Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 41
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Oncogene ; 20(50): 7334-41, 2001 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-11704863

RESUMEN

Ras mutations occur at high frequency in thyroid cancer. In vitro, the effects of Ras in thyroid cells are pleiotropic in that expression of activated Ras has been reported to stimulate proliferation and apoptosis. An understanding of the factors that contribute to the survival versus demise of Ras-transformed cells is essential to our understanding of the contribution of Ras to thyroid neoplasia and other cancers. Constitutive expression of oncogenic H-Ras sensitized Wistar rat thyroid (WRT) cells to apoptosis stimulated by multiple insults. When deprived of matrix attachment, Ras-transformed cells perished by apoptotic cell death at a high frequency. In contrast, parental cells were more resistant to suspension-induced cell death. Ras effects on anchorage-independent cell death were reproduced by a mutant protein that signals selectively to Raf-1, but not by mutant Ras that preferentially binds to RalGDS. Expression of a Ras mutant that selectively activates PI3K resulted in substantial protection from detachment-induced cell death. MAPK activity was increased in adherent Ras12V- and Ras12V35S-expressing cells, but abolished upon detachment. Interestingly, impaired MAPK activity was sufficient to stimulate apoptosis in adherent Ras-transformed cells, but not in parental cells. Treatment with a PI3K inhibitor also stimulated apoptosis selectively in Ras-transformed cells. These results demonstrate that constitutive expression of activated Ras elicits differential effects on the survival of thyroid cells. Moreover, Ras expression results in a greater dependence of thyroid cells on MAPK and PI3K activity for their survival.


Asunto(s)
Apoptosis/genética , Transformación Celular Neoplásica/genética , Genes ras , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Glándula Tiroides/citología , Sustitución de Aminoácidos , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/métodos , Línea Celular Transformada/citología , Medio de Cultivo Libre de Suero , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Humanos , Sistema de Señalización de MAP Quinasas , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Mutación Puntual , Proteínas Proto-Oncogénicas c-raf/fisiología , Ratas , Ratas Wistar , Suspensiones , Factor de Intercambio de Guanina Nucleótido ral/fisiología
2.
Mol Cell Biol ; 21(6): 1921-9, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11238928

RESUMEN

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ras-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyrotropin (TSH) on cell proliferation and differentiation, we examined the regulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Both cAMP and protein kinase A (PKA) contribute to the regulation of Rap1 activity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and PKA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner. Interference with PKA activity blocked phosphorylation but not the activation of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expression of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated Rap1 activity in some cells. The dual regulation of Rap1 by cAMP and PKA extends to downstream effectors. The ability of TSH to stimulate Akt phosphorylation was markedly enhanced by the expression of activated Rap1A and was repressed in cells expressing a putative dominant-negative Rap1A mutant. Although the expression of activated Rap1A was sufficient to stimulate wortmannin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylation in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutant that could be activated but not phosphorylated. These findings indicate that dual signals, Rap1 activation and phosphorylation, contribute to TSH-stimulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulated differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing activated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.


Asunto(s)
Diferenciación Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Genes ras , Mutación , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Wistar , Glándula Tiroides/efectos de los fármacos , Tirotropina/metabolismo , Tirotropina/farmacología , Proteínas de Unión al GTP rap1/genética
3.
Oncogene ; 19(32): 3609-15, 2000 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-10951566

