RESUMEN
The serine/threonine kinase Rho-kinase was recently identified as a downstream effector of the small GTPase Rho, mediating effects of Rho on the actin cytoskeleton. Also phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) has been implicated in the regulation of actin polymerization. As the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, we investigated whether Rho-kinase is involved in the control of PI(4,5)P(2) levels. Overexpression of RhoA in HEK-293 cells increased phosphatidylinositol 4-phosphate (PI4P) 5-kinase activity and concomitantly enhanced cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreased both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA could be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase had no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulated PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA was reversed by the Rho-kinase inhibitor HA-1077. Our data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/genéticaRESUMEN
In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Células 3T3 , Citoesqueleto de Actina/química , Citoesqueleto de Actina/enzimología , Actinas/análisis , Animales , Transporte Biológico , Endosomas/química , Endosomas/enzimología , Activación Enzimática , Receptores ErbB/genética , Ratones , Mutación , Proteínas Proto-Oncogénicas pp60(c-src)/análisis , Transducción de Señal/fisiologíaRESUMEN
Epidermal growth factor (EGF) and platelet-derived growth (PDGF) are suggested to be involved in the proliferation of human gliomas. We examined the effects of these growth factors on two human malignant glioma cell lines. Treatment of the A172 glioblastoma and the Hs683 glioma cell line with EGF and PDGF resulted in the tyrosine autophosphorylation, and hence activation, of the respective growth factor receptors. In addition, both cell lines responded to EGF and PDGF with increased deoxyribonucleic acid (DNA) synthesis. Because the intrinsic protein tyrosine kinase activity of this class of growth factor receptors is indispensable for their functioning, we tested the effects of specific protein tyrosine kinase inhibitors on growth factor-induced DNA synthesis and glioma cell proliferation. Genistein inhibited both EGF- and PDGF-stimulated autophosphorylation of the receptors and induction of DNA synthesis. However, genistein seemed to be cytotoxic to the cells. The tyrphostins RG 50875 and RG 13022 dose-dependently inhibited DNA synthesis induced by EGF, PDGF, and serum. RG 13022 completely blocked the EGF- and PDGF-induced DNA synthesis at a concentration of 50 mumol/L. The tyrphostins showed no selectivity in blocking either EGF or PDGF signaling. With concentrations up to mumol/L, no cytotoxic side effects of the tyrphostins were observed. Both tyrphostins also inhibit serum-driven cell growth in a dose-dependent manner. These results support the hypothesis that activated protein tyrosine kinase receptors are involved in the proliferation of A172 and Hs683 glioma cells. Selective inhibitors of protein tyrosine kinases, therefore, might have the potential to contribute to the treatment of growth factor-dependent gliomas.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Glioblastoma/patología , Glioma/patología , Isoflavonas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Genisteína , Humanos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Tirosina Quinasas/fisiología , Células Tumorales Cultivadas/patologíaRESUMEN
To study the relationship between the tyrosine kinase c-Src and the epidermal growth factor receptor (EGF-R), we used the breast cancer cell line ZR75-1, which was transfected with the EGF-R. The EGF-R transfected cell line expressed 60 times more EGF-R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF-R over-expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c-Src. However, the cell line over-expressing the EGF-R showed a twofold enhancement of c-Src kinase activity after EGF stimulation. The activation of c-Src kinase by EGF was confirmed in other EGF-R expressing cell types.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Neoplasias de la Mama , Línea Celular , Membrana Celular/enzimología , Citosol/enzimología , Receptores ErbB/biosíntesis , Humanos , Cinética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Three cell lines established from human gliomas were found to differ in the capacity to phosphorylate the glycolytic enzyme pyruvate kinase in vitro. Phosphorylation in the glioblastoma cell line U-138 was more pronounced than in the glioma cell line Hs 683 and in the glioblastoma cell line A-172. All 3 cell lines showed similar pyruvate kinase isozyme patterns and expressed about 90% K-type and 10% M-type subunits. So, differences in pyruvate kinase phosphorylation could not be explained by differences in the availability of the appropriate substrate, being pyruvate kinase type K. As in gliomas, phosphorylation could specifically and almost completely be inhibited by fructose-1,6-bisphosphate. In order to investigate a potential physiological significance of the phosphorylation of pyruvate kinase, we have characterized these cell lines for several glycolytic parameters. In U-138 cells, the production of lactate appeared to be 2 times higher as compared with A-172 and Hs 683 cells under normal growth conditions and even 4 times higher under low glucose culture regime. The efflux of lactate correlated with the pyruvate kinase phosphorylation pattern in the cell lines. In none of the cell lines could the lactate production be stimulated by glutamine as additional energy source under low glucose culture conditions. The higher glycolytic flux in U-138 cells was not accompanied by higher glycolytic enzyme activities. The isozyme patterns of hexokinase, pyruvate kinase, aldolase, enolase and lactate dehydrogenase in the cell lines were nearly identical and resembled the patterns previously described for solid gliomas. However, the isozyme composition of phosphofructokinase in the cell lines differed from the situation in gliomas. While in gliomas the expression of L-type phosphofructokinase is favored, in the glioma cell lines, we found an increase in the expression of C-type subunits.
Asunto(s)
Glioma/metabolismo , Glucólisis , Piruvato Quinasa/metabolismo , Adenosina Trifosfato/análisis , Humanos , Isoenzimas/análisis , L-Lactato Deshidrogenasa/análisis , Lactatos/metabolismo , Ácido Láctico , Fosforilación , Piruvato Quinasa/análisis , Células Tumorales CultivadasRESUMEN
It has been suggested that Ag-specific T cell factors play a role in the early phase of cellular immune responses. Two of these factors are studied in this paper. The first factor is specific macrophage arming factor (SMAF), that binds to (arms) macrophages and renders them specifically cytotoxic against tumor cells. The second factor is involved in the induction of an early (2 h) mast cell-dependent hypersensitivity reaction, that precedes the delayed-type hypersensitivity response (mast cell arming T cell factor; MTCF). In this study we compare both factors in an allogeneic murine tumor system (C57BL (H-2b) mice sensitized against SL2 (H-2d) lymphoma cells), both factors were: 1) dependent on T lymphocytes for their production, 2) detectable in serum 2 to 3 days after immunization, and 3) MHC (H-2)-Ag specific. Immunochemical studies showed that both factors have a molecular mass between 45 and 90 kDa and bind to the mAb 14-30 (directed against specific T cell factors), but not to anti-kappa/lambda L chain antibodies. Furthermore, it was shown that SMAF produced in vitro could induce a mast cell-dependent early 2-h hypersensitivity reaction against SL2 tumor cells, and resembled in this way MTCF. We concluded that the biologic activities and immunochemical characteristics of SMAF and MTCF are similar. Both factors are produced during the early stages of the immune response and seem to play a role in the initiation of the cell-mediated immune response.
Asunto(s)
Linfocinas/fisiología , Mastocitos/inmunología , Linfocitos T/trasplante , Animales , Citotoxicidad Inmunológica , Edema/inmunología , Hipersensibilidad Tardía/etiología , Hipersensibilidad Tardía/inmunología , Inmunidad Celular , Inmunización Pasiva , Linfocinas/biosíntesis , Linfocinas/sangre , Activación de Macrófagos , Factores Activadores de Macrófagos , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2-type holoenzyme of both rat and human origin, and did not cross-react with the M1-type isozyme. This was established by immunoblot analysis, both under dissociating and non-dissociating conditions.