RESUMO
High-deflection strain gauges show potential as economical and user-friendly sensors for capturing large deformations. The interpretation of these sensors is much more complex than that of conventional strain gauges due to the viscoelastic nature of strain gauges. This research endeavor developed and tested a model for interpreting sensor outputs that includes the time-dependent nature of strain gauges. A model that captures the effect of quasi-static strains was determined by using a conventional approach of fitting an equation to observed data. The dynamic relationship between the strain and the resistance was incorporated by superimposing dynamic components onto the quasi-static model to account for spikes in resistances that accompany each change in sensor strain and subsequent exponential decays. It was shown that the model can be calibrated for a given sensor by taking two data points at known strains. The resulting sensor-specific model was able to interpret strain-gauge electrical signals during a cyclical load to predict strain with an average mean absolute error (MAE) of 1.4% strain, and to determine the strain rate with an average MAE of 0.036 mm/s. The resulting model and tuning procedure may be used in a wide range of applications, such as biomechanical monitoring and analysis.
Assuntos
ViscosidadeRESUMO
The activity of growth factors is crucial for tumor progression. We previously characterized a secreted fibroblast growth factor-binding protein (FGF-BP1) as a chaperone molecule, which enhances the biological functions of FGFs by releasing FGFs from the extracellular matrix. Here, we characterize the frequency and pattern of FGF-BP1 expression during the malignant progression of pancreas and colorectal carcinoma. For this, we generated monoclonal antibodies that detect FGF-BP1 protein in formalin-fixed, paraffin-embedded tissues and applied in situ hybridization to detect FGF-BP1 mRNA in adjacent tissue sections. FGF-BP1 protein and mRNA were found up-regulated (>70% positive) in parallel (r = 0.70, P < 0.0001) in colon adenoma (n = 9) as well as primary (n = 46) and metastatic (n = 71) colorectal cancers relative to normal colon epithelia (all P < 0.0001, versus normal). Similarly, pancreatitis (n = 17), pancreatic intraepithelial neoplasia (n = 80), and pancreatic adenocarcinoma (n = 67) showed a significant up-regulation of FGF-BP1 compared with normal pancreas (n = 42; all P < 0.0001, relative to normal). Furthermore, the biological activity of FGF-BP1 is neutralized by one of the antibodies, suggesting the potential for antibody-based therapeutic targeting. We propose that the up-regulation of the secreted FGF-BP1 protein during initiation of pancreas and colon neoplasia could make this protein a possible serum marker indicating the presence of high-risk premalignant lesions.
Assuntos
Adenocarcinoma/genética , Proteínas de Transporte/biossíntese , Neoplasias do Colo/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/patologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Proteínas de Transporte/fisiologia , Transformação Celular Neoplásica , Neoplasias do Colo/patologia , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias Pancreáticas/patologia , Pancreatite/genética , Pancreatite/patologia , Pancrelipase/fisiologia , RNA Mensageiro/biossíntese , Medição de Risco , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Carcinoembryonic antigen (CEA) has been shown to participate in the progression and metastatic growth of colorectal cancer. However, its biological function remains elusive. Recently, we found that CEA protects colon cancer cells from undergoing apoptosis, suggesting a complex role that includes signal transduction activity. Additionally, it was reported that CEA binds to Kupffer cells and macrophages to a membrane-anchored homolog of heterogeneous nuclear protein M4 (hnRNP M4), which subsequently was named CEA-receptor (CEAR). Cytoplasmatic and membranous expression of CEAR in CEA-positive colon cancer tissues prompted us to analyze the CEA-CEAR interaction in HT29 colon cancer cells. Both, CEA and CEAR were found on the cell surface of HT29 cells, as demonstrated by confocal microscopy. Imaging analysis suggested co-localization and, thus, interaction of both molecules. To confirm this observation, immunoprecipitation experiments and Western blot analysis were performed and indicated binding of CEA and CEAR. Immunoprecipitation of CEA resulted in a pull down of CEAR. The pull down of CEAR correlated with the amount of CEA as demonstrated by ribozyme targeting of CEA. Finally, external treatment of HT29 cells with soluble CEA induced tyrosine phosphorylation of CEAR, suggesting a CEA-dependent role of CEAR in signal transduction. Future experiments will elucidate whether the CEA-CEAR interaction is involved in CEA's antiapoptotic role and mediates the prometastatic properties of CEA in colon cancer cells.