RESUMEN

In addition to protein kinase A (PKA), cAMP regulates the activity of cAMP-gated channels and Rap1-specific guanine nucleotide exchange factors. We tested the hypothesis that the targets of cAMP might also include regulators of the Ras protooncogene. In rat thyroid cells, thyrotropin (TSH) stimulates proliferation through a cAMP-mediated pathway that requires Ras activity. Interference with Ras impairs DNA synthesis stimulated by TSH as well as cAMP elevating agents and analogs, demonstrating that the requirement for Ras lies down-stream of cAMP. Although cAMP stimulates proliferation, microinjection of the purified PKA catalytic subunit failed to do so, suggesting that factors in addition to PKA are required for cAMP-stimulated cell cycle progression. When added to thyroid cells expressing human Ha-Ras, TSH rapidly and markedly increased the proportion of GTP-bound Ras. Ras activity was increased within 1 min of TSH addition, maximal at 5-15 min, and declined to basal levels 30-60 min after hormone treatment. Cyclic AMP elevating agents elicited similar effects on Ras, indicating that TSH activates Ras through a cAMP-mediated pathway. Although cAMP-mediated, Ras activation by TSH and cAMP was independent of PKA activity. Moreover, cAMP-stimulated Ras activation was not impaired by tyrosine kinase inhibitors. These results indicate that cAMP activates targets in addition to PKA in thyroid cells, and that these targets may include regulators of Ras. The ability of cAMP elevating agents to activate Ras in addition to PKA may explain the inability of the PKA catalytic subunit to stimulate DNA synthesis in thyroid cells.


Asunto(s)
AMP Cíclico/metabolismo , Proteínas ras/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Línea Celular , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN/biosíntesis , Humanos , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Wistar , Glándula Tiroides/citología , Tirotropina/farmacología , Proteínas ras/genética
4.
J Cell Sci ; 113 ( Pt 9): 1535-42, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10751145

RESUMEN

Reduced gap junction activity has long been implicated in tumorigenesis. To elucidate the potential role of intercellular communication in melanoma development, we examined gap junctional capability of melanocytic cells from various stages of tumor progression in coculture models using dye transfer assays. Normal melanocytes coupled with keratinocytes by gap junctional formation, whereas melanoma cells did not. Instead, melanoma cells communicated among themselves and with fibroblasts. This switch in communication partners coincided with a shift from E-cadherin to N-cadherin expression during melanoma development. Forced expression of E-cadherin by adenoviral gene transfer in N-cadherin-expressing melanoma cells restored gap junctional compatibility with keratinocytes. Our data suggest that (1) melanocyte transformation is associated with loss of the pre-existing gap junctional activity with keratinocytes but a concomitant gain of communication with a newly juxtaposed cell type, the fibroblasts, (2) the specificity of gap junctional formation during melanoma development is determined by the cadherin profile on the melanocytic cells and (3) the overall gap junctional activity of melanocytic cells is not reduced with transformation.


Asunto(s)
Cadherinas/metabolismo , Comunicación Celular , Uniones Comunicantes , Melanoma/metabolismo , Melanoma/patología , Adenoviridae/genética , Cadherinas/genética , Células Cultivadas , Conexinas/metabolismo , Técnicas de Transferencia de Gen , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Células Tumorales Cultivadas
5.
Oncogene ; 19(7): 924-32, 2000 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-10702801

RESUMEN

Hormones are specialized mitogens that stimulate proliferation in their differentiated target cells. Thyrotropin (TSH), the physiologic regulator of thyroid cells, stimulates cAMP-mediated proliferation and thyroid-specific gene expression. The mitogenic effects of TSH require Ras, therefore Ras activation should be compatible with the maintenance of thyroid differentiation. However, expression of activated Ras extinguishes the differentiated phenotype of thyroid cells. One explanation for this apparent paradox is the selective utilization of Ras effector pathways. We tested the hypothesis that Ras signaling through PI3K mediates the mitogenic effects of TSH in cells which retain their differentiated character. Expression of a Ras effector mutant (RasV12S35) that signals preferentially through Raf-1, although sufficient to confer TSH-independent proliferation, abolished hormone-regulated expression of thyroglobulin and the sodium/iodide symporter. In contrast, expression of a Ras mutant (RasV12C40) that binds selectively to PI3K conferred TSH-independent proliferation without marked effects on thyroid-specific gene expression. Unlike the inhibitory effects of TSH on the proliferation of RasV12S35-expressing cells, TSH enhanced RasV12C40-stimulated proliferation by further increasing the activity of p70s6k, an important mediator of the mitogenic effects of TSH and RasV12C40. These results demonstrate that channeling Ras-dependent signals to PI3K confers TSH with the ability to stimulate proliferation in differentiated cells. Oncogene (2000) 19, 924 - 932.


