RESUMEN

23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts. Surprisingly, while expression of either G12V:H-Ras or F156L:H-Ras results in constitutive signaling through PI3-kinase, only cells expressing G12V:H-Ras additionally possess constitutive signaling through MAPK, and NFkappaB. Pharmacological abrogation of the Ras-induced constitutive PI3-kinase signal, however, is not responsible for the impaired differentiation or apoptosis in either A2:G12V:H-Ras myoblasts or A2:F156L:H-Ras myoblasts. Thus, our data suggest that a pathway distinct from those that signals through MAPK, NFkappaB or PI3-kinase is responsible for the impaired differentiation and apoptosis in 23A2 skeletal myoblasts expressing constitutively active Ras.

Asunto(s)

Apoptosis , Diferenciación Celular , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animales , Transformación Celular Neoplásica , Células Cultivadas , Expresión Génica , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , Proteína MioD/genética , Mioblastos Esqueléticos/enzimología , FN-kappa B/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Transducción de Señal

RESUMEN

The proteins that compose the Ras family of small guanosine triphosphatases share a remarkably high degree of sequence similarity, yet recent evidence indicates that they may have unique biological properties. How is it that similar proteins carry out different jobs in the cell? Wolfman addresses this question by surveying recent reports that indicate that different biological roles may be born out of distinct subcellular localizations of the Ras proteins. It appears that the small differences in their amino acid sequences and their different posttranslational modifications may be all that is necessary to direct various Ras proteins to different sites.

Asunto(s)

Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo

RESUMEN

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.

Asunto(s)

Transformación Celular Neoplásica , Células Epiteliales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas ras/metabolismo , Movimiento Celular , Fibronectinas , Genes ras , Mucosa Intestinal , Intestino Delgado , Proteínas Quinasas Activadas por Mitógenos , Mutación , Unión Proteica , Proteínas ras/genética

RESUMEN

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

Asunto(s)

Regulación Neoplásica de la Expresión Génica/fisiología , Quinasa 1 de Quinasa de Quinasa MAP , Sistema de Señalización de MAP Quinasas/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas ras/fisiología , Células 3T3 , Animales , Fibrosarcoma/enzimología , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/fisiología , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética

RESUMEN

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.

Asunto(s)

Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Animales , Línea Celular , Línea Celular Transformada , Membrana Celular/metabolismo , Medio de Cultivo Libre de Suero , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos , Genes ras , Guanosina Trifosfato/metabolismo , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Cinética , Ratones , Oligodesoxirribonucleótidos Antisentido/farmacología , Proteína Quinasa C/genética , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C-epsilon , Proteínas Proto-Oncogénicas c-raf/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras)/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección

RESUMEN

We report that c-N-Ras possesses an isoform-specific, functional role in cell survival under steady-state conditions. This function includes protection from programmed cell death by serum deprivation or upon treatment with apoptosis-inducing agents. The data demonstrate that c-N-Ras may play a functional role in the regulation of steady-state phosphorylated Akt and serine 136-phosphorylated Bad (Ser(136)-pBad). Immortalized N-Ras knockout fibroblasts possess nearly undetectable levels of steady-state Ser(136)-pBad. In contrast, wild-type control cells and the N-Ras knockout cells ectopically expressing c-N-Ras at control levels maintained easily detectable levels of Ser(136)-pBad both at steady-state and following treatment with tumor necrosis factor alpha. Similar results were seen with Ser(112)-pBad. These differences did not arise from differences in total Bad protein levels. These data correlate with the observation that the N-Ras knockout cells exhibit a heightened susceptibility to the induction of apoptosis. Ectopic expression of c-N-Ras in the N-Ras knockout cells at endogenous levels, compared with control cells, significantly rescues the apoptotically sensitive phenotype. Elevated expression of either c-Kirsten A-Ras or c-Kirsten B-Ras did not reverse the apoptotic sensitivity of the N-Ras knockout cells or result in increased levels of either phospho-Akt or phospho-Bad. Our results indicate that, at steady state, c-N-Ras possesses an isoform-specific, functional role in cell survival.

Asunto(s)

Apoptosis/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Transducción de Señal , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas p21(ras)/genética

RESUMEN

TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891-1897) that catalytically active TYK2 was necessary for IFN-beta to induce the beta-R1 gene. We now report IFN-beta-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-beta induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-beta-mediated phosphorylation of interferon-alpha receptor-1 (IFNAR-1) was defective in IFN-beta treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-beta treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-beta-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Interferón beta/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Receptores de Interferón/metabolismo , Transactivadores/metabolismo , Catálisis , Activación Enzimática , Humanos , Proteínas de la Membrana , Fosforilación , Receptor de Interferón alfa y beta , Factor de Transcripción STAT3 , Transducción de Señal , TYK2 Quinasa , Células Tumorales Cultivadas

