled conditions in potato (Solanum tuberosum L.) plants. This paper accurately defines four stages of tuber development and their correlation to external morphological characteristics and histological structures. Protein kinase activity, assayed in each stage using Historic HAS as substrate, was differentially expressed during the tuberization process. Phosphorylation was maximum in the first stages of tuber formation. The incorporation of [32PO4 -1] to endogenous peptides containing serine/threonine amino acidic residues followed the same pattern that the protein kinase activity did.
RESUMEN
A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.
RESUMEN
The present studies were carried out to characterize the cAMP-phosphodiesterase enzyme (PDE) in luteal cells recovered from pseudopregnant rats with streptozotocin-induced diabetes. A significant increase in the specific activity of the enzyme was detected in luteal cells from diabetic rats (Group D) with respect to control rats (Group C). This increase could not be prevented by insulin therapy (Group I). Luteal cells from Groups C and D rats responded in vitro to insulin by increasing their PDE activity (% of stimulus of specific activity: C = 75%, D = 110%). However, in cells isolated from Group I, the hormone caused an inhibition of PDE activity (% of inhibition of specific activity: 48%). When cytosolic fractions from Groups C, D and I were submitted to ion exchange chromatography, two PDE activity peaks could be observed and the activity of the different fractions was increased in the presence of Ca2+ and calmodulin. Nevertheless, the Ca(2+)-calmodulin effect was much lower in the extracts from Groups D and I than for controls. Kinetic studies of luteal PDE showed nonlinear Lineweaver-Burk graphs with two apparent ATP hydrolysis sites. Similar K(m) values were found for PDE from groups C, D, and I, whereas the Vmax2 for the enzyme was higher in Groups D and I. The endogenous concentration of cAMP, measured by RIA, showed no significant differences among Groups C, D, and I. On the basis of these results, we conclude that the specific activity of PDE is significantly increased in luteal cells from streptozotocin-induced diabetic animals, which could explain the previously described reduction in LH-stimulated progesterone production by luteal cells in diabetic rats.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Diabetes Mellitus Experimental/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/aislamiento & purificación , Animales , Calmodulina/farmacología , Fraccionamiento Celular , Cromatografía por Intercambio Iónico , Femenino , Cinética , Ovario/enzimología , Seudoembarazo/metabolismo , Ratas , Ratas Sprague-Dawley , SuperovulaciónRESUMEN
A multifunctional Ca2+/calmodulin-dependent protein kinase (TcCaM K) was purified and characterized from the cytosol of Trypanosoma cruzi epimastigote forms. Like mammalian CaM KII, TcCaM K has a broad substrate specificity and a similar subunit composition. Western blot analysis revealed that this TcCaM K possesses two subunits of 50 and 60 kDa, which exhibited autophosphorylating activity. A panel of monoclonal and polyclonal antibodies raised against rat brain CaM KII could also recognize TcCaM K. However, experimental evidence suggests a different conformational arrangement of the TcCaM K subunits. Like its mammalian counterpart, two highly active autonomous, Ca(2+)-independent, states of TcCaM K can be isolated. These states, caused by high phosphate incorporation, differ only in their extent of Ca2+/CaM-dependence. About 15-20% of the autophosphorylated TcCaM K can be reverted using protein phosphatase 2A, and, consequently, its Ca(2+)-dependent activity is also partially restored. The situation is somewhat different when the enzyme is linked to the cytoskeleton, as we have previously shown. The membrane-bound form is present only in the native form. Activation increases its protein kinase activity from 5- to 14-fold. In this study, we provide evidence of another form of TcCaM K present in soluble fractions of the parasite that can be isolated in autonomous states. Our results suggest that autophosphorylation of membrane-bound TcCaM K may be responsible for kinase release in a Ca2+/CaM-independent state. These properties of TcCaM K may play an important role in regulating Ca(2+)-dependent processes in the parasite.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Peso Molecular , Péptidos/química , Fosforilación , Conformación Proteica , Ratas , Solubilidad , Especificidad por Sustrato , Trypanosoma cruzi/inmunologíaRESUMEN
A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.