RESUMEN
Paget's disease of bone (PDB) is a common disorder characterized by focal areas of increased and disorganized osteoclastic bone resorption, leading to bone pain, deformity, pathological fracture, and an increased risk of osteosarcoma. Genetic factors play an important role in the pathogenesis of Paget's disease. In some families, the disease has been found to be linked to a susceptibility locus on chromosome 18q21-22, which also contains the gene responsible for familial expansile osteolysis (FEO)--a rare bone dysplasia with many similarities to Paget's disease. Insertion mutations of the TNFRSF11A gene encoding Receptor Activator of NF kappa B (RANK) have recently been found to be responsible for FEO and rare cases of early onset familial Paget's disease. Loss of heterozygosity (LOH) affecting the PDB/FEO critical region has also been described in osteosarcomas suggesting that TNFRSF11A might also be involved in the development of osteosarcoma. In order to investigate the possible role of TNFRSF11A in the pathogenesis of Paget's disease and osteosarcoma, we conducted mutation screening of the TNFRSF11A gene in patients with familial and sporadic Paget's disease as well as DNA extracted from Pagetic bone lesions, an osteosarcoma arising in Pagetic bone and six osteosarcoma cell lines. No specific abnormalities of the TNFRSF11A gene were identified in a Pagetic osteosarcoma, the osteosarcoma cell lines, DNA extracted from Pagetic bone lesions, or DNA extracted from peripheral blood in patients with familial or sporadic Paget's disease including several individuals with early onset Paget's disease. These data indicate that TNFRSF11A mutations contribute neither to the vast majority of cases of sporadic or familial PDB, nor to the development of osteosarcoma.
Asunto(s)
Neoplasias Óseas/genética , Predisposición Genética a la Enfermedad , Glicoproteínas/genética , Osteítis Deformante/genética , Osteosarcoma/genética , Receptores Citoplasmáticos y Nucleares/genética , Adulto , ADN/análisis , Análisis Mutacional de ADN , Cartilla de ADN/química , Pruebas Genéticas , Humanos , Osteoprotegerina , Mutación Puntual , Reacción en Cadena de la Polimerasa , Receptores del Factor de Necrosis TumoralRESUMEN
DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transactivadores/metabolismo , Adenocarcinoma/patología , Biomarcadores , Western Blotting , Regulación de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Neoplasias Pancreáticas/patología , Sensibilidad y Especificidad , Proteína Smad4RESUMEN
The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.
Asunto(s)
Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Mutación , Transactivadores , Proteína de la Poliposis Adenomatosa del Colon , Alelos , Factor de Transcripción CDX2 , Neoplasias Colorrectales/metabolismo , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Genes Supresores de Tumor/genética , Genes Supresores de Tumor/fisiología , Proteínas de Homeodominio/metabolismo , Homeostasis , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , beta CateninaRESUMEN
The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.
Asunto(s)
Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Genes APC , Genes myc , Transactivadores , Proteína de la Poliposis Adenomatosa del Colon , Sitios de Unión , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Genes Reporteros , Células HT29 , Humanos , Mutación , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , Transcripción Genética , beta CateninaRESUMEN
Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.
Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/genética , Genes APC , Transactivadores , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Desmoplaquinas , Humanos , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , beta Catenina , gamma CateninaRESUMEN
The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Péptidos/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Portadoras/química , Proteínas de Ciclo Celular , Biología Computacional , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Biblioteca de Péptidos , Fosfoproteínas/química , Unión Proteica , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.
Asunto(s)
Distrofina/química , Ligandos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular/métodos , Secuencia Conservada , ADN Complementario , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/químicaRESUMEN
Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.
Asunto(s)
Neoplasias del Colon/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Genes APC , Transactivadores , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína de la Poliposis Adenomatosa del Colon , Neoplasias del Colon/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Mutación de Línea Germinal , Humanos , Mutación , Fosforilación , Transducción de Señal , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Transfección , Células Tumorales Cultivadas , beta CateninaRESUMEN
The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteína Quinasa C/aislamiento & purificación , Transducción de Señal , Células 3T3 , Secuencia de Aminoácidos , Animales , Femenino , Regulación Enzimológica de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Transcripción Genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich , Dominios Homologos srcRESUMEN
Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.
Asunto(s)
Bacteriófago lambda/metabolismo , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Bacteriófago lambda/genética , Clonación Molecular , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Unión Proteica/genética , Dominios Homologos src/genéticaRESUMEN
Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.
Asunto(s)
Proteínas/genética , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Péptidos/metabolismo , Transducción de Señal , Dominios Homologos src , Familia-src Quinasas , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Secuencia de Consenso , Cortactina , Proteína Adaptadora GRB2 , Isoenzimas/metabolismo , Ligandos , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Fosfolipasa C gamma , Unión Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-crk , Proteínas Proto-Oncogénicas c-yes , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Fosfolipasas de Tipo C/metabolismoAsunto(s)
Péptidos/genética , Dominios Homologos src/genética , Secuencia de Aminoácidos , Animales , Bacteriófago M13/genética , Femenino , Biblioteca de Genes , Técnicas Genéticas , Glutatión Transferasa/genética , Glutatión Transferasa/aislamiento & purificación , Ligandos , Datos de Secuencia Molecular , Oocitos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Transducción de Señal , Xenopus laevisRESUMEN
We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.
Asunto(s)
Bacteriófagos/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Ligandos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Xenopus laevisRESUMEN
The F-type subfamily of LINE-1 or L1 retroposons [for long interspersed (repetitive) element 1] was dispersed in the mouse genome several million years ago. This subfamily appears to be both transcriptionally and transpositionally inactive today and therefore may be considered evolutionarily extinct. We hypothesized that these F-type L1s are inactive because of the accumulation of mutations. To test this idea we used phylogenetic analysis to deduce the sequence of a transpositionally active ancestral F-type promoter, resurrected it by chemical synthesis, and showed that it has promoter activity. In contrast, F-type sequences isolated from the modern genome are inactive. This approach, in which the automated DNA synthesizer is used as a "time machine," should have broad application in testing models derived from evolutionary studies.