RESUMEN

Amplification of several chromosomal regions have been observed in human breast carcinomas. One such region, 8p11, is amplified in 10-15% of tumor samples. Although the FGFR1 gene is located close to this region, and is often included within the amplicon, the observation that tumors exhibiting 8p11 amplification do not always overexpress FGFR1 suggests that another gene located close to FGFR1 is involved in the tumorigenic process. We now report the precise location of four expressed sequence tags (ESTs) within this region and the cloning of a novel gene, designated TACC1 (transforming acidic coiled coil gene 1), which encodes an 8 kb transcript and which is expressed at high levels during early embryogenesis. Constitutive expression of this gene under the control of the cytomegalovirus (CMV) promoter in mouse fibroblasts, results in cellular transformation and anchorage independent growth, suggesting that inappropriate expression can impart a proliferative advantage. This observation raises the possibility that amplification of TACC1 could promote malignant growth, thereby making TACC1 an attractive candidate for the gene promoting tumorigenicity as a result of the 8p11 amplification in human breast cancers.

Asunto(s)

Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 8/genética , Amplificación de Genes , Proteínas Asociadas a Microtúbulos , Proteínas Nucleares , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transformación Celular Neoplásica/química , Transformación Celular Neoplásica/patología , Clonación Molecular , Proteínas Fetales/biosíntesis , Proteínas Fetales/química , Proteínas Fetales/genética , Genes Relacionados con las Neoplasias , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Especificidad de Órganos/genética , Estructura Secundaria de Proteína

RESUMEN

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Comunicación Autocrina , Transformación Celular Neoplásica , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Ligandos , Ratones , Modelos Biológicos , Transducción de Señal , Transformación Genética

RESUMEN

Both insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) are key modulators of skeletal myoblast differentiation. The critical signaling pathways used by either IGF-1 or FGF-2 to inhibit differentiation have not been determined. In this study, we show that both IGF-1 and FGF-2 inhibit the differentiation of 23A2 myoblasts and that both stimulate signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK roughly 8-fold in 23A2 myoblasts. We used the selective chemical inhibitor of MEK, PD 098059, to determine if signaling by MEK is required by IGF-1 or FGF-2 to inhibit differentiation. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. Addition of PD 098059 to the culture medium 10 min before the addition of IGF-1 or FGF-2 completely blocked the signal from MEK to MAPK and restored the ability of the 23A2 myoblasts to differentiate in the presence of either IGF-1 or FGF-2. The peak of signaling through MEK to MAPK in response to either IGF-1 or FGF-2 occurred within the first hour with maximal activation observed after 10 min. This signal remained elevated (at roughly 70% above basal) for at least 48 h. PD 098059 was added to the culture 60 min after IGF-1 or FGF-2 to test whether this initial peak of signaling was sufficient for the inhibition of differentiation. The restoration of myogenic potential seen when cells were preincubated with PD 098059 was essentially identical to that seen when PD 098059 was added to cultures after the initial peak of signaling from MEK to MAPK, suggesting that persistent signaling through MEK is required for the inhibition of differentiation by either IGF-1 or FGF-2.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Músculo Esquelético/citología , Proteínas Quinasas/metabolismo , Línea Celular , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Inhibidores de Proteínas Quinasas , Transducción de Señal

RESUMEN

The Ras GTPases function as molecular switches, regulating a multiplicity of biological events. However the contribution, if any, of a specific c-Ras isoform (Ha-, N-, or Ki-ras A or B) in the regulation of a given biological or biochemical process, is unknown. Murine C3H1OT1/2 fibroblasts transformed with activated (G12V)Ha-ras or (Q61K)N-ras proliferate in serum-free media and have constitutive MAPK activity. The growth factor antagonist, suramin, inhibited the serum-independent proliferation of Ha-ras transformed fibroblasts, but not the serum-independent proliferation of N-ras transformed cells. The inhibition of cell proliferation was concomitant with the abrogation of the constitutive MAPK activity in the Ha-ras transformed fibroblasts. Analysis of the Ras-signalling complexes in immunoprecipitates from Ha-ras transformed cells revealed that Raf-1 co-immunoprecipitated with endogenous c-N-ras but not (G12V)Ha-ras. Pretreatment with suramin resulted in the loss of Raf-1 from c-N-ras immunoprecipitates. A c-N-ras antisense oligonucleotide, which down-regulated c-N-ras protein levels, abrogated the constitutive MAPK activity and serum-independent proliferation of (G12V)Ha-ras transformed cells. The data suggest that Raf-1 has a higher affinity for N-ras then Ha-ras in vivo, and c-N-ras function is required for the serum-independent proliferation of Ha-ras transformed cells.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Animales , Sangre , División Celular/efectos de los fármacos , Línea Celular Transformada , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Sueros Inmunes , Ratones , Ratones Endogámicos C3H , Oligonucleótidos Antisentido/farmacología , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/inmunología , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-raf/metabolismo , Suramina/farmacología

