RESUMEN
Integrins, matrix metalloproteases (MMPs), and the cytokine TGF-beta have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-beta exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-beta. Because the latent domain of TGF-beta1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-beta (SLC) to the cell surface where TGF-beta activation could be locally controlled. Here, we show that SLC binds to alpha(v)beta8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP-dependent release of active TGF-beta, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell-matrix interactions.
Asunto(s)
Integrinas/metabolismo , Metaloendopeptidasas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , División Celular/fisiología , Línea Celular , Homeostasis , Humanos , Neoplasias Pulmonares , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismo , Factor 2 Asociado a Receptor de TNF , Transfección , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
We used a spheroid model of colon carcinoma to analyze integrin dynamics as a function of the epithelial-mesenchymal transition (EMT), a process that provides a paradigm for understanding how carcinoma cells acquire a more aggressive phenotype. This EMT involves transcriptional activation of the beta6 integrin subunit and a consequent induction of alphavbeta6 expression. This integrin enhances the tumorigenic properties of colon carcinoma, including activation of autocrine TGF-beta and migration on interstitial fibronectin. Importantly, this study validates the clinical relevance of the EMT. Kaplan-Meier analysis of beta6 expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high beta6 expression. Elevated receptor expression did not simply reflect increasing tumor stage, since log-rank analysis showed a more significant impact on the survival of patients with early-stage, as opposed to late-stage, disease. Cox regression analysis confirmed that this integrin is an independent variable for these tumors. These findings define the alphavbeta6 integrin as an important risk factor for early-stage disease and a novel therapeutic candidate for colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Carcinoma/metabolismo , Carcinoma/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Integrina alfa5/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Animales , Comunicación Autocrina , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mucosa Intestinal/patología , Mesodermo/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica , Pronóstico , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity. METHODS AND RESULTS: Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation. CONCLUSIONS: These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.
Asunto(s)
Endotelio Vascular/metabolismo , Hemorreología , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Endotelio Vascular/citología , Humanos , Inflamación , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa Quinasa 3 , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Cordón Umbilical/citologíaRESUMEN
OBJECTIVE: A new member of the MAP kinase family, big MAP kinase-1 (BMK1), has been recently identified to promote cell growth and attenuate apoptosis. P90 ribosomal S6 kinase (p90RSK), one of the potentially important substrates of extracellular signal regulated kinase (ERK), regulates gene expression in part via phosphorylation of CREB and the Na(+)/H(+) exchanger. Recently, we have demonstrated that the activity of BMK1, Src (the upstream regulator of BMK1) and p90RSK was increased in hypertrophied myocardium induced by pressure-overload in the guinea pig. However, the abundance and activity of these kinases in human hearts are unknown. METHODS: In addition to the three classical MAP kinases (ERK, p38 kinase, and c-Jun NH(2)-terminal kinase (JNK)), we examined the protein expression and activity of Src, BMK1, and p90RSK in explanted hearts from patients with dilated cardiomyopathy (n=9). Normal donor hearts, which were not suitable for transplant for technical reasons, were used as controls (n=5). RESULTS: There were no significant differences in the levels of protein expression of these kinases between normal and failing hearts. ERK1/2 and p90RSK were activated in heart failure compared to control (P<0.01 and P<0.03, respectively), while the activity of p38 kinase was decreased (P<0.05) and the activity of JNK was unchanged in heart failure. By contrast, the activities of Src and BMK1 were significantly reduced in end-stage heart failure compared to normal donor hearts (P<0.05). CONCLUSION: These data suggest that multiple MAP kinases, p90RSK, and Src are differentially regulated in human failing myocardium of patients with idiopathic dilated cardiomyopathy and may be involved in the pathogenesis of this complex disease.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Adulto , Cardiomiopatía Dilatada/complicaciones , Estudios de Casos y Controles , Activación Enzimática , Insuficiencia Cardíaca/etiología , Humanos , MAP Quinasa Quinasa 4 , Masculino , Persona de Mediana Edad , Proteína Quinasa 7 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/análisis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteínas Quinasas S6 Ribosómicas/análisis , Proteínas Quinasas p38 Activadas por Mitógenos , Familia-src Quinasas/análisis , Familia-src Quinasas/metabolismoRESUMEN
Activated Src kinase may contribute to the progression and spread of breast cancers and recent in vitro evidence suggests a role for Src in acquired endocrine resistance. The purpose of this study was to investigate whether modulation of Src activity in endocrine-sensitive and endocrine-resistant breast cancer cells directly affected their phenotype and sensitivity to 4-hydroxy Tamoxifen (tamoxifen) and to determine whether Src activity in breast cancer tissue affected patient outcome. Expression of constitutively active Src in ER-positive, endocrine-sensitive MCF7 breast cancer cells resulted in the development of an aggressive phenotype, akin to that previously observed in cell models of Tamoxifen resistance, and, significantly, attenuated their response to tamoxifen. Conversely, expression of dominant negative-Src in tamoxifen-resistant MCF7 cells resensitized them to tamoxifen. An exploratory immunohistochemical study of an archival primary breast tumor series (n = 75) with parallel clinicopathological data and in normal breast tissues (n = 19) revealed higher levels of activated Src in the cytoplasm (p < 0.01) and lower levels of nuclear Src (p < 0.01) in tumor tissue compared with normal tissue. Whereas elevated levels of activated-Src in the cytoplasm of tumors was significantly associated with reduced survival in ER+ patients (p = 0.031), elevated levels of activated Src within the nucleus appeared to associate with an improved hormonal response. Together these data are further suggestive of a role for Src in breast cancer where it may alter response to endocrine therapy.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Tamoxifeno/farmacología , Familia-src Quinasas/metabolismo , Adulto , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Persona de Mediana Edad , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/patología , Pronóstico , Receptores de Estrógenos/biosíntesis , Tasa de Supervivencia , Tamoxifeno/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Human cytomegalovirus (CMV) infection is a major cause of morbidity in immunosuppressed individuals, and congenital CMV infection is a leading cause of birth defects in newborns. Infection with pathogenic viral strains alters cell-cell and cell-matrix interactions, affecting extracellular matrix remodeling and endothelial cell migration. The multifunctional cytokine transforming growth factor (TGF)-beta1 regulates cell proliferation, differentiation, and extracellular matrix remodeling. Secreted as a latent protein complex, TGF-beta1 requires activation before binding to receptors that phosphorylate intracellular effectors. TGF-beta1 is activated by integrin alphavbeta6, which is strongly induced in the epithelium by injury and inflammation but has not previously been found in endothelial cells. Here, we report that CMV infection induces integrin alphavbeta6 expression in endothelial cells, leading to activation of TGF-beta1, signaling through its receptor ALK5, and phosphorylation of its intracellular effector Smad3. Infection of endothelial cells was also found to stimulate collagen synthesis through a mechanism dependent on both TGF-beta1 and integrin alphavbeta6. Immunohistochemical analysis showed integrin alphavbeta6 up-regulation in capillaries proximal to foci of CMV infection in lungs, salivary glands, uterine decidua, and injured chorionic villi of the placenta, demonstrating both its induction in endothelium and up-regulation in epithelium in vivo. Our results suggest that activation of TGF-beta1 by integrin alphavbeta6 contributes to pathological changes and may impair endothelial cell functions in tissues that are chronically infected with CMV.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Colágeno/biosíntesis , Infecciones por Citomegalovirus/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células Epiteliales/metabolismo , Integrinas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animales , Células Cultivadas , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/virología , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Humanos , Cadenas beta de Integrinas/metabolismo , Visón , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína smad3/metabolismo , Venas Umbilicales/patología , Venas Umbilicales/virología , Regulación hacia Arriba , Replicación ViralRESUMEN
Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1beta (IL-1beta) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1beta has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1beta would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1beta on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1beta significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1beta decreased basal and dexamethasone-induced epithelial sodium channel alpha-subunit (alpha ENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1beta on alpha ENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of alpha v beta6 integrin and transforming growth factor-beta. These results indicate that IL-1beta may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in alpha ENaC expression through p38 MAPK-dependent inhibition of alpha ENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-1/fisiología , Alveolos Pulmonares/metabolismo , Canales de Sodio/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Transporte Biológico , Biotinilación , Western Blotting , Membrana Celular/metabolismo , Supervivencia Celular , Células Cultivadas , Cartilla de ADN/química , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Edema/metabolismo , Electrofisiología , Canales Epiteliales de Sodio , Epitelio/metabolismo , Humanos , Integrinas/metabolismo , Interleucina-1/metabolismo , Luciferasas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sodio/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Agua/metabolismoRESUMEN
In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.
Asunto(s)
Membrana Celular/metabolismo , Luz , Retina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Familia-src Quinasas/metabolismo , Animales , Arrestina/metabolismo , Fraccionamiento Celular , Membrana Celular/química , Citoesqueleto/metabolismo , Immunoblotting , Modelos Biológicos , Fosforilación , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Retina/citología , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/química , Segmento Externo de la Célula en Bastón/citologíaRESUMEN
In order to investigate whether c-Src is involved in carcinogenesis and progression of breast carcinoma, we examined the expression of activated c-Src in tissue sections from surgically resected human breast specimens. First, we confirmed the specificity of the antibody against activated c-Src (Clone 28) using six cell lines established from human breast carcinomas by western blotting. As expected, activated c-Src was detected as a 60 kDa band in all cell lines tested. Immunofluorescence analysis demonstrated that the activated c-Src was mainly observed in cytoplasms of these cells. Then, we designed an immunohistochemical study with 73 human breast carcinoma tissues. Glandular epithelial and myoepithelial cells in normal mammary glands adjacent to carcinoma nests and infiltrating stromal cells were negative for activated c-Src. In contrast, 37 of the 73 breast carcinoma tested (50.7%) were positive for activated c-Src, and this positive staining was inversely correlated with Ki-67 labeling index (p < 0.0001), TNM stage (p < 0.0001), tumor size (p < 0.0001), an d histological grade (p = 0.0002). These results strongly suggest that the activation of c-Src would be related to the progression of breast carcinomas with low aggressiveness.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales , Western Blotting , Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Transformación Celular Neoplásica/metabolismo , Activación Enzimática , Células Epiteliales/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Invasividad NeoplásicaRESUMEN
Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.
Asunto(s)
Líquidos Corporales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Alveolos Pulmonares/metabolismo , Canales de Sodio/genética , Sodio/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Activación Enzimática , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Luciferasas/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta/genética , Canales de Sodio/análisis , Transfección , Factor de Crecimiento Transformador beta1RESUMEN
We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse alpha(v)beta(6) integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of alpha(v)beta(6) to the transforming growth factor-beta1 latency-associated peptide with IC(50) values as low as 18 pm, and prevented the subsequent alpha(v)beta(6)-mediated activation of transforming growth factor-beta1. The antibodies also inhibited alpha(v)beta(6) binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by alpha(v)beta(6)-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for alpha(v)beta(6) and showed no cross-reactivity with the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(8),or alpha(IIb)beta(3). The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to alpha(v)beta(6), suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for alpha(v)beta(6) and resemble those described for alpha(IIb)beta(3). We also report for the first time the relative abilities of divalent cations to promote alpha(v)beta(6) binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of alpha(v)beta(6) as well as the role of alpha(v)beta(6) in vivo.
Asunto(s)
Antígenos de Neoplasias/química , Integrinas/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/inmunología , Unión Competitiva , Cationes , Adhesión Celular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Epítopos/química , Fibronectinas/química , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Inmunoensayo , Concentración 50 Inhibidora , Integrinas/inmunología , Cinética , Ligandos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Células 3T3 NIH , Oligopéptidos/química , Péptidos/química , Agregación Plaquetaria , Unión Proteica , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca(2+) channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca(2+) channel blocker, and chelation of extracellular Ca(2+). These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.