RESUMO
Ephs and FGFRs belong to a superfamily of receptor tyrosine kinases, playing important roles in stem cell biology. We previously reported that EphA4 and FGFR form a heterodimer following stimulation with ligands, trans-activating each other and signaling through a docking protein, FRS2α, that binds to both receptors. Here, we investigated whether the interaction between EphA4 and FGFRs can be generalized to other Ephs and FGFRs, and, in addition, examined the downstream signal mediating their function in embryonic neural stem/progenitor cells. We revealed that various Ephs and FGFRs interact with each other through similar molecular domains. When neural stem/progenitor cells were stimulated with FGF2 and ephrin-A1, the signal transduced from the EphA4/FGFR/FRS2α complex enhanced self-renewal, while stimulation with ephrin-A1 alone induced neuronal differentiation. The downstream signal required for neuronal differentiation appears to be MAP kinase mainly linked to the Ras family of G proteins. MAP kinase activation was delayed and sustained, distinct from the transient activation induced by FGF2. Interestingly, this effect on neuronal differentiation required the presence of FGFRs. Specific FGFR inhibitor almost completely abolished the function of ephrin-A1 stimulation. These findings suggest that the ternary complex of EphA, FGFR and FRS2α formed by ligand stimulation regulates self-renewal and differentiation of mouse embryonic neural stem/progenitor cells by ligand-specific fine tuning of the downstream signal via FRS2α.
Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Receptor EphA1/metabolismo , Receptor EphA4/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Embrião de Mamíferos/citologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células-Tronco Neurais/citologia , Receptor EphA1/genética , Receptor EphA4/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
EphA4 belongs to a superfamily of receptor tyrosine kinases and interacts with several molecules including fibroblast growth factor receptors (FGFRs) as we reported earlier. Several receptor tyrosine kinases, FGFRs, Trks, Alk and Ret, are currently known to transduce a signal through a docking protein, fibroblast growth factor receptor substrate 2α (FRS2α). However, nothing has been reported about the interaction of FRS2α with EphA4. Using the yeast two-hybrid system and the in vitro binding and kinase assays, we found that the mid-kinase region of EphA4 directly interacts with the FRS2α PTB domain upon tyrosine phosphorylation of the EphA4 juxtamembrane (JM) domain and EphA4 directly phosphorylates FRS2α. We also found that the FRS2α PTB domain and the amino-terminal region of EphA4 bind to the amino- and carboxy-terminal regions of the FGFR JM domain, respectively, suggesting that FRS2α and EphA4 interact with FGFR simultaneously. Furthermore, a kinase-dead EphA4 mutant that constitutively binds to FGFR functions as a dominant-negative molecule for signaling through both EphA4 and FGFR, and so does the truncated FRS2α lacking multiple tyrosine phosphorylation sites. These dominant-negative mutants similarly inhibit the ligand-dependent proliferation of the mouse embryonic neural stem/progenitor cells. These results suggest the formation of a ternary complex comprising EphA4, FGFR and FRS2α. The signaling complex appears to integrate the input from FGFR and EphA4, and release the output signal through FRS2α.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Neurais/metabolismo , Receptor EphA4/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Complexo Ternário/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Células-Tronco Neurais/citologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Transdução de SinaisRESUMO
BACKGROUND: DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied. MATERIALS AND METHODS: A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene. RESULTS: Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not. CONCLUSION: Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Receptores ErbB/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação/genética , Transdução de Sinais , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Rim/citologia , Rim/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
The aim of this study was to identify glycobiological biomarkers that indicate sensitivity to trastuzumab, a humanized monoclonal antibody against HER2 in plasma samples from breast cancer patients. Plasma samples were obtained from 24 breast cancer patients treated with trastuzumab monotherapy. The catalytic activities of plasma alpha1-6, fucosyltransferase (FUT8) and alpha-L fucosidase (FUCA) were analyzed using high-performance liquid chromatography (HPLC) and spectrophotometer, respectively. The plasma N-glycan profiles were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Plasma FUT8 activity was not significantly correlated with either the clinical response or progression-free survival (PFS). On the other hand, plasma FUCA activity was significantly correlated with PFS (p < 0.05). The MALDI-TOF-MS analysis of the plasma N-glycan profile revealed that the expression of 2534 m/z N-glycan was lower in patients with progressive disease (PD) and was correlated with PFS. Low expression of 2534 m/z N-glycan discriminated between PD and non-PD with 75% sensitivity and 82% specificity. We demonstrated that the plasma FUCA activity and 2534 m/z N-glycan may be predictive biomarkers of sensitivity to trastuzumab. Our results suggest that glycosylation analysis may provide useful information for determining clinical cancer therapy and provide novel insight into biomarker studies using glycobiological tools in the field of breast cancer.
