RESUMO
Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Mieloma Múltiplo/patologia , Pirazóis/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/fisiologia , Ureia/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Humanos , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Ureia/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have identified a new protease in Escherichia coli, which is required for its viability under normal growth conditions. This protease is anchored in the inner membrane and the gene encoding it has been named ecfE, since it is transcribed by Esigma(E) polymerase. Multicopy expression of the ecfE gene was found to turn down expression of both Esigma(E)- and Esigma(32)-transcribed promoters. Purified EcfE degrades both heat shock sigma factors RpoE and RpoH in vitro. EcfE has a zinc binding domain at the N-terminus, a PDZ-like domain in the middle and a highly conserved tripeptide, LDG, at the C-terminus. These features are characteristic of members of a new class of proteases whose activity occurs close to the inner membrane or within the inner membrane. Temperature-sensitive mutants of this gene were isolated mapping to the catalytic site and other domains that exhibited constitutively elevated levels of both heat shock regulons.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Membrana/metabolismo , Alelos , Arabinose/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/fisiologia , Eletroforese em Gel de Poliacrilamida , Endopeptidases/genética , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Estabilidade Enzimática/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/genética , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Polissacarídeos/metabolismo , Estrutura Terciária de Proteína/fisiologia , Fator sigma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Fatores de Transcrição/metabolismo , Zinco/metabolismoRESUMO
The elucidation of whole-genome sequences is expected to have a revolutionary impact on the discovery of novel medicines. With the availability of complete genome sequences of more than 30 different species, the field of antimicrobial drug discovery has the opportunity to access a remarkable diversity of genomic information. In this review, I summarize how microbial genomics has changed strategies of drug discovery by applying bioinformatics, novel genetic approaches and genomics-based technologies, including analysis of gene expression using DNA microarrays.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Desenho de Fármacos , Genoma Bacteriano , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Biotecnologia/métodos , Biologia Computacional/métodosRESUMO
The genomics era is providing us with vast amounts of information derived from whole-genome sequencing. This will doubtlessly revolutionise biology and the way novel medicines will be discovered. To leverage this information efficiently, however, technologies in addition to high-throughput sequencing are required. DNA microarray technology is one technology that has already shown great potential for both basic research and drug discovery. With particular emphasis on antibacterial research we will summarise in this review the key technological aspects and most important applications of DNA microarrays demonstrated so far.
Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Farmacogenética/métodos , Bactérias/efeitos dos fármacos , Mapeamento Cromossômico , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Farmacogenética/estatística & dados numéricos , Farmacogenética/tendênciasRESUMO
We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open reading frames or y-genes) and conserved in the compact genome of Mycoplasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to screen for essential E. coli genes. To verify this phenotype, we constructed conditional mutants in genes for which no null mutants could be obtained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ). The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Bacillus subtilis. This low number of essential genes was unexpected and might be due to a characteristic of the versatile genomes of E. coli and B. subtilis that is comparable to the phenomenon of nonorthologous gene displacement. The gene ygjD, encoding a sialoglycoprotease, was eliminated from a minimal genome computationally derived from a comparison of the Haemophilus influenzae and M. genitalium genomes. We show that ygjD and its ortholog ydiE are essential in E. coli and B. subtilis, respectively. Thus, we include this gene in a minimal genome. This study systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown function required for survival on complex media.
Assuntos
Escherichia coli/genética , Genoma Bacteriano , Sequência de Aminoácidos , Bacillus subtilis/genética , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
TlpA, the membrane-anchored, thioredoxin-like protein from Bradyrhizobium japonicum, is essential for cytochrome aa3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline-109 in its active-site sequence C107 V108 P109 C110 by a histidine residue. The resulting active-site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild-type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild-type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild-type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.
