RESUMO
Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Metaboloma , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositóis/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Cromatografia de Afinidade , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Exossomos/química , Exossomos/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Espectrometria de Massas , Camundongos , Células PC-3 , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositóis/química , Fosfatidilinositóis/classificação , Fosfatidilinositóis/isolamento & purificação , Próstata/química , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , EstereoisomerismoRESUMO
In this work, AuPd core-shell nanoparticles (NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (Au-Pd/AtPCS1-E. coli) with catalytic enhanced chemiluminescence (CL) and benzyl alcohol oxidation (BAO) was investigated. Such biosynthesis of AuPd core-shell NPs was obviously enhanced due to insertion of the gene sequence of Arabidopsis thaliana phytochelatin synthase (AtPCS1) to a plasmid vector (pET-28b) of Escherichia coli (E. coli). The obtained Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (AtPCS1-E. coli) could generate phytochelatins (PCs, (γ-Glu-Cys)n-Gly, nâ¯>â¯1) for efficient capture and enrichment of Au3+. The component and morphology of AuPd core-shell NPs were checked through X-ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). Catalytic CL (in H2O2-luminol system) and BAO (in H2O2-benzyl alcohol system) effect with different experimental conditions were examined, respectively. These results revealed that multifunctional PCs could effectively facilitate biosynthetic process of AuPd core-shell NPs with better distribution, higher yield and lower cost while stronger CL intensity and higher conversion could be obtained for further quantitative analysis and application.
Assuntos
Álcool Benzílico/química , Escherichia coli/metabolismo , Ouro/química , Nanopartículas/química , Nanopartículas/metabolismo , Paládio/química , Arabidopsis/metabolismo , Catálise , Luminescência , Microscopia Eletrônica de Varredura , Oxirredução , Fitoquelatinas/química , Fitoquelatinas/metabolismo , Difração de Raios XRESUMO
In this work, CdS nanoparticles (CdS NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (CdS/AtPCS1-E. coli) with fluorescence (FL) performance for detection of pyrogallol and gallic acid was investigated. Through expression of the AtPCS1 gene inside E. coli cells by pET28b vector, biosynthesis of CdS NPs was greatly enhanced due to generation of phytochelatins (PCs, (γ-Glu-Cys)n-Gly, nâ¯≥â¯2) for efficient capture of Cd2+. The expression of AtPCS1 and concentration of glutathione (GSH) and PCs were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC), respectively. The morphology and component were checked through scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). FL effect with different experimental conditions were examined. In addition, it is also applied to determination of pyrogallol and gallic acid. These results revealed that multifunctional PCs could effectively facilitate biosynthesis of CdS NPs with higher yield, better distribution and lower cost while stronger FL intensity could be obtained for quantitative analysis.
Assuntos
Aminoaciltransferases , Arabidopsis/enzimologia , Compostos de Cádmio , Escherichia coli , Ácido Gálico , Nanopartículas , Pirogalol , Compostos de Selênio , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Compostos de Cádmio/química , Compostos de Cádmio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Ácido Gálico/análise , Ácido Gálico/química , Nanopartículas/química , Nanopartículas/metabolismo , Pirogalol/análise , Pirogalol/química , Compostos de Selênio/química , Compostos de Selênio/metabolismoRESUMO
Aluminium ions inhibit growth of the budding yeast Saccharomyces cerevisiae. Disruption of the SSO2 gene increased the susceptibility to aluminium. Sso2p belongs to the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. SSO2 has one paralogue, SSO1, which encodes Sso1p. The SNARE complex containing Sso1/2p plays a role in the recognition of plasma membrane targeted vesicle transport. The susceptibility to aluminium stress was not increased in the Δsso1 strain. The phenotype of aluminium ion influx between the wild-type and Δsso2 strains was not different, suggesting that Sso2p was involved in the elimination of cellular aluminium. However, the cellular lipid constitution of Δsso2 was richer in unsaturated fatty acids than the wild type, indicating that Sso2p is associated with lipid homeostasis of the plasma membrane. Aluminium treatment increased the production of reactive oxygen species (ROS) during proliferation. ROS production was increased in the Δsso2 strain after 3 h of aluminium treatment compared with the wild type. These results suggested that Sso2p plays a role in maintaining the lipid composition of the plasma membrane and the increase in unsaturated fatty acids amplified the production of ROS in the acute phase of aluminium stress. ROS derived from aluminium stress inhibited growth and resulted in the susceptibility of the Δsso2 strain.
Assuntos
Alumínio/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Qa-SNARE/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Sequência de Aminoácidos/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/genética , Proteínas de Membrana/genética , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/química , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors.
Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/metabolismo , Ácido Etacrínico/farmacologia , Proteínas Fúngicas/metabolismo , Glutationa Transferase/metabolismoRESUMO
Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin production of Aspergillus flavus became stronger as the length of the alkyl chains on the esters became longer. Pentyl, hexyl, heptyl, and octyl syringates showed strong activity at 0.05 mM. Heptyl and octyl parabens, and octyl gallate also inhibited aflatoxin production as strongly as octyl syringate. Alkyl parabens and alkyl gallates inhibit the complex II activity of the mitochondrial respiration chain; thus, whether alkyl syringates inhibit complex II activity was examined. Inhibitory activities of alkyl syringates toward complex II also became stronger as the length of the alkyl chains increased. The complex II inhibitory activity of octyl syringate was comparable to that of octyl paraben and octyl gallate. These results suggest that alkyl syringates, alkyl parabens, and alkyl gallates, including commonly used food additives, are useful for aflatoxin control.
Assuntos
Aflatoxinas/antagonistas & inibidores , Aspergillus flavus/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Gálico/análogos & derivados , Aflatoxinas/biossíntese , Aspergillus flavus/efeitos dos fármacos , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Gálico/síntese química , Ácido Gálico/química , Ácido Gálico/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacosRESUMO
Mycotoxin contamination of crops is a serious problem throughout the world because of its impact on human and animal health as well as economy. Inhibitors of mycotoxin production are useful not only for developing effective methods to prevent mycotoxin contamination, but also for investigating the molecular mechanisms of secondary metabolite production by fungi. We have been searching for mycotoxin production inhibitors among natural products and investigating their modes of action. In this article, we review aflatoxin and trichothecene production inhibitors, including our works on blasticidin S, methyl syringate, cyclo(L-Ala-L-Pro), respiration inhibitors, and precocene II.
Assuntos
Aflatoxinas/antagonistas & inibidores , Aspergillus/efeitos dos fármacos , Contaminação de Alimentos/prevenção & controle , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Tricotecenos/antagonistas & inibidores , Aflatoxinas/biossíntese , Aspergillus/patogenicidade , Aspergillus/fisiologia , Benzopiranos/farmacologia , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/microbiologia , Fusarium/patogenicidade , Fusarium/fisiologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Humanos , Nucleosídeos/farmacologia , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Peptídeos Cíclicos/farmacologia , Doenças das Plantas/microbiologia , Relação Estrutura-Atividade , Tricotecenos/biossínteseRESUMO
Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H(2)O(2)-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCdelta at Tyr332 in vitro. PKCdelta was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H(2)O(2)-induced tyrosine phosphorylation of PKCdelta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKCdelta at Tyr332 in the H(2)O(2)-stimulated but not in the growth-factor treated cells, and suggest that PKCdelta in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteína Quinase C-delta/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fosforilação , Proteína Quinase C-delta/química , Ratos , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/metabolismoRESUMO
Toll-like receptor (TLR) family members recognize specific molecular patterns within pathogens. Signaling through TLRs results in a proximal event that involves direct binding of adaptor proteins to the receptors. We observed that TIRAP/Mal, an adaptor protein for TLR2 and TLR4, binds protein kinase Cdelta (PKCdelta). TIRAP/Mal GST-fusion protein and a TIRAP/Mal antibody were able to precipitate PKCdelta from rat peritoneal macrophage and THP1 cell lysates. Truncation mutants of TIRAP/Mal showed that the TIR domain of TIRAP/Mal is responsible for binding. TLR2- and TLR4-mediated phosphorylation of p38 MAPK, IKK, and IkappaB in RAW264.7 cells were abolished by depletion of PKCdelta. These results suggest that PKCdelta binding to TIRAP/Mal promotes TLR signaling events.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Feminino , Glutationa Transferase/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/química , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Ratos , Receptores de Interleucina-1/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PKCdelta was revealed to make a homologous protein complex that shows a high protein kinase activity upon H(2)O(2) stimulation by expressing the enzymes having different epitope tags in COS-7 cells. The association of the endogenous PKCdelta in the cells was observed by sucrose density gradients. Analysis using the mutant replacing the tyrosine phosphorylation sites showed that PKCdelta is activated without tyrosine phosphorylation in the stimulated cells, and the time course of the activation was parallel with that of the complex formation. The binding sites were identified as the C1 and C2-like regions in the regulatory domain using a series of deletion mutants. The binding between the C1 and C2-like region fragments was induced by cell stimulation, whereas the association of the C1 region fragments by itself and that of the C2-like region fragments were observed even without stimulation. These results suggest that the protein complexes of PKCdelta through the association between the C1 and C2-like regions by different combinations are generated in the H(2)O(2)-treated cells, that may show an enhanced protein kinase activity.
Assuntos
Peróxido de Hidrogênio/farmacologia , Rim/metabolismo , Proteína Quinase C-delta/biossíntese , Proteína Quinase C-delta/química , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Humanos , Rim/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
This protocol describes biochemical procedures to monitor the activation of the protein kinase C (PKC) family using PKCdelta as the representative. The PKC family is composed of ten isoforms divided into cPKC, nPKC and aPKC groups, and their catalytic activity is regulated by multiple mechanisms. For example, PKCdelta in the nPKC group is activated by diacylglycerol as a second messenger in the receptor-coupled manner, through tyrosine phosphorylation and protein complex formation in stress-stimulated cells, and by the caspase-catalyzed cleavage during apoptosis. The isoform is immunoprecipitated from cultured cells, the protein kinase activity is measured by in vitro kinase assay and the tyrosine phosphorylation and protein complex formation are characterized by immunoblot, whereas the generation of the catalytic fragment is detected by immunoblot in the cell extract. The combination of these procedures is useful to evaluate the activation states of the PKC family in cells. This protocol can be completed in 3-5 d.
Assuntos
Bioensaio/métodos , Proteína Quinase C-delta/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Humanos , Células JurkatRESUMO
FOXO4 transcription factor, also referred to AFX, contains three putative phosphorylation motif sites for protein kinase B (PKB), Thr32, Ser197, and Ser262, and it is proposed that phosphorylated FOXO4 stays in the cytosol and is imported to the nucleus through dephosphorylation to induce target gene expression. These three sites were revealed to be phosphorylated by PKB in vitro on phosphopeptide analysis, and in cultured cells on immunoblotting with phosphorylation-site specific antibodies. The mutants with either Thr32 or Ser197 replaced by Ala were found mostly in the nuclear but not the cytosol fraction, and treatment with platelet-derived growth factor did not change their distributions in the cells. FOXO4 proteins mutated at these two sites showed 3- to 5-fold higher transcriptional activity than that of the wild type. In contrast, the replacement of Ser262 did not alter the localization or transcriptional activity. These results indicate that phosphorylation at Thr32 and Ser197 is indispensable, whereas that at Ser262 is not critical, for regulation of the nuclear localization and transcriptional activity of FOXO4. These properties are similar to those of FOXO1 and FOXO3, and thus FOXO transcription factors seem to be regulated through a common mechanism by PKB in the growth factor signaling pathway.
Assuntos
Sinais de Localização Nuclear/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular , Células Cultivadas , Fatores de Transcrição Forkhead , Vetores Genéticos , Humanos , Fosforilação , Plasmídeos/genética , Transdução de SinaisRESUMO
Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.
Assuntos
Resposta ao Choque Térmico , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células COS , Análise Mutacional de DNA , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , Resposta ao Choque Térmico/genética , Temperatura Alta , Camundongos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Ratos , Serina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Treonina/metabolismo , TransfecçãoRESUMO
Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.
Assuntos
Apoptose , Ceramidas/farmacologia , Complexo de Golgi/enzimologia , Proteína Quinase C/química , Acetofenonas/farmacologia , Animais , Benzopiranos/farmacologia , Ceramidas/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mutação , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , Estrutura Terciária de Proteína , Transporte Proteico , RNA/química , RNA Interferente Pequeno/metabolismo , Ratos , Fatores de Tempo , Tirosina/química , Tirosina/metabolismoRESUMO
Protein kinase C (PKC)delta was the first new/novel PKC isoform to be identified by the screening of mammalian cDNA libraries, based on the structural homology of its nucleotide sequences with those of classical/conventional PKC isoforms. PKC delta is expressed ubiquitously among cells and tissues. It is activated by diacylglycerol produced by receptor-mediated hydrolysis of membrane inositol phospholipids as well as by tumor-promoting phorbol ester through the binding of these compounds to the C1 region in its regulatory domain. It is also cleaved by caspase to generate a catalytically active fragment, and it is converted to an active form without proteolysis through the tyrosine phosphorylation reaction. Various lines of evidence indicate that PKC delta activated in distinct ways plays critical roles in cellular functions such as the control of growth, differentiation, and apoptosis. This article briefly summarizes the regulatory mechanisms of PKC delta activity and its functions in cell signaling.