Assuntos
Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Biomarcadores/química , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , alfa-L-Fucosidase/sangue , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Biomarcadores/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Intervalo Livre de Doença , Feminino , Fucosiltransferases/metabolismo , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estudos Prospectivos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Trastuzumab , Resultado do Tratamento , alfa-L-Fucosidase/químicaRESUMO
Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR.
Assuntos
Inibidores Enzimáticos/farmacologia , Receptores ErbB/química , Receptores ErbB/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Western Blotting , Linhagem Celular , Receptores ErbB/metabolismo , Humanos , Imunoprecipitação , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas , Transfecção , Tirosina/química , Tirfostinas/farmacologiaRESUMO
SRPX2 (Sushi repeat containing protein, X-linked 2) was first identified as a downstream molecule of the E2A-HLF fusion gene in t(17;19)-positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real-time RT-PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned-medium obtained from SRPX2-overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU-16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU-16 and HSC-39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Neoplasias Gástricas/patologia , Adulto , Idoso , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citoplasma/metabolismo , Feminino , Quinase 1 de Adesão Focal/metabolismo , Humanos , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Proteínas de Neoplasias , Proteínas do Tecido Nervoso/genética , Fosforilação , RNA Mensageiro/análise , Neoplasias Gástricas/metabolismoRESUMO
The Eph receptor tyrosine kinases and their ephrin ligands form a unique cell-cell contact-mediated bidirectional signaling mechanism for regulating cell localization and organization. High expression of Eph receptors in a wide variety of human tumors indicates some roles in tumor progression, which makes these proteins potential targets for anticancer therapy. For this purpose, we did gene expression profiling for 47 surgical specimens of brain tumors including 32 high-grade glioma using a microarray technique. The analysis, focused on the receptor tyrosine kinases, showed that EphA4 mRNA in the tumors was 4-fold higher than in normal brain tissue. To investigate the biological significance of EphA4 overexpression in these tumors, we analyzed EphA4-induced phenotypic changes and the signaling mechanisms using human glioma U251 cells. EphA4 promoted fibroblast growth factor 2-mediated cell proliferation and migration accompanied with enhancement of fibroblast growth factor 2-triggered mitogen-activated protein kinase and Akt phosphorylation. In addition, active forms of Rac1 and Cdc42 increased in the EphA4-overexpressing cells. Furthermore, we found that EphA4 formed a heteroreceptor complex with fibroblast growth factor receptor 1 (FGFR1) in the cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling. Thus, our results indicate that EphA4 plays an important role in malignant phenotypes of glioblastoma by enhancing cell proliferation and migration through accelerating a canonical FGFR signaling pathway.
Assuntos
Movimento Celular , Glioma/metabolismo , Glioma/patologia , Receptor EphA4/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/enzimologia , Glioma/genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor EphA4/genética , Transdução de Sinais/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The glycosylation of cell surface proteins is important for cancer biology processes such as cellular proliferation or metastasis. alpha1,6-Fucosyltransferase (FUT8) transfers a fucose residue to n-linked oligosaccharides on glycoproteins. Herein, we study the effect of fucosylation on epidermal growth factor receptor (EGFR) activity and sensitivity to an EGFR-specific tyrosine kinase inhibitor (EGFR-TKI). The increased fucosylation of EGFR significantly promoted EGF-mediated cellular growth, and the decreased fucosylation by stable FUT8 knockdown weakened the growth response in HEK293 cells. The overexpression of FUT8 cells were more sensitive than the control cells to the EGFR-TKI gefitinib, and FUT8 knockdown decreased the sensitivity to gefitinib. Finally, to examine the effects in a human cancer cell line, we constructed stable FUT8 knockdown A549 cells, and found that these cells also decreased EGF-mediated cellular growth and were less sensitive than the control cells to gefitinib. In conclusion, we demonstrated that the modification of EGFR fucosylation affected EGF-mediated cellular growth and sensitivity to gefitinib. Our results provide a novel insight into how the glycosylation status of a receptor may affect the sensitivity of the cell to molecular target agents.
Assuntos
Receptores ErbB/metabolismo , Fucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Gefitinibe , Glicosilação/efeitos dos fármacos , HumanosRESUMO
Fibroblast growth factor (FGF) signal is implicated in not only cell proliferation, but cell migration and morphological changes. Several different Rho family GTPases downstream of the Ras/ERK pathway are postulated to mediate the latter functions. However, none have been recognized to be directly coupled to FGF receptors (FGFRs). We have previously reported that EphA4 and FGFRs hetero-oligomerize through their cytoplasmic domains, trans-activate each other, and transduce a signal for cell proliferation through a docking protein, FRS2alpha (Yokote, H., Fujita, K., Jing, X., Sawada, T., Liang, S., Yao, L., Yan, X., Zhang, Y., Schlessinger, J., and Sakaguchi, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 18866-18871). Here, we have found that ephexin1, a guanine nucleotide exchange factor for Rho family GTPases, constitutes another downstream component of the receptor complex. Ephexin1 directly binds to the kinase domain of FGFR mainly through its DH and PH domains. The binding appears to become weaker and limited to the DH domain when FGFRs become activated. FGFR-mediated phosphorylation of ephexin1 enhances the guanine nucleotide exchange activity toward RhoA without affecting the activity to Rac1 or Cdc42. The FGFR-mediated tyrosine phosphorylation includes, but is not limited to, the residue (Tyr-87) phosphorylated by Src family kinase, which is known to be activated following EphA4 activation. The Tyr-to-Asp mutations that mimic the tyrosine phosphorylation in some of the putative FGFR-mediated phosphorylation sites increase the nucleotide exchange activity for RhoA without changing the activity for Rac1 or Cdc42. From these results, we conclude that ephexin1 is located immediately downstream of the EphA4-FGFR complex and the function is altered by the FGFR-mediated tyrosine phosphorylation at multiple sites.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neuropeptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Fatores de Crescimento de Fibroblastos , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , Neuropeptídeos/genética , Células PC12 , Fosforilação , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Receptor EphA4/genética , Receptor EphA4/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Tirosina/genética , Tirosina/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
A humanized anti-HER2 monoclonal antibody pertuzumab (Omnitarg, 2C4), binding to a different HER2 epitope than trastuzumab, is known as an inhibitor of heterodimerization of the HER receptors. Potent antitumor activity against HER2-expressing breast and prostate cancer cell lines has been clarified, but this potential is not clear against lung cancers. The authors investigated the in vitro anti-tumor activity of pertuzumab against eight non-small cell lung cancer cells expressing various members of the HER receptors. A lung cancer 11_18 cell line expressed a large amount of HER2 and HER3, and its cell growth was stimulated by an HER3 ligand, heregulin (HRG)-alpha. Pertuzumab significantly inhibited the HRG-alpha-stimulated cellular growth of the 11_18 cells. Pertuzumab blocked HRG-alpha-stimulated phosphorylation of HER3, mitogen-activated protein kinase (MAPK), and Akt. In contrast, pertuzumab failed to block epidermal growth factor (EGF)-stimulated phosphorylation of EGF receptor (EGFR) and MAPK. Immunoprecipitation showed that pertuzumab inhibited HRG-alpha-stimulated HER2/HER3 heterodimer formation. HRG-alpha-stimulated HER3 phosphorylation was also observed in the PC-9 cells co-overexpressing EGFR, HER2, and HER3, but the cell growth was neither stimulated by HRG-alpha nor inhibited by pertuzumab. The present results suggest that pertuzumab is effective against HRG-alpha-dependent cell growth in lung cancer cells through inhibition of HRG-alpha-stimulated HER2/HER3 signaling.
Assuntos
Anticorpos Monoclonais/fisiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Inibidores do Crescimento/fisiologia , Neoplasias Pulmonares/patologia , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/fisiologia , Transdução de Sinais/fisiologia , Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Dimerização , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/metabolismo , Humanos , Ligantes , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neuregulina-1/antagonistas & inibidores , Neuregulina-1/fisiologia , Fosforilação , Receptor ErbB-3/metabolismo , Transdução de Sinais/imunologiaRESUMO
PURPOSE: AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer. EXPERIMENTAL DESIGN: We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo. RESULTS: AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC(50) of 0.15 and 0.37 micromol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus-truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 micromol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar K(i) values ( approximately 0.05 micromol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts. CONCLUSIONS: AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.
Assuntos
Antineoplásicos/uso terapêutico , Quinazolinas/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Gefitinibe , Humanos , Camundongos , Camundongos Endogâmicos , Quinazolinas/administração & dosagem , Quinazolinas/farmacologia , RNA Interferente Pequeno/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/análise , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias Gástricas/enzimologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The existence of an in-frame deletion mutant correlates with the sensitivity of lung cancers to EGFR (epidermal growth factor receptor)-targeted tyrosine kinase inhibitors. We reported previously that the in-frame 15-bp deletional mutation (delE746-A750 type deletion) was constitutively active in cells. Kinetic parameters are important for characterizing an enzyme; however, it remains unclear whether the kinetic parameters of deletion mutant EGFR are similar to those of wild-type EGFR. We analysed autophosphorylation in response to ATP and inhibition of gefitinib for deletion mutant EGFR and wild-type EGFR. Kinetic studies, examining autophosphorylation, were carried out using EGFR fractions extracted from 293-pDelta15 and 293-pEGFR cells transfected with deletion mutant EGFR and wild-type EGFR respectively. We demonstrated the difference in activities between unstimulated wild-type (K(m) for ATP=4.0+/-0.3 microM) and mutant EGFR (K(m) for ATP=2.5+/-0.2 microM). There was no difference in K(m) values between EGF-stimulated wild-type EGFR (K(m) for ATP=1.9+/-0.1 microM) and deletion mutant EGFR (K(m) for ATP=2.2+/-0.2 microM). These results suggest that mutant EGFR is active without ligand stimulation. The K(i) value for gefitinib of the deletion mutant EGFR was much lower than that of wild-type EGFR. These results suggest that the deletion mutant EGFR has a higher affinity for gefitinib than wild-type EGFR.
Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Quinazolinas/farmacologia , Linhagem Celular , Gefitinibe , Humanos , Cinética , Mutação , Fosforilação , Deleção de SequênciaRESUMO
A yeast two-hybrid analysis has shown that the juxtamembrane region of FGF receptor 3 (FGFR3) interacts with the cytoplasmic domain of EphA4, which is a member of the largest family of receptor tyrosine kinases. Complex formation between the two receptors was shown to be mediated by direct interactions between the juxtamembrane domain of FGFR1, FGFR2, FGFR3, or FGFR4 and the N-terminal portion of the tyrosine kinase domain of EphA4. Activation of FGFR1 in transfected cells resulted in tyrosine phosphorylation of a kinase-negative EphA4 mutant and activation of EphA4 led to tyrosine phosphorylation of a kinase-negative FGFR1 mutant. Moreover, both receptors stimulate tyrosine phosphorylation of the docking protein FRS2alpha and induce mitogen-activated protein kinase stimulation with a time course and intensity that depends on the ligand that is applied. We also demonstrate that FGF-receptor-mediated mitogen-activated protein kinase stimulation is potentiated in cells costimulated with ephrin-A1. The direct interaction between EphA4 and FGFRs and the potentiation of FGF response that is induced by ephrin-A1 stimulation may modulate the biological responses that are mediated by these receptor families in cells or tissues in which the two receptors are coexpressed.
