RESUMEN
BACKGROUND: Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers. METHODS: Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest. RESULTS: PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%. CONCLUSION: We identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.
RESUMEN
The Wnt ligands are a family of secreted signaling proteins which play key roles in a number of cellular processes under physiological and pathological conditions. Wnts bind to their membrane receptors and initiate a signaling cascade which leads to the nuclear localization and transcriptional activity of ß-catenin. The development of purified recombinant Wnt ligands has greatly aided in our understanding of Wnt signaling and its functions in development and disease. In the current study, we identified non-Wnt related signaling activities which were present in commercially available preparations of recombinant Wnt3a. Specifically, we found that treatment of cultured fibroblasts with recombinant Wnt3a induced immediate activation of TGF-ß and BMP signaling and this activity appeared to be independent of the Wnt ligand itself. Therefore, while purified recombinant Wnt ligands continue to be a useful tool for studying this signaling pathway, one must exercise a degree of caution when analyzing the results of experiments that utilize purified recombinant Wnt ligands.
Asunto(s)
Artefactos , Biotecnología/normas , Factor de Crecimiento Transformador beta1/agonistas , Proteína Wnt3A/farmacología , Animales , Anticuerpos Neutralizantes/farmacología , Biotecnología/métodos , Proteína Morfogenética Ósea 7/antagonistas & inhibidores , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Ratones , Células 3T3 NIH , Proteínas/genética , Proteínas/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Control de Calidad , Quinolinas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor ß (PDGFRß) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFRß in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRß. In contrast, ligand-induced internalization of PDGFRß was unaffected by HD-PTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFRß accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRß and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.
Asunto(s)
Movimiento Celular/genética , Proliferación Celular/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Becaplermina , Línea Celular , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ratones , Fosfoproteínas/metabolismo , Fosforilación/genética , Fosforilación/fisiología , Transporte de Proteínas/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/genéticaRESUMEN
We have investigated whether the recently discovered transcription factor, zinc finger BED domain-containing protein 6 (ZBED6), is expressed in insulin-producing cells and, if so, to what extent it affects beta cell function. ZBED6 was translated from a ZC3H11A transcript in which the ZBED6-containing intron was retained. ZBED6 was present in mouse ßTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. We propose that ZBED6 supports proliferation and survival of beta cells, possibly at the expense of specialized beta cell function-i.e., insulin production-because (i) the nuclear ZBED6 were the predominant forms in rapidly proliferating ßTC-6 cells, but not in human islet cells; (ii) down-regulation of ZBED6 in ßTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell-cycle arrest, increased expression of beta cell-specific genes, and higher rates of apoptosis; (iii) silencing of ZBED6 in the human PANC-1 duct cell line reduced proliferation rates; and (iv) ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis, and apoptosis. Furthermore, it is possible that beta cells, by switching from full length to a truncated form of ZBED6, can decide the subcellular localization of ZBED6, thereby achieving differential ZBED6-mediated transcriptional regulation.
Asunto(s)
Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/genética , Línea Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Citometría de Flujo , Regulación de la Expresión Génica/genética , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Espectrometría de Masas , Ratones , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARNRESUMEN
Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF ß-receptor (PDGFRß) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRß kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias/metabolismo , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-sis/farmacología , Factor de Transcripción STAT3/metabolismo , Animales , Becaplermina , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células 3T3 NIH , Neoplasias/genética , Neoplasias/patología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Tirosina Quinasas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
CD44 is a cell surface receptor for hyaluronan which affects cell adhesion and migration, and has been implicated in chronic inflammation and in tumorigenesis. To elucidate the molecular mechanisms underlying its multiple functions, we used a peptide-based pull-down assay to identify proteins that interact with CD44. Nonphosphorylated or phosphorylated peptides from the intracellular CD44 C-terminus, were immobilized and used as baits. Interacting proteins were subjected to SDS-gel electrophoresis and were identified by MALDI-TOF mass spectrometry. Several interaction partners were identified, including proteins involved in cytoskeletal reorganization, transcription, endocytosis, and intracellular transport. An endogenous complex between CD44 and one of the interacting proteins, the actin binding protein IQGAP1, was demonstrated in several normal and transformed cell types.
Asunto(s)
Receptores de Hialuranos/metabolismo , Transducción de Señal/fisiología , Línea Celular , Transformación Celular Neoplásica , Células Cultivadas , Femenino , Humanos , Ácido Hialurónico/metabolismo , Masculino , Unión Proteica , Estructura Terciaria de Proteína/fisiología , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.
Asunto(s)
Amiloide/química , Antígenos de Superficie/química , Proteínas de la Leche/química , Algoritmos , Secuencia de Aminoácidos , Amiloide/ultraestructura , Humanos , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) beta-receptor (PDGFRbeta) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRbeta removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRbeta. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRbeta. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRbeta. Our data suggest that Alix inhibits down-regulation of PDGFRbeta by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/fisiología , Regulación hacia Abajo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ubiquitina/química , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Datos de Secuencia Molecular , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Homología de Secuencia de Aminoácido , Porcinos , Tirosina/químicaRESUMEN
We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.