Assuntos
Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Receptores de N-Acetilglucosamina/metabolismo , Animais , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/farmacologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Neoplasias do Colo/imunologia , Células HT29 , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/biossíntese , Humanos , Imuno-Histoquímica , Imunoprecipitação , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Receptores de N-Acetilglucosamina/biossínteseRESUMO
Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein-protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Colesterol/metabolismo , Clonagem Molecular , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/química , Células HeLa/química , Humanos , Lisossomos/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Vesículas Transportadoras/químicaRESUMO
A major obstacle to achieving constitutive ribozyme expression in cells is that expression or elimination of the target gene may provide either a growth advantage or disadvantage to the cells that express ribozyme. Many approaches have been used to overcome this problem, mostly based on the effort to create conditional or inducible expression systems. In this chapter, we describe the most common choice for overcoming this problem, tetracycline-regulated ribozyme expression. This system consists of two central components: transcriptional transactivators that interact specifically with bacterial cis-regulatory elements and antibiotics that can modulate the binding of the transactivators at low and nontoxic doses. Here, we summarize protocols to generate cell lines expressing tetracycline-regulated ribozyme constructs.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , RNA Catalítico/genética , Tetraciclina/farmacologia , Transcrição Gênica/genética , Animais , Sequência de Bases , Linhagem Celular , Células Clonais , Códon/genética , Vetores Genéticos , Conformação de Ácido Nucleico , Plasmídeos , Transcrição Gênica/efeitos dos fármacos , Transfecção/métodosRESUMO
Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.
Assuntos
Proteínas de Transporte , Citocinas , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Quinase do Linfoma Anaplásico , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Junções Célula-Matriz/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Gravidez , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic.
Assuntos
Inibidores da Angiogênese/farmacologia , Fibrossarcoma/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Dibenzazepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Invadopodia are filopodia-like projections possessing protease activity that participate in tumor cell invasion. We demonstrate that co-localization of cortactin and phosphotyrosine identifies a subset of cortactin puncta termed "invadopodial complexes" that we find to be closely associated with the plasma membrane at active sites of focal degradation of the extracellular matrix in MDA-MB-231 breast cancer cells. Manipulation of c-Src activity in cells by transfection with kinase activated c-Src(527) or kinase inactive c-Src(295) results in a dramatic increase or decrease, respectively, in the number of these structures associated with changes in the number of sites of active matrix degradation. Overexpression of kinase-inactive c-Src(295) does not prevent localization of cortactin at the membrane; however, co-localized phosphotyrosine staining is decreased. Thus, elevated phosphotyrosine at invadopodial complexes is specifically associated with the proteolytic activity of invadopodia. Further, invadopodial complexes are spatially, morphologically and compositionally distinct from focal adhesions as determined by localization of focal adhesion kinase (FAK), which is not present in invadopodial complexes. Expression of kinase-inactive c-Src(295) blocks invadopodia activity, but does not block filopodia formation. Thus, invadopodia, but not filopodia, are highly correlated with matrix invasion, and sites of invadopodial activity can be identified by the formation of invadopodial complexes.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Cortactina/metabolismo , Matriz Extracelular/metabolismo , Invasividade Neoplásica/fisiopatologia , Fosfotirosina/metabolismo , Pseudópodes/metabolismo , Neoplasias da Mama/fisiopatologia , Proteína Tirosina Quinase CSK , Carcinoma/fisiopatologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Regulação para Baixo/fisiologia , Ativação Enzimática/fisiologia , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Peptídeo Hidrolases/metabolismo , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transfecção , Regulação para Cima/fisiologia , Quinases da Família srcRESUMO
The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.
Assuntos
Neoplasias da Mama/fisiopatologia , Histona Acetiltransferases/genética , Transativadores/genética , Ubiquitina-Proteína Ligases/fisiologia , Neoplasias da Mama/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/fisiopatologia , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Rim , Coativador 3 de Receptor Nuclear , Plasmídeos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Transfecção , Ubiquitina/genéticaRESUMO
Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.
Assuntos
Proteínas de Transporte/química , Fatores de Crescimento de Fibroblastos/química , Sequência de Aminoácidos , Animais , Aorta/metabolismo , Ligação Competitiva , Western Blotting , Bovinos , Proliferação de Células , Células Cultivadas , Quimiotaxia , Cisteína/química , DNA Complementar/metabolismo , Dissulfetos/química , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Fases de Leitura Aberta , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
Fibroblast growth factors (FGFs) have been implicated in a variety of physiologic and pathologic processes from embryonic development to tumor growth and angiogenesis. FGFs are immobilized in the extracellular matrix of different tissues and require release from this storage site to trigger a response. Secreted FGF-binding proteins (FGF-BPs) can release immobilized FGFs, enhance the activity of locally stored FGFs and can thus serve as an angiogenic switch molecule in cancer. Here, we report on the effect of human FGF-BP transgene expression in chicken embryos. To establish the transgenic model, plasmid-based reporter vectors expressing luciferase, beta-galactosidase or green fluorescent protein were introduced through different routes into 4- to 5-day-old embryos grown outside their egg shell on top of the yolk sac. This allows for easy manipulation and continuous observation of phenotypic effects. Expression of human FGF-BP induced dose-dependent vascular permeability, hemorrhage and embryonic lethality. Light and electron microscopic studies indicate that this hemorrhage results from compromised microvascular structure. An FGF-1 expression vector with an added secretory signal mimicked this vascular leakiness phenotype whereas wild-type FGF-1 required coexpression of a threshold amount of FGF-BP. This model is a powerful tool for real-time monitoring of the effects of transient transgene expression during embryogenesis.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Neoplasias/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Animais , Embrião de Galinha , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Cinética , Neovascularização Patológica/patologiaRESUMO
The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Quinase do Linfoma Anaplásico , Animais , Separação Celular , Sobrevivência Celular , Meios de Cultura Livres de Soro/farmacologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Immunoblotting , Ligantes , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , RNA Catalítico/metabolismo , Receptores Proteína Tirosina Quinases , Transdução de Sinais , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP- and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rho-induced serum response element transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2-expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active Rho mutant reversed the disassembly phenotype, in which the coexpressing cells were more spread and less contracted than Rho alone-expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Citoesqueleto/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteínas de Transporte/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Biblioteca Gênica , Genes Reporter , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , Transfecção , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Midkine (MK) is a developmentally regulated, secreted growth factor homologous to pleiotrophin (PTN). To investigate the potential role of MK in tumor growth, we expressed MK in human SW-13 cells and studied receptor binding, signal transduction, and activity of MK. The MK protein stimulates soft agar colony formation in vitro and tumor growth of SW-13 cells in athymic nude mice, as well as proliferation of human endothelial cells from brain microvasculature and umbilical vein (HUVEC) in the low ng/ml range. MK binds to anaplastic lymphoma kinase (ALK), the receptor for PTN, with an apparent K(d) of 170 pm in intact cells, and this receptor binding of MK is competed by PTN with an apparent K(d) of approximately 20 pm. Monoclonal antibodies raised against the extracellular ligand-binding domain of ALK inhibit ALK receptor binding of MK as well as MK-stimulated colony formation of SW-13 cells. Furthermore, MK stimulates ALK phosphorylation in WI-38 human fibroblasts and activates PI3-kinase and MAP kinase signal transduction in WI-38, HUVEC, neuroblastoma (SH SY-5Y) and glioblastoma (U87MG) cells that express the ALK protein. We conclude that MK can act as a growth, survival, and angiogenic factor during tumorigenesis and signals through the ALK receptor.