Assuntos
Receptor EphA4/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Ativação Transcricional , Anticorpos Monoclonais/química , Diferenciação Celular , Linhagem Celular , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Efrina-A1/química , Deleção de Genes , Vetores Genéticos , Humanos , Immunoblotting , Imunoprecipitação , Mutação , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptor EphA4/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/química , Retroviridae/genética , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Tirosina/químicaRESUMO
Nuclear localization of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in the process of apoptosis. To study the function of GAPDH, we expressed GAPDH C-terminally fused with or without nuclear localization signal (NLS) in SH-SY5Y and NB41A3 cells using a retrovirus expression system. GAPDH carrying NLS (GAPDH-NLS) was expressed mainly in the nucleus. However, expression of GAPDH-NLS did not cause any difference in cell survival rate as compared to that of the vector alone or GAPDH without NLS. Treatment with 1-Methyl-4-phenyl-pyridium iodide (MPP+) caused no difference in the cell survival rate or in the pattern or extent of apoptosis among the three transductants. In the cells expressing GAPDH without NLS, MPP+ did not cause visible translocation of GAPDH into nucleus before the onset of apoptosis. Since GAPDH is known to comprise a CRM1-mediated nuclear export signal, we blocked the nuclear export of GAPDH by treatment with leptomycin B, an inhibitor of CRM1-mediated nuclear export. The treatment did not cause any difference in apoptosis among the three transductants. An additional treatment with MPP+ induced no apoptotic difference in these cells. Thus, we have concluded that a simple nuclear localization of GAPDH does not induce apoptosis, and that MPP+-induced apoptosis is not caused by nuclear translocation of GAPDH.
Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Apoptose/fisiologia , Núcleo Celular/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Humanos , Carioferinas/metabolismo , Carioferinas/farmacologia , Cinética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteína Exportina 1RESUMO
Fibroblast growth factor (FGF) 23 is an important phosphaturic factor that inhibits inorganic phosphate (Pi) reabsorption from the renal proximal tubule. Its overproduction and proteolysis-resistant mutation such as R179Q cause tumor-induced osteomalacia and autosomal dominant hypophosphatemic rickets, respectively. To clarify the signaling mechanisms of FGF23 that mediate the reduction of Pi reabsorption, we inhibited the function of the known FGFRs in opossum kidney (OK-E) cells by expressing a dominant-negative (DN) form of FGFR. OK-E cells, which represent the renal proximal tubular cells, expressed all four known FGFRs. FGF23(R179Q) bound to and activated FGFR2, a prominent FGFR expressed in OK-E cells. The activated receptor transmitted a signal to increase the expression of type IIa Na(+)/Pi co-transporter and the Pi uptake. Expression of FGFR2(DN), which suppresses the major FGFR-mediated signal through the FRS2alpha-ERK pathway, reversed the function of FGF23(R179Q). When FGF23(R179Q) was applied to the basolateral side of polarized OK-E cells, regardless of the FGFR2(DN) expression, the apical Pi uptake decreased significantly. The apical application of FGF23(R179Q) in the polarized cells did not show such decrease but increase. The exogenously expressed FGFR2 was detectable only at the apical membrane. These results suggest that an FGF23 receptor, which is functionally distinct from the known FGFRs, is expressed at the basolateral membrane of OK-E cells.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Simportadores/genética , Animais , Fator de Crescimento de Fibroblastos 23 , Regulação da Expressão Gênica/fisiologia , Humanos , Túbulos Renais Proximais/metabolismo , Gambás , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Simportadores/biossínteseRESUMO
Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.