Asunto(s)
Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal/fisiología , Tirotropina/fisiología , Proteínas ras/fisiología , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , ADN/biosíntesis , Mitógenos/metabolismo , Ratas , Ratas Wistar , Glándula Tiroides/citología , Glándula Tiroides/metabolismo
6.
Endocrinology ; 141(2): 606-14, 2000 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10650941

RESUMEN

TSH stimulates proliferation and maintains differentiated function in thyroid follicular cells. The mitogenic activity and the stimulatory effects of TSH on thyroid-specific gene expression are impaired by interferon-gamma (IFNgamma); however, the mechanisms for these effects have not been elucidated in detail. We examined the effects of IFNgamma on acute responses to TSH in rat thyroid cells. IFNgamma did not impair TSH-stimulated p70/p85 ribosomal protein S6 kinase (p70/p85s6k) activity or cAMP response element (CRE)-regulated gene expression, although it inhibited DNA synthesis and thyroglobulin expression, effects measured over a more prolonged time course than those on kinase activity and reporter gene expression. Unexpectedly, when cells were chronically exposed to IFNgamma, CRE-lacZ promoter activity was decreased, whereas other cAMP-mediated signals, such as p70/p85s6k activity and CRE-binding protein phosphorylation, were unaffected. Activating protein-1-regulated promoters were also impaired by IFNgamma treatment, but with kinetics that differed from those of CRE-regulated promoters. Neither acute nor chronic treatment with interleukin-1beta impaired cAMP signaling, indicating that the effects of IFNgamma are specific. These studies identify CRE- and activating protein-1-regulated promoters as targets of IFNgamma in thyroid cells and fibroblasts. IFNgamma-mediated inhibition of these promoters, in addition to those containing thyroid-specific transcription factor-1-binding sites, may contribute to the profound effects of IFNgamma on thyroid cells.


Asunto(s)
AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Genes MHC Clase I , Interferón gamma/farmacología , Glándula Tiroides/fisiología , Tirotropina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Interleucina-1/farmacología , Cinética , Ratas , Ratas Wistar , Proteínas Recombinantes , Proteínas Quinasas S6 Ribosómicas/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo
7.
Endocrinology ; 141(1): 146-52, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10614633

RESUMEN

Several reports have indicated that protein kinase C (PKC) is an important regulator of proliferation in thyroid cells. Unlike TSH, the mitogenic effects of phorbol esters are accompanied by de-differentiation. The role of individual PKC isoforms in thyroid cell proliferation and differentiation has not been examined. Recent studies have implicated the atypical PKCzeta, a phorbol ester-unresponsive isozyme, in cell proliferation, death, and survival. We overexpressed PKCzeta in Wistar rat thyroid (WRT) cells and determined that PKCzeta conferred TSH-independent DNA synthesis and cell proliferation. Cells overexpressing PKCzeta show higher levels of phosphorylated p42/p44 MAPK compared with vector-transfected cells. Experiments using a luciferase reporter for Elk-1 revealed that PKCzeta overexpressing cells exhibit higher basal Elk-1 transcriptional activity than vector-transfected control cells. Interestingly, stimulation of Elk-1 transcriptional activity by MEK1, a p42/p44 MAPK kinase, was significantly enhanced in cells overexpressing PKCzeta. Strikingly, TSH retained the ability to stimulate Tg expression in cells expressing PKCzeta. These results suggest that PKCzeta stimulates TSH-independent mitogenesis through a p42/p44 MAPK-dependent pathway. Unlike overexpression of Ras or phorbol ester treatment, PKC overexpression does not impair thyroglobulin (Tg) expression.


Asunto(s)
Proteínas de Unión al ADN , Proteína Quinasa C/fisiología , Glándula Tiroides/citología , Tirotropina/fisiología , Animales , Western Blotting , División Celular , Células Cultivadas , ADN/biosíntesis , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Vectores Genéticos/genética , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Wistar , Factores de Transcripción/genética , Proteína Elk-1 con Dominio ets
8.
Mol Cell Biol ; 19(9): 5882-91, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10454535

RESUMEN

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.


