RESUMEN

An increase in the activity of mitogen-activated protein kinase (MAPK) has been correlated with the progression of prostate cancer to advanced disease in humans. The serine/threonine protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of MAPK but its role in prostate cancer has not previously been examined. Increasing RSK isoform 2 (RSK2) levels in the human prostate cancer line, LNCaP, enhanced prostate-specific antigen (PSA) expression, an important diagnostic marker for prostate cancer, whereas inhibiting RSK activity using a RSK-specific inhibitor, 3Ac-SL0101, decreased PSA expression. The RSK2 regulation of PSA expression occurred via a mechanism involving both RSK2 kinase activity and its ability to associate with the coactivator, p300. RNA interference of the androgen receptor (AR) showed that the AR was important in the RSK2-mediated increase in PSA expression. RSK levels are higher in approximately 50% of human prostate cancers compared with normal prostate tissue, which suggests that increased RSK levels may participate in the rise in PSA expression that occurs in prostate cancer. Furthermore, 3Ac-SL0101 inhibited proliferation of the LNCaP line and the androgen-independent human prostate cancer line, PC-3. These results suggest that proliferation of some prostate cancer cells is dependent on RSK activity and support the hypothesis that RSK may be an important chemotherapeutic target for prostate cancer.

Asunto(s)

Neoplasias de la Próstata/enzimología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Humanos , Masculino , Antígeno Prostático Específico/biosíntesis , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/genética , Receptores Androgénicos/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/biosíntesis , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Activación Transcripcional , Transfección

RESUMEN

We describe a novel mechanism for transcriptional regulation, in which docking of p90 ribosomal S6 kinase 2 (Rsk2) to the hormone-binding domain (HBD) of estrogen receptor alpha (ERalpha) induces a conformational change that enhances the transcriptional activation function contained in the HBD. A constitutively active mutant of Rsk2 specifically enhances ERalpha-mediated transcription by phosphorylation of Ser167 in ERalpha and by physically associating with residues 326-394 of the ERalpha HBD. The anti-estrogen 4-hydroxytamoxifen blocks Rsk2-mediated activation of ERalpha, by inducing a conformation of ERalpha in which the Rsk2 docking site is masked. Transcriptional activation and docking are specific for ERalpha and do not occur with the related isoform, ERbeta. ERalpha phosphorylation, docking and transcriptional activation are regulated by the Rsk2 N-terminal kinase domain. The allosteric regulation of a target protein, independent of phosphorylation, may be paradigmatic of a general function for protein kinase docking sites.

Asunto(s)

Estradiol/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Receptores de Estrógenos/metabolismo , Transcripción Genética , Activación Transcripcional/fisiología , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Sitios de Unión , Neoplasias de la Mama , Línea Celular , Cricetinae , Estradiol/farmacología , Receptor alfa de Estrógeno , Femenino , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Fosfoserina/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Estructura Secundaria de Proteína , Receptores de Estrógenos/química , Proteínas Recombinantes/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Células Tumorales Cultivadas

RESUMEN

Mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) lie immediately downstream of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38 MAPK. Although the family of MAPKAPKs shares sequence similarity, it demonstrates selectivity for the upstream activator. Here we demonstrate that each of the ERK- and p38 MAPK-regulated MAPKAPKs contains a MAPK docking site positioned distally to the residue(s) phosphorylated by MAPKs. The isolated MAPK docking sites show specificity for the upstream activator similar to that reported for the full-length proteins. Moreover, replacement of the ERK docking site of p90 ribosomal S6 kinase with the p38 MAPK docking site of MAPKAPK2 converts p90 ribosomal S6 kinase into a stress-activated kinase in vivo. It is apparent that mechanisms controlling events downstream of the proline-directed MAPKs involve specific MAPK docking sites within the carboxyl termini of the MAPKAPKs that determine the cascade in which the MAPKAPK functions.