Assuntos
Proteínas de Bactérias/metabolismo , Rhizobiaceae/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Ditiotreitol/metabolismo , Genes Bacterianos , Glutationa/metabolismo , Histidina/química , Insulina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Prolina/química , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
Curli, an adhesive surface fibre produced by Escherichia coli and salmonellae, was proposed on the basis of genetic evidence to follow a distinct assembly pathway involving an extracellular intermediate of the fibre subunit CsgA, the polymerization of which can be induced at the cell surface by a 'nucleator' protein (CsgB). Here we show biochemically that CsgA is actively secreted to the extracellular milieu and that CsgB is surface located. We demonstrate that the putative curli assembly factor CsgG is an outer membrane-located lipoprotein. CsgG is highly resistant to protease digestion both in vivo and in vitro. During curli assembly, CsgG is required to maintain the stability of CsgA and CsgB. In line with this, it is possible to modulate the steady-state levels of CsgA and CsgB by varying intracellular levels of CsgG. This suggests that, in the absence of CsgG, CsgA and CsgB are proteolytically degraded. Moreover, curli production and steady-state levels of CsgA and CsgB can be increased above wild-type levels by overexpression of CsgG, meaning that the quantity of assembled curli fibres can be controlled by this lipoprotein.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Fibronectinas/metabolismo , Lipoproteínas/metabolismo , Peptídeos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Endopeptidase K/metabolismo , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ácido Palmítico/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Dodecilsulfato de Sódio/farmacologia , Ureia/farmacologiaRESUMO
The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-negative soil bacterium Bradyrhizobium japonicum was initially discovered due to its essential role in the maturation of cytochrome aa3. A soluble form of TlpA lacking the N-terminal membrane anchor acts as a protein thiol:disulfide oxidoreductase. TlpA possesses an active-site disulfide bond common to all members of the thiol:disulfide oxidoreductase family. In addition, it contains two non-active-site cysteines that form a structural disulfide bond (Loferer, H., Bott, M., and Hennecke, H. (1993) EMBO J. 12, 3373-3383; Loferer, H., and Hennecke, H. (1994) Eur. J. Biochem. 223, 339-344). Here, we compare the far- and near-UV CD spectra of TlpA before and after reduction of both disulfides by dithiothreitol and show that the non-active-site disulfide bond is not required for the integrity of TlpA's native conformation. In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the active-site disulfide and leaves the non-active-site disulfide bond intact, even at high molar excess over TlpA. The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA at 355 nm, which may be interpreted as a quenching of tryptophan fluorescence by the active-site disulfide bond. Using the specific fluorescence of TlpA as a measure of its redox state, a value of 1.9 +/- 0.2 M was determined for the TlpA:glutathione equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant, like cytoplasmic thioredoxins. The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia coli, is not capable of oxidizing the active-site cysteines of TlpA. This suggests that TlpA's primary role in vivo is keeping the thiols of certain proteins reduced and that TlpA's active, reduced state may be maintained owing to its kinetically restricted oxidation by other periplasmic disulfide oxidoreductases such as DsbA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Conformação Proteica , Rhizobiaceae/metabolismo , Tiorredoxinas/metabolismo , Proteínas de Bactérias/biossíntese , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Cisteína , Primers do DNA , Ditiotreitol/farmacologia , Escherichia coli , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Dissulfeto de Glutationa , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por RestriçãoRESUMO
Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.
Assuntos
Grupo dos Citocromos c/metabolismo , Nitratos/metabolismo , Rhizobiaceae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Grupo dos Citocromos c/genética , DNA Bacteriano/genética , Transporte de Elétrons , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Mapeamento por Restrição , Rhizobiaceae/genética , Rhizobiaceae/crescimento & desenvolvimento , Glycine max/metabolismo , Glycine max/microbiologia , Simbiose/genética , Simbiose/fisiologiaRESUMO
A total of nine genes potentially concerned with the biosynthesis of c-type cytochromes have been identified recently in the bacteria Bradyrhizobium japonicum and Rhodobacter capsulatus, and homologous counterparts appear to be present also in Escherichia coli. Most of the respective gene products are membrane-bound, while others are located in the periplasmic space. As inferred from sequence analyses, several of these proteins may play roles in membrane transport or redox processes, both functions being consistent with the required steps in cytochrome c formation (membrane translocation of heme; covalent linkage of protoheme IX to cysteine thiols). Further genes of B. japonicum, E. coli, Bacillus subtilis and Paracoccus denitrificans have been studied whose products are necessary for the formation of intact heme/copper oxidases. Some of them are probably required in protein folding and assembly whereas others appear to be enzymes catalyzing steps in the biosynthesis of the heme cofactors.