Asunto(s)
División Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal/fisiología , Células 3T3 , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Aminoácidos , Animales , División Celular/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos
9.
J Biol Chem ; 273(35): 22442-52, 1998 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-9712868

RESUMEN

Prostaglandin receptors may be activated by their cognate ligand or by free radical catalyzed isoprostanes, products of arachidonic acid peroxidation. For example, prostaglandin F2alpha (PGF2alpha) causes hypertrophy of neonatal rat ventricular myocytes, via the PGF2alpha receptor (FP). However, the FP may also be activated by the isoprostane, 8,12-iso-iPF2alpha-III (Kunapuli, P., Lawson, J. A., Rokach, J., and FitzGerald, G. A. (1997) J. Biol. Chem. 272, 27147-27154). Both ligands induce myocyte hypertrophy with overlapping potencies. Interestingly, the hypertrophic effects of these two agonists on cardiomyocytes are additive. Furthermore, the preference of these two agonists for activation of intracellular signal transduction pathways differs in several respects. Thus, PGF2alpha and 8,12-iso-iPF2alpha-III stimulate inositol phosphate formation with EC50 values of 50 +/- 12 nM and 3.5 +/- 0.6 microM, respectively. Moreover, PGF2alpha causes a robust activation ( approximately 50-fold) of Erk2, whereas 8,12-iso-iPF2alpha-III has no effect. Similarly, PGF2alpha causes translocation of cytosolic phospholipase A2 and also results in a 7-fold increment in the formation of 6-keto-PGF1alpha, whereas 8,12-iso-iPF2alpha-III exerts no effect on this pathway. On the other hand, both agonists are equally potent in activating JNK1 and c-Jun, whereas neither activates the p38 kinase. Both PGF2alpha and 8,12-iso-iPF2alpha-III activate the p70S6 kinase (p70(S6K)), but not Akt, downstream of phosphatidylinositol-3-kinase (PI3K). However, both wortmannin, a PI3K inhibitor, and rapamycin, an inhibitor of p70(S6K) activity, inhibit 8,12-iso-iPF2alpha-III -induced myocyte hypertrophy, with IC50 values of 60 +/- 12 and 3 +/- 1.7 nM, respectively, whereas neither compound abrogates the PGF2alpha-mediated response. Thus, both PGF2alpha and 8,12-iso-iPF2alpha-III induce myocyte hypertrophy via discrete signaling pathways. Although both agonists signal via the JNK pathway to initiate changes in c-Jun-dependent gene transcription, PGF2alpha preferentially activates the MEK-Erk2- cytosolic phospholipase A2 pathway. In contrast, the PI3K-p70(S6K) pathway appears to be essential for 8,12-iso-iPF2alpha-III-induced myocyte hypertrophy.


Asunto(s)
Cardiomegalia/inducido químicamente , Dinoprost/análogos & derivados , Dinoprost/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Activación Enzimática , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato
10.
Mol Cell Biol ; 18(7): 3718-26, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9632754

RESUMEN

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular , Línea Celular , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Mutagénesis Sitio-Dirigida , Fosforilación , Ratas , Ratas Wistar , Tiroglobulina/genética , Glándula Tiroides , Proteínas ras/genética
11.
Endocrinology ; 139(4): 1991-8, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9528986

RESUMEN

cAMP exerts differential effects on mitogenic signaling pathways. In many cells, cAMP inhibits growth factor-stimulated MAPK activity and proliferation. In others, cAMP promotes growth. TSH stimulates proliferation through elevations in cAMP in thyroid follicular cells. This mitogenic pathway is dependent upon both protein kinase A and Ras, but not upon Raf-1, mitogen-activated protein kinase kinase, or mitogen-activated protein kinase. We report that TSH, acting through cAMP, activates pp70s6k and that this activity is required for TSH-stimulated DNA synthesis. A similar role for pp70s6k in cAMP-mediated mitogenesis was observed in secondary rat Schwann cells and in Swiss3T3 fibroblasts, two additional cell types that respond to cAMP with growth. In contrast, cAMP elevation did not activate pp70s6k in NIH3T3 or REF52 fibroblasts, cells in which cAMP fails to stimulate proliferation. Together, these results suggest that pp70s6k plays an important and general role in cAMP-mediated proliferation.