RESUMEN

Expression of oncogenic H-Ras in 23A2 myoblasts (A2:H-Ras cells) is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype. Because oncogenic Ras is known to induce the secretion of several different growth factors involved in maintaining the transformed phenotype of both fibroblast and epithelial cells, we explored the possibility that expression of oncogenic Ras in 23A2 myoblasts might lead to the secretion of a factor which inhibits differentiation. The differentiation of 23A2 myoblasts was inhibited (i) by coculture with an equal number of A2:H-Ras cells, (ii) by culture with an equal number of A2:H-Ras cells in the same tissue culture medium on an insert which allowed equilibration of molecules smaller than 1 micron, and (iii) by culture in media previously conditioned by A2:H-Ras cells. Similar results were obtained when 23A2 myoblasts expressing oncogenic N-Ras were substituted for A2:H-Ras cells in each assay. No inhibition of differentiation was observed, however, when differentiation-defective E1A-expressing 23A2 cells or C3H10T1/2 fibroblasts were substituted for A2:H-Ras cells. The differentiation inhibitor(s) in media conditioned by A2:H-Ras cells is heat stable, larger than 3 kD, and sensitive to the non-specific growth factor antagonist, suramin. Western analyses failed to detect either FGF-2 or TGFbeta (the known inhibitors of myoblast differentiation) in media conditioned by A2:H-Ras cells. Furthermore, while FGF-2 is a potent activator of MAP kinase and TGFbeta is a potent inhibitor of mink lung epithelial cell (CCL64) growth, conditioned media from A2:H-Ras cells does not activate MAP kinase and does not inhibit the growth of CCL64 cells. These results indicate that expression of oncogenic Ras induces the secretion of a novel inhibitor of skeletal myoblast differentiation. Furthermore, these results are the first to implicate an autocrine/paracrine mechanism in the inhibition of differentiation by oncogenic Ras.

Asunto(s)

Genes ras/fisiología , Músculo Esquelético/citología , Animales , Diferenciación Celular , Medios de Cultivo Condicionados , Factor 2 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Ratones , Ratones Endogámicos C3H , Suramina/farmacología , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/fisiología

RESUMEN

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.

Asunto(s)

Músculo Esquelético/fisiología , Transducción de Señal/fisiología , Transformación Genética , Proteínas ras/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Expresión Génica , Genes ras , Ratones , Ratones Endogámicos C3H , Músculo Esquelético/citología , Músculo Esquelético/enzimología , Fenotipo , Proteínas ras/biosíntesis

RESUMEN

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.

Asunto(s)

Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Proto-Oncogénicas p21(ras)/química , Secuencia de Aminoácidos , Animales , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , MAP Quinasa Quinasa 1 , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neurofibromina 1 , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Ratas , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

We demonstrated previously that v-Src activates a phospholipase D (PLD) activity (Song, J., Pfeffer, L.M., and Foster, D.A. (1991) Mol. Cell. Biol. 11, 4903-4908) and that this activation is dependent upon a G protein(s) (Jiang H., Alexandropoulos, K., Song, J., and Foster, D.A. (1994) Mol. Cell. Biol. 14, 3676-3682). An in vitro PLD assay was developed to study G protein involvement in v-Src-induced PLD activity. Maximal PLD activity in membranes isolated from v-Src-transformed cells was dependent upon both GTP and cytosol. In this report, we present three lines of evidence demonstrating that v-Src-induced PLD activity is mediated by Ras. First, a neutralizing Ras monoclonal antibody (Y13-259) inhibits PLD activity in membranes isolated from v-Src-transformed Balb/c 3T3 cells. Second, immobilized Ras protein depleted cytosol of the ability to stimulate PLD activity. This effect was dependent upon preloading immobilized Ras with GTP. Last, expression of a dominant negative Ras mutant in v-Src-transformed cells reduced PLD activity to the level observed in the nontransformed parental cells. These data establish a novel role for Ras in the regulation of PLD activity.

Asunto(s)

Proteína Oncogénica pp60(v-src)/metabolismo , Fosfolipasa D/metabolismo , Proteínas ras/metabolismo , Células 3T3 , Animales , Ácido Araquidónico/metabolismo , Membrana Celular/enzimología , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/farmacología , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Transfección , Proteínas ras/genética

RESUMEN

Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

Asunto(s)

Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Unión Competitiva , Ciclo Celular , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal

RESUMEN

We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.

Asunto(s)

Guanilil Imidodifosfato/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Técnicas In Vitro , MAP Quinasa Quinasa 1 , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-raf , Ratas , Proteínas Recombinantes/metabolismo

RESUMEN

The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Transformación Celular Neoplásica , Activación Enzimática , Isoenzimas/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Proteínas Oncogénicas v-raf , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Especificidad por Sustrato

RESUMEN

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Factores de Crecimiento Nervioso/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Sistema Libre de Células , Activación Enzimática , MAP Quinasa Quinasa 1 , Datos de Secuencia Molecular , Células PC12 , Fosforilación , Proteínas Proto-Oncogénicas c-raf , Ratas , Proteínas Recombinantes/metabolismo

RESUMEN

Cellular Ras proteins are essential elements in normal signal transduction pathways while activated Ras proteins are prevalent in many different forms of human cancers. Here, we discuss the mechanism through which Ras proteins, either cellular or activated, transmit a proliferative signal by activating cytoplasmic serine/threonine kinases.

Asunto(s)

División Celular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Citoplasma/enzimología , Genes ras , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal