RESUMO
We recently demonstrated that cyclic GMP (cGMP)-dependent protein kinase (G-kinase) activates the human fos promoter in a strictly cGMP-dependent manner (T. Gudi et al., J. Biol. Chem. 271:4597-4600, 1996). Here, we demonstrate that G-kinase translocates to the nucleus by an active transport mechanism which requires a nuclear localization signal (NLS) and is regulated by cGMP. Immunofluorescent staining of G-kinase was predominantly cytoplasmic in untreated cells, but intense nuclear staining appeared in 8-bromo (Br)-cGMP-treated cells. We identified a putative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1alpha precursor. Fusion of the G-kinase NLS to the N terminus of beta-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K407-->E) within the G-kinase NLS produced an enzyme with normal cGMP-dependent activity in vitro which did not translocate to the nucleus and did not transactivate the fos promoter in the presence of 8-Br-cGMP in vivo. In contrast, N-terminally truncated versions of G-kinase with constitutive, cGMP-independent activity in vitro localized to the nucleus and transactivated the fos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is required for transcriptional activation of the fos promoter and suggest that a conformational change of the kinase, induced by cGMP binding or by removal of the N-terminal autoinhibitory domain, functionally activates an otherwise cryptic NLS.
Assuntos
Núcleo Celular/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação da Expressão Gênica , Sinais Direcionadores de Proteínas/metabolismo , Animais , Células Cultivadas , Cricetinae , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I , Genes fos , Humanos , Microscopia Confocal , Regiões Promotoras Genéticas , beta-Galactosidase/metabolismoRESUMO
We have shown that nitric oxide (NO) regulates c-fos gene expression via cGMP-dependent protein kinase (G-kinase), but NO's precise mechanism of action is unclear. We now demonstrate that: (1) NO targets two transcriptional elements in the fos promoter, i.e., the fos AP-1 binding site and the cAMP-response element (CRE); (2) NO activation of these two enhancer elements requires the CRE binding protein CREB because a dominant negative CREB fully inhibits NO transactivation of reporter genes whereas dominant negative Fos or CCAAT enhancer binding proteins have no effect; (3) CREB is phosphorylated by G-kinase in vitro and its phosphorylation increases in vivo when G-kinase is activated either directly by cGMP or indirectly by NO via soluble guanylate cyclase; (4) NO activation of fos promoter elements requires nuclear translocation of G-kinase but not activation of mitogen-activated protein kinases.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Genes fos , Sistema de Sinalização das MAP Quinases , Óxido Nítrico/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/genética , Elementos Facilitadores Genéticos , Genes Reporter , Guanilato Ciclase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The rhesus erythrocytes were examined for the presence of protein(s) similar to the 70-kDa class of heat shock proteins (hsp 70). Also, interactions of these proteins with the erythrocyte membrane were studied under heat stress. These cells in their cytosol contained at least two proteins of about 70 kDa molecular mass; one of which closely resembled the hsp 70 family of proteins. This protein under normal conditions localized mainly in the cytosol, but it had a strong tendency to bind the membrane under heat stress. The binding was almost exclusively restricted to the membrane skeleton and seemed to involve primarily the hydrophobic interactions. A 70-kDa protein immunologically similar to the above protein(s) was detected also in the membranes of rhesus erythrocytes harboring the schizont stage of the simian malarial parasite Plasmodium knowlesi. From these results, we conclude that hsp 70-like proteins in the mature mammalian erythrocytes could perhaps play an important role in protecting the cells under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.
Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Eritrócitos/metabolismo , Proteínas de Choque Térmico/metabolismo , Animais , Eritrócitos/parasitologia , Temperatura Alta , Humanos , Técnicas In Vitro , Macaca mulatta , Plasmodium knowlesi , Especificidade da EspécieRESUMO
Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase-deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser(169); Ala substitution for Ser(169) resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore beta-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser (169)--> Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , AMP Cíclico/fisiologia , Fatores de Ligação de DNA Eritroide Específicos , Eritropoese , Eritropoetina/fisiologia , Histona Acetiltransferases , Camundongos , Mutagênese Sítio-Dirigida , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Coativador 3 de Receptor Nuclear , Fosfosserina/metabolismo , Relação Estrutura-Atividade , Transativadores/fisiologia , Ativação TranscricionalRESUMO
Nitric oxide (NO) and cGMP have been implicated in many neuronal functions, including regulation of gene expression, but little is known about the downstream targets of NO/cGMP in the nervous system. We found that type II cGMP-dependent protein kinase (G-kinase), which is widely expressed in the brain, mediated NO- and cGMP-induced activation of the fos promoter in cells of neuronal and glial origin; the enzyme was ineffective in regulating gene expression in fibroblast-like cells. The effect of G-kinase II on gene expression did not require calcium uptake but was synergistically enhanced by calcium. G-kinase II was membrane associated and did not translocate to the nucleus; however, a soluble G-kinase II mutant translocated to the nucleus and regulated gene expression in fibroblast-like cells. Soluble G-kinase I also regulates fos promoter activity, but membrane targeting of G-kinase I prevented the enzyme from translocating to the nucleus and regulating transcription in multiple cell types, including glioma cells; this suggests that cell type-specific factor(s) that mediate the transcriptional effects of extranuclear G-kinase II are not regulated by G-kinase I. Our results suggest that G-kinase I and II control gene expression by different mechanisms and that NO effects on neuronal plasticity may involve G-kinase II regulation of gene expression.-Gudi, T., Hong, G. K.-P., Vaandrager, A. B., Lohmann, S. M., Pilz, R. B. Nitric oxide and cGMP regulate gene expression in neuronal and glial cells by activating type II cGMP-dependent protein kinase.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Óxido Nítrico/fisiologia , Células 3T3 , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Cálcio/metabolismo , Células Cultivadas , Cricetinae , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteína Quinase Dependente de GMP Cíclico Tipo II , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Proteínas Oncogênicas v-fos/genética , Regiões Promotoras Genéticas/fisiologia , Transdução de Sinais , Ativação TranscricionalRESUMO
Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B(12)) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [(14)C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [(14)C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.
Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/antagonistas & inibidores , Carbono/metabolismo , Ácido Fólico/metabolismo , Óxido Nítrico/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Cricetinae , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Metionina/biossíntese , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Nucleotídeos de Purina/biossíntese , Ratos , Serina/biossíntese , Vitamina B 12/metabolismoRESUMO
The cAMP/cAMP-dependent protein kinase (A-kinase) and Ca2+/calmodulin-dependent protein kinase (Cam-kinase) signal transduction pathways are well known to regulate gene transcription, but this has not been demonstrated directly for the cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway. Here we report that transfection of G-kinase into G-kinase-deficient cells causes activation of the human c-fos promoter in a strictly cGMP-dependent manner. The effect of G-kinase appeared to be mediated by several sequence elements, most notably the serum response element (SRE), the AP-1 binding site (FAP), and the cAMP response element (CRE). The magnitude of G-kinase transactivation of the fos promoter was similar to that of A-kinase, but there were significant differences between G-kinase and A-kinase activation of single enhancer elements and of a chimeric Gal4-CREB transcription factor. Our results indicate that G-kinase transduces signals to the nucleus independently of A-kinase or Ca2+, although it may target some of the same transcription factors as A-kinase and Cam-kinase.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/biossíntese , Regulação Enzimológica da Expressão Gênica , Genes fos , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Cricetinae , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Humanos , Rim , Cinética , Mutagênese Insercional , Proteínas Recombinantes/biossíntese , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.