Assuntos
Proteínas de Bactérias/genética , Grupo dos Citocromos c/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Genes Bacterianos , Aerobiose , Sequência de Aminoácidos , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Escherichia coli , Dados de Sequência Molecular , Rhodobacter capsulatusRESUMO
The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane-anchored, periplasmic thioredoxin-like protein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A soluble form of TlpA, which lacks its N-terminal membrane anchor, was overexpressed in E. coli and purified by a two-step procedure. Pure TlpA was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RNaseA. Evidence is presented that two non-active-site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins.
Assuntos
Proteínas de Bactérias/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Western Blotting , Clonagem Molecular , Cisteína/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Plasmídeos , Rhizobiaceae , Ribonucleases/metabolismo , Solubilidade , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificaçãoRESUMO
Thioredoxins and eukaryotic protein disulphide isomerases were, until recently, the only enzymes known to catalyse reversible oxidation and reduction of cysteine residues of a wide spectrum of protein substrates. Genetic and biochemical investigations on different bacterial systems have now led to the discovery of novel prokaryotic protein disulphide oxidoreductases that are located either in the periplasm or in the cytoplasmic membrane.
Assuntos
Bactérias/enzimologia , Dissulfetos/metabolismo , Oxirredutases/metabolismo , Cisteína/metabolismo , Glutarredoxinas , Bactérias Gram-Negativas/enzimologia , Isomerases/genética , Isomerases/metabolismo , Oxirredutases/genética , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Isomerases de Dissulfetos de Proteínas , Proteínas , Tiorredoxinas/metabolismoRESUMO
We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.
Assuntos
Proteínas de Bactérias/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Genes Bacterianos , Rhizobiaceae/metabolismo , Simbiose , Tiorredoxinas/biossíntese , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Clonagem Molecular , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Escherichia coli , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Plasmídeos , Biossíntese de Proteínas , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Homologia de Sequência de Aminoácidos , Glycine max/microbiologia , Glycine max/ultraestrutura , Tiorredoxinas/química , Tiorredoxinas/genética , beta-Galactosidase/metabolismoRESUMO
We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.
Assuntos
Grupo dos Citocromos c/genética , Genes Bacterianos , Rhizobiaceae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fenótipo , Plasmídeos , Mapeamento por Restrição , Rhizobiaceae/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation-induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s).
Assuntos
Dano ao DNA/genética , Fatores de Transcrição/efeitos da radiação , Animais , Reparo do DNA/genética , Elementos Facilitadores Genéticos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos da radiaçãoRESUMO
Expression of the transforming Ha-ras oncogene in MMTV-LTR transfected NIH 3T3 cells leads to a growth factor independent activation of the Na+/H(+)-antiporter. The activation of the antiporter is insensitive to the protein kinase inhibitor staurosporine and equally expressed in protein kinase C-depleted cells. It is concluded that the Ha-ras induced activation of the antiporter occurs by a protein kinase C-independent mechanism. An inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of the extracellular [Na+] concentration results in a depression of the bombesin induced release of Ca2+ from intracellular stores. These results are explained by a steep pH-dependence of the Ca2(+)-mobilizing system which exhibits a maximum at pH 7.1 in the system studied here. Stimulation by growth factors of quiescent cells with a resting pH below 7 results in a shift of the cytosolic pH towards the optimum for the Ca2+ release. In agreement with the proposed interrelationship, pHi and [Ca2+]i rise and peak simultaneously after addition of bombesin to G0 arrested cells.
Assuntos
Proteínas de Transporte/metabolismo , Genes ras , Alcaloides/farmacologia , Animais , Bombesina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Citosol/metabolismo , Dexametasona/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Proteína Quinase C/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.