RESUMO
We describe the identification and DNA-binding properties of nuclear proteins from rat L6 myoblasts which recognize an interspecies conserved 3' untranslated segment of pro alpha 1 (I) collagen cDNA. Levels of the two pro alpha 1 (I) collagen RNAs, present in L6 myoblasts, decreased drastically between 54 and 75 h after induction of myotube formation in serum-free medium. Both mRNAs contained a conserved sequence segment of 135 nucleotides (termed tame sequence) in the 3' untranslated region that had 96% homology to the human and murine pro alpha 1 (I) collagen genes. The cDNA of this tame sequence was specifically recognized by nuclear protein(s) from L6 myoblasts, as judged by gel retardation assays and DNase I footprints. The tame-binding protein(s) was able to recognize its target sequence on double-stranded DNA but bound also to the appropriate single-stranded oligonucleotide. Protein that bound to the tame sequence was undetectable in nuclear extracts of L6 myotubes that did not accumulate the two collagen mRNAs. Therefore, the activity of this nuclear protein seems to be linked to accumulation of the sequences that it recognizes in vitro. The collagen RNAs and the nuclear tame-binding proteins reappeared after a change of medium, which further suggests that the RNAs and the protein(s) are coordinately regulated.
Assuntos
Colágeno/genética , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Músculos/citologia , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Diferenciação Celular , Linhagem Celular , Colágeno/metabolismo , DNA/genética , Sondas de DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Dados de Sequência Molecular , Músculos/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosRESUMO
Gastrin, cholecystokinin (CCK), and CCK-related peptides comprise a hormonal family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium ([Ca2+]i). Furthermore, gastrin acts as a direct growth factor through CCKB/gastrin receptors. We report here that the expression of the mRNA coding for CCKB/gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of Ca2+ mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expression of CCKB/gastrin receptor mRNA. Accordingly, gastrin (1-100 nM) did not cause any measurable increase in [Ca2+]i. In contrast, the addition of cholecystokinin residues 26-33 (CCK-8) caused a rapid and transient increase in [Ca2+]i in this cell line. CCK-8 mobilized Ca2+ in a dose-dependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nM). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 3 nM) in GLC19 but not in H510 cells. The selective CCKB/gastrin antagonist blocked the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate Ca2+ mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.
Assuntos
Cálcio/metabolismo , Carcinoma de Células Pequenas/metabolismo , Gastrinas/farmacologia , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/metabolismo , Receptores da Colecistocinina/biossíntese , Sincalida/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bradicinina/farmacologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Células Clonais , Primers do DNA , Cães , Expressão Gênica , Humanos , Cinética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Muridae , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/biossíntese , RNA Neoplásico/isolamento & purificação , Ratos , Receptores da Colecistocinina/antagonistas & inibidores , Receptores da Colecistocinina/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.
Assuntos
Fibroblastos/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Células 3T3 , Acetofenonas/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Benzopiranos/farmacologia , Briostatinas , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fixadores/farmacologia , Glutaral/farmacologia , Humanos , Isoenzimas/química , Lactonas/farmacologia , Macrolídeos , Camundongos , Mitógenos/farmacologia , Ligação Proteica , Isoformas de Proteínas , Proteína Quinase C/química , Proteína Quinase C-delta , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RA-induced apoptosis was drastically reduced to 3 to 15% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 microM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mzl cultures, were able to grow as single cells.
Assuntos
Apoptose , Linhagem da Célula , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Células-Tronco/fisiologia , Animais , Biomarcadores/análise , Caspase 8 , Caspase 9 , Caspases/farmacologia , Diferenciação Celular , Divisão Celular , Ceramidas/farmacologia , DNA Complementar , Eletroforese em Gel de Ágar , Fibroblastos/citologia , Camundongos , Neuroglia/citologia , Neurônios/citologia , Transfecção , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
We used primary cultures of rat hippocampal neurons and PCC7-Mz1 cells to correlate the expression of the protein kinase C (PKC) gene family with specific events during neural differentiation. Multipotent PCC7-Mz1 embryonic carcinoma stem cells develop into a tissue-like pattern of neuronal, fibroblast-like and astroglial cells by all-trans retinoic acid (RA) treatment. Western blot analyses demonstrate that PKCalpha, betaI, gamma, theta, mu, lambda, and zeta were constitutively expressed but the expression of PKCbetaII, delta, epsilon, and eta was up-regulated three days after addition of RA when cells mature morphologically. While the protein levels of the PKC isoforms betaII, delta and eta decreased after d6, when the major phenotypical alterations of the developing neurons were completed, PKCepsilon expression remained at a high level. Immunofluorescence studies demonstrated that PKCalpha, lambda and zeta were constantly expressed in stem cells and the arising cell types. PKCdelta was detected in all differentiated cell types, whereby PKCbetaII, gamma, epsilon, and zeta were solely found in the neuronal derivatives with PKCgamma predominantly located in the nuclei. PKCeta was weakly expressed at the Golgi complex of stem cells but expanded throughout the entire somata of all developing neurons. In contrast, PKCbetaII was abundant only in the somata of a minor fraction of all neurons (approximately 2.5%). Also, PKCepsilon was exclusively synthesized by a subpopulation of neurons (40+/-5%), where it was localized in the somata and in the axons. PKCzeta was persistently expressed in two forms, the full-length PKCzeta and the constitutively active, proteolytic product PKMzeta, reasoning that permanent PKCzeta activity is important for PCC7-Mz1 physiology. Fractionation of extracts from undifferentiated and differentiating PCC7-Mz1 cells revealed that the conventional cPKCalpha was partly and the cPKCbetaI and the novel nPKCs delta and epsilon were mainly membrane bound, implying that they were also in an active state. However, when using the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate) to monitor cellular PKC activity, we observed that activation of PKC by phorbol ester was required for complete MARCKS phosphorylation and its translocation from the membrane to the cytoplasm. Our data show that the cell type-specific expression, subcellular localization and activation of PKCs are regulated in an isoform-specific manner during neurogenesis suggesting that they are involved in the control of neural development and in particular in neuronal differentiation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Neurônios/enzimologia , Proteína Quinase C/genética , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Diferenciação Celular , Células Cultivadas , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Neurônios/citologia , Biossíntese de Proteínas , Proteína Quinase C/imunologia , Ratos , Frações Subcelulares , Tretinoína/farmacologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
We determined the nucleotide sequences of five members of the rbcS gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of potato. The genomic organization, structure and expression of the genes is compared to the features of the rbcS genes in tomato. Within the two species, Lycopersicon esculentum and Solanum tuberosum, both members of the Solanaceae, the rbcS genes share more interspecific sequence identity, especially in the 5'- and 3'-untranslated regions and the intron sequences, than within one species. However, the expression data of orthologous rbcS genes containing highly identical regulatory cis-acting elements were found to be different, suggesting that the simple finding of these motifs does not automatically imply similar transcriptional and/or post-transcriptional gene regulation.
Assuntos
Família Multigênica , Proteínas de Plantas/genética , Ribulose-Bifosfato Carboxilase/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Sequência de Bases , DNA , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/enzimologia , Verduras/genéticaRESUMO
It is demonstrated here that p42 MAPKinase (p42 MAPK) phosphorylates the Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) at Ser-113. In permeabilised Swiss 3T3 cells activation of protein kinase C (PKC) leads to p42 MAPK activation, but only the protein kinase C sites in MARCKS become phosphorylated and not Ser-113. The mitogen platelet-derived growth factor (PDGF) elicits the same response. These results demonstrate that while Ser-113 is a substrate for p42 MAPK in vitro and can be phosphorylated in vivo as shown by Taniguchi et al. [(1994) J. Biol. Chem. 269, 18299-18302], its phosphorylation is not subject to acute regulation by p42 MAPK in Swiss 3T3 cells.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Serina/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Mitógenos/farmacologia , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Substrato Quinase C Rico em Alanina Miristoilada , Fragmentos de Peptídeos/química , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Haplorrinos , Rim/citologia , Dados de Sequência Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência , Transdução de SinaisRESUMO
To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Proteínas de Membrana , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Vetores Genéticos , Humanos , Insetos , Isoenzimas/biossíntese , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Peso Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Oligopeptídeos/metabolismo , Dibutirato de 12,13-Forbol/metabolismo , Fosforilação , Proteína Quinase C/biossíntese , Proteína Quinase C/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TransfecçãoRESUMO
The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Anticorpos Monoclonais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Humanos , Ativação do Canal Iônico , Cinética , Canal de Potássio Kv1.1 , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio/imunologia , Canais de Potássio/fisiologia , Proteína Quinase C/metabolismo , Transporte ProteicoRESUMO
Potent activators of protein kinase C in fibroblasts: This property was determined for several indolactam V analogues (1) with a new cell-based assay system. This tumor-promoting indole alkaloid and analogues thereof can be synthesized efficiently on the solid phase. The key steps of the combinatorial approach are a regioselective amination of the indole ring and an enantioselective enzymatic reaction.
Assuntos
Cálcio/metabolismo , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Colecistocinina/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptores da Colecistocinina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Células Clonais , Clonagem Molecular , Cães , Gastrinas/farmacologia , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Receptores da Colecistocinina/biossíntese , Receptores da Colecistocinina/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
We analyzed the effect of growth factors on the localization of the 80-kDa acidic myristoylated alanine-rich C-kinase substrate (80-kDa MARCKS), the major protein kinase C (PKC) substrate, in Swiss 3T3 fibroblasts. Virtually all 80-kDa MARCKS of quiescent cultures of these cells was membrane bound. However, within 40 min after addition of bombesin (10 nM) to these cells, the content of 80-kDa MARCKS in the cytoplasmic fraction increased 25-fold. Phosphorylated 80-kDa MARCKS was detectable in the cytoplasmic fraction as early as 30 s after addition of bombesin and the translocation was sustained for 6 h i.e. until 80-kDa MARCKS became down-regulated. The ability of bombesin to stimulate translocation of 80-kDa MARCKS was dose-dependent (concentration required to produce 50% of the effect was 0.6 nM bombesin) and was abolished by the specific antagonist [Leu14,13 psi 14CH2NH]bombesin. Furthermore, platelet-derived growth factor (PDGF) stimulated a dose-dependent (concentration required to produce 50% of the effect was 3 ng/ml) translocation which was comparable to that induced by bombesin in terms of kinetics and magnitude. Translocation was independent of continuous protein synthesis, but dependent on active PKC. Depletion or inhibition of PKC activity abolished the 80-kDa MARCKS translocation induced by either bombesin or PDGF. Furthermore, the neuropeptides beta-endothelin, bradykinin, and vasopressin, which are known to stimulate PKC activity, also promoted translocation. In contrast, epidermal growth factor, insulin and forskolin, which do not activate PKC, failed to cause such an effect. Translocation of 80-kDa MARCKS was also observed in Rat1 cells treated with phorbol ester, PDGF and beta-endothelin. We conclude that the translocation of 80-kDa MARCKS from the membrane to the cytosol is an early response to a variety of growth-promoting factors that stimulate PKC through different signal-transduction pathways.
Assuntos
Bombesina/farmacologia , Endotelinas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas/metabolismo , Células 3T3/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Proteína Quinase C/metabolismoRESUMO
The acidic 80-kDa myristoylated alanine-rich C-kinase substrate protein (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80-kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1-80K, expressed 4.5 +/- 0.8-fold and 9.5 +/- 1.5-fold higher levels of 80-kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern-blot and Northern-blot analyses of Rat1-80K showed intact integration and correct transcription of the introduced 80-kDa MARCKS gene. The overexpressed 80-kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80-kDa MARCKS has been described as a calmodulin-binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and triflouperazine on the entry into the S-phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K cells was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that overexpression of the 80-kDa MARCKS protein reduces the free concentration of calmodulin in the cell.
Assuntos
Calmodulina/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína Quinase C/metabolismo , Proteínas/metabolismo , Animais , Transporte Biológico , Ciclo Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Fibroblastos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Proteína Quinase C/genética , Proteínas/genética , Ratos , Sulfonamidas/farmacologia , Transfecção , Trifluoperazina/farmacologiaRESUMO
Three RNA species (5, 2, 0.15 kb) characterized by the repetitive identifier (ID) sequence, expressed constitutively, and at low abundance have been identified in rat L6 muscle cells by hybridization to cDNA pL6-411. Comigration of these three RNAs with 28, 18, and 5.8 S ribosomal RNAs (rRNAs) has suggested the possibility that pL6-411 RNAs are related to ribosomes or ribosome-like structures. Subsequent experiments showed that pL6-411-related RNAs could indeed be found in ribosome-like particles which were indistinguishable from ribosomes when separated on sucrose gradients under native (low salt, isolation of intact ribosomes) or denaturing conditions (detergent, high salt, isolation of ribosome subunits). Furthermore, we demonstrate that pL6-411-related RNAs are cytoplasmic in L6 cells, may be transcribed in nucleoli, and, based on their nucleotide sequence, have the potential of inter- and intramolecular hybridization. Expression of pL6-411 RNAs was also shown in adult as well as in fetal rat tissues after Day 14 of gestation. These above findings provide supportive evidence for the hypothesis that pL6-411 5- and 2-kb RNAs could exist in a subset of ribosomes. These ribosome-like pL6-411 particles nevertheless differ from ribosomes in that their associated RNAs have different nucleotide sequences, are of lower abundance, and are up-regulated later in development than rRNAs. We discuss our results in the context of a postulated ribosome subset containing RNAs other than rRNAs. These ribosome-like particles might be involved in the translational control of ID-positive mRNAs.