Asunto(s)

Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ingeniería de Proteínas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Cricetinae , Activación Enzimática , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Fosforilación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Ratas , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Quinasas S6 Ribosómicas/química , Proteínas Quinasas S6 Ribosómicas/genética , Especificidad por Sustrato , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

p90 ribosomal S6 kinases (RSKs), containing two distinct kinase catalytic domains, are phosphorylated and activated by extracellular signal-regulated kinase (ERK). The amino-terminal kinase domain (NTD) of RSK phosphorylates exogenous substrates, whereas the carboxyl-terminal kinase domain (CTD) autophosphorylates Ser-386. A conserved putative autoinhibitory alpha helix is present in the carboxyl-terminal tail of the RSK isozymes ((697)HLVKGAMAATYSALNR(712) of RSK2). Here, we demonstrate that truncation (Delta alpha) or mutation (Y707A) of this helix in RSK2 resulted in constitutive activation of the CTD. In vivo, both mutants enhanced basal Ser-386 autophosphorylation by the CTD above that of wild type (WT). The enhanced Ser-386 autophosphorylation was attributed to disinhibition of the CTD because a CTD dead mutation (K451A) eliminated Ser-386 autophosphorylation even in conjunction with Delta alpha and Y707A. Constitutive activity of the CTD appears to enhance NTD activity even in the absence of ERK phosphorylation because basal phosphorylation of S6 peptide by Delta alpha and Y707A was approximately 4-fold above that of WT. A RSK phosphorylation motif antibody detected a 140-kDa protein (pp140) that was phosphorylated upon epidermal growth factor or insulin treatment. Ectopic expression of Delta alpha or Y707A resulted in increased basal phosphorylation of pp140 compared with that of WT, presenting the possibility that pp140 is a novel RSK substrate. Thus, it is clear that the CTD regulates NTD activity in vivo as well as in vitro.

Asunto(s)

Proteínas Quinasas S6 Ribosómicas/metabolismo , Ribosomas/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Dominio Catalítico , Células Cultivadas , Cricetinae , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Datos de Secuencia Molecular , Mutación , Fosforilación , Estructura Secundaria de Proteína , Proteínas Quinasas S6 Ribosómicas/química , Proteínas Quinasas S6 Ribosómicas/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser36 --> Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no discernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was inhibited by wild type HA-p190, but JNK activity was enhanced by the S36N mutant. Co-expression of HA-p190 with a fragment containing only the mutated GTP binding domain partially inhibited the beaded extension phenotype, suggesting that it may sequester a factor required for p190 function. Taken together these data demonstrate that within the cell, the Rho/Rac GAP activity of p190 can be regulated by the N-terminal GTP binding domain.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido , Guanosina Trifosfato/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Asparagina , Sitios de Unión , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carbacol/farmacología , Secuencia Conservada , Proteínas de Unión al ADN , Activación Enzimática , Proteínas Activadoras de GTPasa , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras , Serina , Transfección

RESUMEN

Phosphorylation of Ser118 of human estrogen receptor alpha (ER) enhances ER-mediated transcription and is induced by hormone binding and by activation of the mitogen-activated protein kinase (MAPK) pathway. We discovered that phosphorylation of Ser118 reduces the electrophoretic mobility of the ER. Using this mobility shift as an assay, we determined the in vivo stoichiometry and kinetics of Ser118 phosphorylation in response to estradiol, ICI 182,780, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA). In human breast cancer MCF-7 cells, estradiol induced a steady state phosphorylation of Ser118 within 20 min with a stoichiometry of 0.67 mol of phosphate/mol of ER. Estradiol did not activate p42/p44 MAPK, and basal p42/p44 MAPK activity was not sufficient to account for phosphorylation of Ser118 in response to estradiol. In contrast, both EGF and PMA induced a rapid, transient phosphorylation of Ser118 with a stoichiometry of approximately 0. 25, and the onset of Ser118 phosphorylation correlated with the onset of p42/p44 MAPK activation by these agents. Either the EGF- or PMA-induced Ser118 phosphorylation could be inhibited without influencing estradiol-induced Ser118 phosphorylation. The data suggest that a kinase other than p42/p44 MAPK is involved in the estradiol-induced Ser118 phosphorylation. We propose that the hormone-induced change in ER conformation exposes Ser118 for phosphorylation by a constitutively active kinase.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Estradiol/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Estrógenos/efectos de los fármacos , Serina/metabolismo , Animales , Línea Celular , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Mutagénesis Sitio-Dirigida , Fosforilación , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Serina/química , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

The estrogen receptor alpha (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1 increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1 in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

Asunto(s)

Proteínas Cromosómicas no Histona , Proteínas Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa , Serina/metabolismo , Factores de Transcripción , Transcripción Genética , Animales , Células COS , Catálisis , Cricetinae , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Furilfuramida/metabolismo , Chaperonas de Histonas , Humanos , Leucina Zippers , Fosforilación , Receptores de Interferón/metabolismo , Células Tumorales Cultivadas

RESUMEN

This report describes a novel yeast one-hybrid system which easily allows for the detection of mutations in the ligand-binding domain of the estrogen receptor. This screen is based on the observation that a fusion protein consisting of the GAL4 DNA-binding domain and the estrogen receptor can interact with a GAL4 upstream activating sequence and induce the expression of an integrated GAL1-lacZ gene only in the presence of estradiol. Various deletion mutants of the estrogen receptor were tested in this assay and activating function 1 which is present in the N-terminus of the estrogen receptor was found to be responsible for the transactivation produced in the assay. To test if the screen could be used to detect random mutants in the ligand-binding domain of the estrogen receptor the region of the human receptor between amino acids 381 to 403 was mutated by oligonucleotide saturation mutagenesis. Two of the mutants generated by this mutagenesis were characterized to demonstrate that the results obtained from the screen in the yeast screen are relevant to mammalian systems. One of the mutants which has a valine at position number 388 instead of a glycine was able to transactivate in both the yeast and a mammalian system. This mutant was a more potent activator of transcription and also appeared to have a higher affinity for [3H]estradiol in vivo than the wild type receptor. The other mutant which was characterized has five amino acid changes from amino acids 390 through 400. This mutant was nonfunctional in the yeast and mammalian transcription assays and did not bind [3H]estradiol in vivo or in vitro.

Asunto(s)

Estradiol/metabolismo , Mutación , Receptores de Estrógenos/genética , Saccharomyces cerevisiae/genética , Animales , Secuencia de Bases , Línea Celular , Cricetinae , ADN Recombinante , Humanos , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Receptores de Estrógenos/metabolismo , Eliminación de Secuencia , Activación Transcripcional

RESUMEN

Serine 118 is definitively identified as a major site of phosphorylation in the human estrogen receptor expressed in COS-1 cells treated with estradiol or phorbol ester. At least 30% of the estrogen receptor appears to be phosphorylated on serine 118 after treatment with estradiol or phorbol ester. Human estrogen receptor was expressed in COS-1 cells and labeled in vivo with [32P]orthophosphate in the presence of estradiol or phorbol ester. Immunopurified receptor was digested with cyanogen bromide. The most heavily labeled peptide (7 kilodaltons) was identified as amino acids 110-174 by microsequencing. Manual Edman degradation released a major portion of the 32P-label in the peptide at serine 118. A mutant with serine 118 replaced by alanine (S118A) had 80% less 32P-label in the 7 kilodalton peptide. Estrogen receptor labeled in vivo with [32P]-orthophosphate in the presence of estradiol or phorbol ester migrates electrophoretically as a doublet. The major difference between the bands is phosphorylation of serine 118 in the upshifted band. The mutant S118A does not show an upshifted band. Labeling of the estrogen receptor with [35S]methionine indicates that > or = 30% of the receptor is upshifted and suggests that > or = 30% of the receptor is phosphorylated on serine 118.

Asunto(s)

Estradiol/farmacología , Fosfoserina/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Secuencia de Bases , Compartimento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Bromuro de Cianógeno/química , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Mapeo Peptídico , Fosfoserina/química , Proteína Quinasa C/fisiología , ARN Mensajero/genética , Receptores de Estrógenos/química , Proteínas Recombinantes , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

The estrogen receptor, a hormone-regulated transcription factor, regulates gene expression by interacting with a specific nucleotide sequence called the estrogen-responsive element (ERE). In this report we demonstrate by potassium permanganate, osmium tetroxide and diethylpyrocarbonate reactivity and S1 nuclease sensitivity that the nucleotides either within or in the immediate region of imperfect and perfect EREs are in a non-B DNA conformation. The presence of nucleotides in a non-B DNA conformation in the ERE is an intrinsic property of the DNA and is independent of whether the ERE is in linear or supercoiled DNA. S1 nuclease sensitivity was peculiar to the ERE as it was not detected in the thyroid hormone-responsive element. Our results suggest that the nucleotides comprising the ERE are structurally labile. We propose that this intrinsic lability of the ERE could be constrained in vivo such that a unique DNA tertiary structure is formed which may facilitate recognition of the ERE by the estrogen receptor.

Asunto(s)

ADN/química , Estrógenos/metabolismo , Receptores de Estrógenos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN/metabolismo , ADN Recombinante , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Prolactina/genética , Regiones Promotoras Genéticas , Ratas , Eliminación de Secuencia , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo

RESUMEN

An initial crucial step in estrogen activation of gene expression is the interaction of the estrogen receptor with a specific nucleotide sequence [estrogen responsive element (ERE)]. Previously, we found that the estrogen receptor binds preferentially and with high affinity to the lower strand of the rat prolactin imperfect ERE which contains tertiary structure (Lannigan DA and Notides AC, Proc Natl Acad Sci USA 86: 863-867, 1989). Using perfect and imperfect EREs from the upstream region of the chicken vitellogenin II gene, we have now extended our findings and have determined that the estrogen receptor preferentially interacts with either perfect or imperfect EREs which contain tertiary structure. A similar structure is present in a synthetic 42 bp oligonucleotide corresponding to the lower strand of a perfect ERE with flanking sequences from the rat prolactin ERE. Moreover, deviations from the ERE consensus sequence decrease the binding of the estrogen receptor to the tertiary-structured ERE. We also have determined that ERE flanking sequences contribute to the affinity of the receptor for the tertiary-structured ERE. Furthermore, ERE flanking sequences can influence the types of interactions that the estrogen receptor makes with the tertiary-structured ERE.

Asunto(s)

Oligonucleótidos/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Femenino , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/aislamiento & purificación , Prolactina/genética , Ratas

Asunto(s)

ADN/genética , Regulación de la Expresión Génica , Receptores de Estrógenos/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/fisiología , Transcripción Genética , Animales , Secuencia de Bases , ADN/ultraestructura , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Homología de Secuencia de Ácido Nucleico

Asunto(s)

Estrógenos/fisiología , Receptores de Estrógenos/metabolismo , Transcripción Genética/fisiología , Regulación Alostérica , Animales , Secuencia de Bases , Proteínas de Unión al ADN/fisiología , Estrógenos/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Modelos Químicos , Datos de Secuencia Molecular

RESUMEN

An initial step in the transcriptional activation of the prolactin gene by estrogen is the binding of the estrogen-receptor complex to a specific nucleotide sequence [estrogen responsive element (ERE)]. Using the gel mobility assay, we examined the binding mechanism of purified estrogen receptor to the ERE contained on a 255-base-pair fragment from the upstream region between nucleotides -1784 to -1531 of the rat prolactin gene. Remarkably, specific high-affinity binding was detected to the dissociated "coding strand" but not to the "noncoding strand" of the ERE-containing fragment. The dissociated strands of this fragment possess unusual secondary structure, as indicated by their anomalous migration in the gel mobility assay. The estrogen receptor binds to the coding strand of the ERE with a 60-fold higher affinity than to the double-stranded ERE. Furthermore, the receptor binds with a 1000-fold greater affinity to the coding strand of the ERE than to a double-stranded nonspecific DNA fragment. We propose that, in vivo, the estrogen receptor initially binds to the double-stranded ERE. Subsequently, the DNA strands separate due to transitory strand separation and supercoiling, allowing folding of the coding strand of the ERE into a structure that is then bound more tightly by the receptor. The formation of this receptor-ERE coding strand complex may be a crucial step in the mechanism of estrogen-stimulated transcription.

Asunto(s)

ADN/metabolismo , Regulación de la Expresión Génica , Genes , Prolactina/genética , Receptores de Estrógenos/metabolismo , Transcripción Genética , Animales , Unión Competitiva , Cinética , Modelos Teóricos , Unión Proteica , Ratas , Mapeo Restrictivo

RESUMEN

Amiloride has been reported to inhibit Friend murine erythroleukemic (MEL) cell commitment to differentiate by inhibiting the MEL cell plasma membrane Na+-Ca2+ antiporter (R. L. Smith, I. G. Macara, R. Levenson, D. Housman, and L. Cantley. J. Biol. Chem. 257: 773-780, 1982). We therefore screened a series of amiloride analogues to determine whether a more potent and specific inhibitor of MEL cell differentiation could be found. In our experiments, as in those of Lubin (J. Cell. Physiol. 124: 539-544, 1985), amiloride itself did not inhibit MEL cell differentiation. However, we did find that the amiloride analogue phenamil reversibly inhibits dimethyl sulfoxide (DMSO)-induced MEL cell commitment to differentiate with a K1/2 of 2.5-5.0 microM (in plasma clot assay). At an extracellular concentration of 15 microM, phenamil inhibits commitment to differentiate by approximately 90% in the plasma clot assay while having a minimal effect on growth. Phenamil is not metabolized but is rapidly taken up by MEL cells. Phenamil was most effective as an inhibitor when present during the first 12 h of DMSO treatment, indicating that phenamil affects the early commitment process rather than later steps involved in hemoglobin synthesis. Phenamil does not, however, inhibit the early differentiation-induced decrease in [Na+]i and the concomitant drop in the Na+-K+ pump rate. A specific binding site for phenamil is suggested because some analogues in which the phenamil structure is slightly modified are unable to inhibit differentiation.

Asunto(s)

Amilorida/análogos & derivados , Leucemia Eritroblástica Aguda/patología , Amilorida/metabolismo , Amilorida/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Dimetilsulfóxido/farmacología , Virus de la Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/metabolismo , Ratones , Concentración Osmolar , Ouabaína/farmacología , Rubidio/metabolismo , Sodio/metabolismo

RESUMEN

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.

Asunto(s)

Leucemia Eritroblástica Aguda/metabolismo , Leucemia Experimental/metabolismo , Proteínas de Neoplasias/biosíntesis , Sodio/metabolismo , Aminoácidos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cicloheximida/farmacología , Dimetilsulfóxido/farmacología , Concentración de Iones de Hidrógeno , Cinética , Leucemia Eritroblástica Aguda/patología , Leucemia Experimental/patología , Ratones , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo

RESUMEN

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.

Asunto(s)

Leucemia Eritroblástica Aguda/metabolismo , Sodio/metabolismo , Animales , Transporte Biológico Activo , Diferenciación Celular , Línea Celular , Virus de la Leucemia Murina de Friend , Cinética , Leucemia Eritroblástica Aguda/patología , Ratones