RESUMO
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.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Solanum tuberosum/metabolismo , Sacarose/metabolismo , Cálcio/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Plantas , Proteínas Quinases/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Protozoários , Proteínas Ribossômicas/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microscopia Eletrônica , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/metabolismoRESUMO
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.
Assuntos
Ciclinas/isolamento & purificação , Proteínas Quinases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2 , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Histonas/metabolismo , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Cinetina , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Piperidinas/farmacologia , Testes de Precipitina , Proteínas Quinases/isolamento & purificação , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/metabolismoRESUMO
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.
Assuntos
Isoenzimas/genética , Isoenzimas/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Morte Celular/genética , Divisão Celular/genética , Células Cultivadas , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Oligonucleotídeos Antissenso/farmacologia , Fosfatidiletanolaminas , Proteína Quinase C-alfa , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteína Quinase C/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Divisão Celular , Células Cultivadas , Embrião de Galinha , Diglicerídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Líquido Intracelular/metabolismo , Isoenzimas/metabolismo , Músculo Esquelético/citologia , Proteína Quinase C/antagonistas & inibidores , Frações Subcelulares/enzimologiaRESUMO
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.
Assuntos
Proteína Quinase C/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Western Blotting , Bovinos , Cromatografia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Durapatita , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ésteres de Forbol/metabolismo , Fosforilação , Proteína Quinase C/imunologia , Proteína Quinase C/isolamento & purificação , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , DNA de Protozoário , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Temperatura , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Isoenzimas/metabolismo , Músculo Esquelético/citologia , Proteína Quinase C/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Creatina Quinase/metabolismo , DNA/biossíntese , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Músculo Esquelético/embriologia , Naftalenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Fatores de TempoRESUMO
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.