Asunto(s)
Amiloide/inmunología , Epítopos/química , Prealbúmina/inmunología , Secuencia de Aminoácidos , Amiloide/química , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Prealbúmina/química , Prealbúmina/genéticaRESUMEN
Regulation of protein tyrosine phosphatases (PTPs) through reversible oxidation of the active site cysteine is emerging as a general, yet poorly characterized, mechanism for control of the activity of this important group of enzymes. This regulatory mechanism was initially described after in vitro treatment of PTPs with oxidizing agents. However, accumulating evidence has substantiated the notion that this mechanism is also operating in vivo, e.g., in association with the transient increase in H(2)O(2) production which occurs after activation of receptor tyrosine kinases. A novel generic antibody-based method for monitoring of PTP oxidation is described. The sensitivity of this strategy has been validated by the demonstration of oxidation of endogenously expressed PTPs after stimulation of cells with growth factors. The method was also instrumental in providing the first evidence for intrinsic differences between PTP domains with regard to sensitivity to oxidation.
Asunto(s)
Anticuerpos/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Dominio Catalítico/inmunología , Extractos Celulares/inmunología , Células Cultivadas , Cisteína/inmunología , Humanos , Oxidación-Reducción , Proteínas Tirosina Fosfatasas/inmunología , Ácidos Sulfónicos/inmunologíaRESUMEN
MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas/química , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Animales , Anticuerpos Monoclonales , Sitios de Unión , Encéfalo/citología , Encéfalo/embriología , Diferenciación Celular , Embrión de Pollo , Lipoproteínas/metabolismo , Lipoproteínas/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuronas/química , Neuronas/citología , Fosfoproteínas , Fosforilación , Retina/química , Retina/citología , Retina/embriología , Serina/inmunología , Serina/metabolismoRESUMEN
Protein tyrosine phosphatases (PTPs) constitute a large enzyme family with important biological functions. Inhibition of PTP activity through reversible oxidation of the active-site cysteine residue is emerging as a general, yet poorly characterized, regulatory mechanism. In this study, we describe a generic antibody-based method for detection of oxidation-inactivated PTPs. Previous observations of oxidation of receptor-like PTP (RPTP) alpha after treatment of cells with H(2)O(2) were confirmed. Platelet-derived growth factor (PDGF)-induced oxidation of endogenous SHP-2, sensitive to treatment with the phosphatidylinositol 3-kinase inhibitor LY294002, was demonstrated. Furthermore, oxidation of RPTPalpha was shown after UV-irradiation. Interestingly, the catalytically inactive second PTP domain of RPTPalpha demonstrated higher susceptibility to oxidation. The experiments thus demonstrate previously unrecognized intrinsic differences between PTP domains to susceptibility to oxidation and suggest mechanisms for regulation of RPTPs with tandem PTP domains. The antibody strategy for detection of reversible oxidation is likely to facilitate further studies on regulation of PTPs and might be applicable to analysis of redox regulation of other enzyme families with active-site cysteine residues.
Asunto(s)
Anticuerpos/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Alquilación , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Becaplermina , Sitios de Unión , Línea Celular , Humanos , Peróxido de Hidrógeno/farmacología , Ácido Yodoacético/farmacología , Ratones , Datos de Secuencia Molecular , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/química , Proteínas Proto-Oncogénicas c-sis , Rayos Ultravioleta , Vanadatos/farmacologíaRESUMEN
The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family.
Asunto(s)
Células Epiteliales/fisiología , Proteínas de la Membrana/fisiología , Uniones Estrechas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Clonación Molecular , Neoplasias del Colon , Secuencia Conservada , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Cartilla de ADN , Bases de Datos de Ácidos Nucleicos , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Filogenia , Receptores Virales , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Receptores ErbB/metabolismo , Proteínas Oncogénicas de Retroviridae/química , Proteínas Oncogénicas de Retroviridae/metabolismo , Ubiquitina-Proteína Ligasas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Arginina/química , Sitios de Unión , Células CHO , Proteínas Portadoras/genética , Línea Celular , Secuencia de Consenso , Cricetinae , Regulación hacia Abajo , Endocitosis , Receptores ErbB/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteína Oncogénica v-cbl , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prolina/química , Proteínas Proto-Oncogénicas c-cbl , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Homología de Secuencia de Aminoácido , Dominios Homologos srcRESUMEN
We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.
Asunto(s)
Proteínas Proto-Oncogénicas c-kit/metabolismo , Tirosina/metabolismo , Dominios Homologos src , Familia-src Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Aorta , Sitios de Unión , Endotelio Vascular/citología , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Fosfopéptidos/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Proteínas Proto-Oncogénicas c-kit/genética , Porcinos , TransfecciónRESUMEN
The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.
Asunto(s)
Proteínas Portadoras/química , Receptores Virales/química , Receptores Virales/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Sitios de Unión , Western Blotting , Proteínas Portadoras/metabolismo , División Celular , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , ADN Complementario/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/metabolismo , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Humanos , Ratones , Plásmidos/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The comparative endocrinology of the 37-amino-acid-residue islet amyloid polypeptide (IAPP) is poorly known, possibly due to the fact that available antisera, raised against mammalian IAPP, fail to give immunoreactivity with islet parenchymal cells of non-mammalian vertebrates. Using reverse transcriptase-linked polymerase chain reaction with degenerate primers, IAPP was identified, and its deduced amino-acid sequence partially characterized, in three species of teleostean fish, i.e. Danio rerio (zebrafish), Salmo salar (Atlantic salmon), and Myoxocephalus (cottus) scorpius (daddy sculpin). The daddy sculpin is a species where the histophysiology of the pancreatic islet parenchyma has previously been comprehensively studied. From the deduced amino-acid sequence, a synthetic peptide, corresponding to positions 20-29 of Salmo IAPP, was synthesized. A mouse antiserum to this peptide gave a distinct immunoreactivity with the insulin-producing beta cells of the sculpin Brockmann bodies and salmon endocrine pancreas. Thus, IAPP belongs to the group of peptide hormones expressed by the islet parenchymal cells in both mammals and non-mammalian vertebrates. Salmo salar IAPP(20-29) was found to give rise to amyloid-like fibrils in vitro.
Asunto(s)
Amiloide/aislamiento & purificación , Islotes Pancreáticos/química , Salmón/genética , Secuencia de Aminoácidos , Amiloide/análisis , Amiloide/química , Animales , Sueros Inmunes , Inmunohistoquímica , Polipéptido Amiloide de los Islotes Pancreáticos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Transforming growth factor-beta (TGFbeta) is a potent regulator of cell proliferation, differentiation, apoptosis, and migration. TGF-beta type I receptor (TbetaR-I), which has intrinsic serine/threonine kinase activity, is a key component in activation of intracellular TGFbeta signaling. We studied two different classes of TbetaR-I inhibitors, i.e., compounds interfering with the ATP-binding site of the kinase and substrate-mimicking peptides. We found that pyridinylimidazole compounds inhibited TbetaR-I kinase at micromolar concentration. A representative compound, SB203580, inhibited in vivo Smad2 phosphorylation by TbetaR-I and affected TGFbeta-dependent transcriptional activation. Peptides mimicking the TbetaR-I phosphorylation sites at the C-terminus of Smad2 also inhibited the autophosphorylation of TbetaR-I and phosphorylation of Smad2 by TbetaR-I in vitro and in vivo, whereas a similar peptide from Smad5 was without effect. The substrate-mimicking peptide, fused to penetratin, inhibited a TGFbeta1-dependent transcriptional response in a luciferase reporter assay and ligand-dependent growth inhibition of Mv1Lu cells. Thus, the substrate-mimetic peptide is a new type of specific inhibitor of the TGFbeta signaling in vivo.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal , Factores de Transcripción , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Sitios de Unión/efectos de los fármacos , Células COS , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Homeodominio/farmacología , Imidazoles/farmacología , Visón , Imitación Molecular , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal/efectos de los fármacos , Proteína Smad2 , Proteína smad3 , Especificidad por Sustrato , Transactivadores/metabolismo , Transactivadores/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.
Asunto(s)
Fosfopéptidos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Cinética , Lisina/química , Lisina/metabolismo , Datos de Secuencia Molecular , Fosfopéptidos/síntesis química , Fosfopéptidos/química , Fosforilación , Proteínas Tirosina Fosfatasas/química , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/química , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Especificidad por SustratoRESUMEN
We have previously shown that the binding site for GTPase activating protein of Ras (RasGAP) in the PDGF beta-receptor, Tyr771, is phosphorylated to a much lower extent in the heterodimeric configuration of PDGF alpha- and beta-receptors, compared to the PDGF beta-receptor homodimer. The decreased recruitment of the RasGAP to the receptor leads to prolonged activation of the Ras/MAP kinase pathway, which could explain the increase in mitogenicity seen upon induction of heterodimers. The molecular mechanism underlying these differences was investigated. We could show that the loss of phosphorylation of Tyr771 was dependent on presence of intact binding sites for the protein tyrosine phosphatase SHP-2 on the PDGF beta-receptor. Thus, in PDGF receptor mutants in which binding of SHP-2 was lost, a higher degree of phosphorylation of Tyr771 was seen, while other phosphorylation sites in the receptor remained virtually unaffected. Thus, SHP-2 appears to play an important role in modulating phosphorylation of Y771, thereby controlling RasGAP recruitment and Ras/MAP kinase signaling in the heterodimeric configuration of the PDGF receptors.