Asunto(s)
AMP Cíclico/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Tirotropina/farmacología , Células 3T3/citología , Células 3T3/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Línea Celular , ADN/biosíntesis , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ratones , Polienos/farmacología , Ratas , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Células de Schwann/citología , Células de Schwann/efectos de los fármacos , Sirolimus , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos
12.
J Biol Chem ; 272(9): 5600-5, 1997 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-9038168

RESUMEN

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.


Asunto(s)
AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Genes ras , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Replicación del ADN/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Microinyecciones , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal , Tirotropina/farmacología , Factor de Intercambio de Guanina Nucleótido ral , Proteínas de Unión al GTP rap
13.
Oncogene ; 13(6): 1305-14, 1996 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-8808705

RESUMEN

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Transducción de Señal/fisiología , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Células 3T3/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Secuencia Conservada , ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Epítopos/metabolismo , Sustancias de Crecimiento/farmacología , Ratones , Microinyecciones , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal/efectos de los fármacos , Proteínas ras/metabolismo , Proteínas ras/fisiología
14.
J Virol ; 70(4): 2350-9, 1996 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-8642662

RESUMEN

The human immunodeficiency virus type 1 Rev trans activator binds directly to unspliced viral mRNA in the nucleus and activates its transport to the cytoplasm. In additon to the sequences that confer RNA binding and nuclear localization, Rev has a carboxy-terminal region, the activation domain, whose integrity is essential for biological activity. Because it has been established that Rev constitutively exits and reenters the nucleus and that the activation domain is required for nuclear exit, it has been proposed that Rev's activation domain is a nuclear export signal (NES). Here, we used microinjection-based assays to demonstrate that the activation domain of human immunodeficiency virus type 1 Rev imparts rapid nuclear export after its transfer to heterologous substrates. NES- mediated export is specific, as it is sensitive both to inactivation by missense mutation and to selective inhibition by an excess of the wild-type, but not mutant, activation domain peptide. Examination of the Rev trans activators of two nonprimate lentiviruses, visna virus and equine infectious anemia virus, revealed that their activation domains are also potent NESs. Taken together, these data demonstrate that nuclear export can be determined by positively acting peptide motifs, namely, NESs, and suggest that Rev proteins activate viral RNA transport by providing export ribonucleoproteins with specific information that targets them to the cytoplasm.


Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/metabolismo , Virus de la Anemia Infecciosa Equina/metabolismo , Señales de Clasificación de Proteína/metabolismo , Virus Visna-Maedi/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Núcleo Celular/metabolismo , Núcleo Celular/virología , Productos del Gen rev/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Temperatura , Productos del Gen rev del Virus de la Inmunodeficiencia Humana
15.
Proc Natl Acad Sci U S A ; 93(7): 2936-40, 1996 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-8610146

RESUMEN

The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Productos del Gen rev/química , Productos del Gen rev/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Bufonidae , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Femenino , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Oocitos/fisiología , ARN Mensajero/metabolismo , ARN Ribosómico 5S/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factor de Transcripción TFIIIA , Factores de Transcripción/biosíntesis , Transcripción Genética , Xenopus laevis , Productos del Gen rev del Virus de la Inmunodeficiencia Humana
16.
Endocrinology ; 137(1): 96-104, 1996 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-8536648