Assuntos
RNA Ribossômico/fisiologia , RNA/fisiologia , Ribossomos/ultraestrutura , Animais , Sequência de Bases , Linhagem Celular , Nucléolo Celular/fisiologia , Regulação da Expressão Gênica , Hibridização de Ácido Nucleico , RNA Citoplasmático Pequeno , Ratos/embriologia , Ratos/crescimento & desenvolvimento , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteínas/fisiologia , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
We have isolated and characterized a cDNA of 1183 bp, pL6-411, from rat L6 muscle cells. This cDNA contains repetitive sequences - including two inverted copies of the previously described identifier sequence - as shown by sequence analysis. Repetitive sequences from pL6-411 characterize a family of RNAs which is specifically induced during L6 myotube formation. Another part of the pL6-411 sequence, existing at low-copy number per haploid rat genome, hybridized to two RNAs of 5 kb and 2 kb from L6 myoblasts as well as from L6 myotubes. A third pL6-411-related RNA of 150 bases was detected which hybridized with the repetitive sequence but did not hybridize with the low-copy number part of pL6-411. It appears that the 'identifier' sequence in this population of small RNAs is complementary to one of the 'identifier' copies in the pL6-411-related RNA. Finally, we identified on cDNA pL6-411 the recognition site for the TGGCA-binding protein and in both orientations a total of four putative promoters for RNA polymerase III.
Assuntos
Clonagem Molecular , DNA/isolamento & purificação , Músculos/citologia , Transcrição Gênica , Animais , Composição de Bases , Diferenciação Celular , Linhagem Celular , Enzimas de Restrição do DNA , Camundongos , Hibridização de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA/isolamento & purificação , Sequências Repetitivas de Ácido NucleicoRESUMO
Two major protein-kinase-C (PKC) substrates have been described in the literature; an 87-kDa bovine and human PKC substrate, called MARCKS, and an acidic 80-kDa PKC substrate, isolated from rat brain and Swiss 3T3 cells, termed 80K. Since there is only 66-74% sequence similarity between MARCKS and 80K, we have further investigated their relationship in this study. Southern-blot experiments with gene-specific probes demonstrated the presence of the 80K, but not MARCKS, gene in the mouse genome. Furthermore, polymerase-chain-reaction (PCR) analyses using three pairs of primers that specifically recognise either 80K, MARCKS or conserved sequences of both genes, revealed the presence of only the 80K gene in the mouse and rat genomes and only the MARCKS gene in the bovine and human genomes with mRNA expression in the corresponding brain tissues. Northern-blot analysis of a variety of tissues indicated that both 80K and MARCKS have similar patterns of expression. Most components of signal-transduction pathways are present in multiple molecular isoforms as members of a gene family. In contrast, the findings presented in this study indicate that rodent 80K and bovine and human MARCKS are not distinct members of a gene family, but represent the equivalent substrates in different species.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteína Quinase C/metabolismo , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA/química , DNA/genética , Sondas de DNA , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Proteínas/química , RNA Mensageiro/genética , Ratos , Homologia de Sequência , Transdução de SinaisRESUMO
Chitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea) chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1-4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.
Assuntos
Arachis/genética , Quitinases/genética , Sequência de Bases , Southern Blotting , Células Cultivadas , DNA/genética , Indução Enzimática , Técnicas In Vitro , Dados de Sequência Molecular , Phytophthora/fisiologia , Plantas Tóxicas , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Solanum tuberosum/genética , Nicotiana/genéticaRESUMO
GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC beta 1 and the novel PKCs delta and epsilon effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC zeta did not. The K(m) values (between 0.6 and 2.3 microM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The V(max) values of PKC beta 1 and PKC epsilon were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC beta 1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.
Assuntos
Isoenzimas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Proteína GAP-43 , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Cinética , Dados de Sequência Molecular , Família Multigênica , Mapeamento de Peptídeos , Fosfopeptídeos/análise , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.