RESUMEN

We previously reported that microinjection of purified Ras protein stimulated DNA synthesis in quiescent Wistar rat thyrocytes and that TSH (TSH)-stimulated DNA synthesis was Ras-dependent. In contrast to these results, microinjection of cellular or oncogenic Ras significantly reduced TSH-stimulated thyroglobulin (Tg) expression, a marker of thyrocyte differentiation. Microinjection of a dominant inhibitory Ras mutant had no effect on TSH-stimulated Tg expression. As the Tg promoter is cAMP-responsive and Ras was previously reported to interfere with entry of catalytic (C) subunit of the cAMP-dependent protein kinase into the nucleus, experiments were performed to assess the effects of Ras on cAMP-mediated signaling. Microinjection of either cellular or oncogenic Ras had no effect on TSH-stimulated entry of C subunit into the nucleus. Consistent with these data, Ras did not reduce TSH-stimulated cAMP response element binding protein phosphorylation, or cAMP response element-regulated gene expression. These results demonstrate that Ras exerts differential effects on TSH signaling; Ras increases TSH-stimulated DNA synthesis and decreases TSH-induced Tg expression. Moreover, the mechanism through which Ras induces Tg expression lies distal to entry of C subunit into the nucleus, cAMP response element binding protein phosphorylation, and cAMP response element-regulated gene expression.


Asunto(s)
AMP Cíclico/fisiología , Transducción de Señal/efectos de los fármacos , Tiroglobulina/antagonistas & inhibidores , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Proteínas ras/farmacología , Animales , Catálisis , Bovinos , Línea Celular , Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Microinyecciones , Ratas , Ratas Wistar , Tiroglobulina/metabolismo , Glándula Tiroides/citología , Tirotropina/farmacología , Factores de Tiempo
17.
Cell ; 82(3): 463-73, 1995 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-7634336

RESUMEN

Active nuclear import of protein is controlled by nuclear localization signals (NLSs), but nuclear export is not understood well. Nuclear trafficking of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is critical for regulation of gene expression. The heat-stable inhibitor (PKl) of cAPK contains a nuclear export signal (NES) that triggers rapid, active net extrusion of the C-PKl complex from the nucleus. This NES (residues 35-49), fused or conjugated to heterologous proteins, was sufficient for rapid nuclear export. Hydrophobic residues were critical. The NES is a slightly weaker signal than the SV40 NLS. A sequence containing only residues 37-46, LALKLAGLDI, is also sufficient for nuclear export. This is an example of a protein-based NES having no obvious association with RNA. A similar sequence, LQLPPLERLTL, from Rev, an RNA-binding protein of HIV-1, also is an NES.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores de Proteínas Quinasas , Secuencia de Aminoácidos , Animales , Transporte Biológico , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
18.
Biochemistry ; 34(25): 7996-8004, 1995 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-7540866

RESUMEN

We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC50 = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat 1 fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C-terminal truncated receptors (delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in delta CT cells, whereas stimulation of MAP kinase activity was almost absent.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes Inmediatos-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces , Insulina/farmacología , Transducción de Señal , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibroblastos/metabolismo , Genes fos , Guanosina Trifosfato/metabolismo , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero , ADN Polimerasa Dirigida por ARN , Ratas , Factores de Transcripción/genética , Activación Transcripcional
19.
FASEB J ; 9(7): 552-8, 1995 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7737465

RESUMEN

Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.


Asunto(s)
GMP Cíclico/fisiología , Óxido Nítrico/fisiología , Regiones Promotoras Genéticas/fisiología , Factor de Transcripción AP-1/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ratas Wistar , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Activación Transcripcional , Transfección
20.
J Biol Chem ; 270(5): 2041-6, 1995 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-7836431

RESUMEN

The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. We report the isolation of a polyclonal antibody raised to purified recombinant PKI alpha. Using this antibody, the intracellular distribution of endogenous PKI alpha was assessed by immunostaining. The PKI alpha expression and intracellular distribution varied as a function of cell cycle progression. PKI alpha expression appeared low in serum-starved cells and in cells in G1 and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G2/M. Nuclear levels of PKI alpha remained high through cell division and decreased again as cells reentered G1. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G2/M phases of the cell cycle. Consistent with this, microinjection of PKI alpha antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G1/S boundary prevented their subsequent division. Together these results support the idea that PKI alpha plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis.


Asunto(s)
Proteínas Portadoras/farmacología , Ciclo Celular , Péptidos y Proteínas de Señalización Intracelular , Animales , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Ratones , Mitosis , Proteínas Recombinantes
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA