RESUMO
Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carbono , Carcinogênese/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Feminino , Ácido Fólico , Humanos , Metotrexato/uso terapêutico , Camundongos , Estudos RetrospectivosRESUMO
Early diagnosis of lung cancer by low-dose computed tomography is an effective strategy to reduce cancer mortality in high-risk individuals. However, recruitment of at-risk individuals with asymptomatic lung cancer still remains challenging. We developed a minimal invasive serum test, based on the detection of circulating microRNAs, which can identify at-risk individuals with asymptomatic early stage non-small cell lung carcinomas with 80% accuracy.
RESUMO
The deliberative ethics guidelines elaborated and implemented by members of the IFOM-IEO Campus (Firc Institute of Molecular Oncology (IFOM) and the European Institute of Oncology (IEO)). These should serve the dual purpose of establishing a minimal set of standard rules for bioethical debate and any ensuing decision-making process, especially for the perspective of providing real instruments to foster public engagement and public awareness on the ethical issues involved in biomedical research. It is shown that these guidelines instantiate the scheme of one of the correct ways of debating formalised by the western thought.
Assuntos
Bioética , Pesquisa Biomédica/ética , Tomada de Decisões/ética , Academias e Institutos/organização & administração , Comissão de Ética , Humanos , Objetivos Organizacionais , Guias de Prática Clínica como AssuntoRESUMO
The Prep1 homeodomain transcription factor is essential for embryonic development. 25% of hypomorphic Prep1(i/i) embryos, expressing the gene at 2% of the normal levels, survive pregnancy and live a normal-length life. Later in life, however, these mice develop spontaneous pre-tumoral lesions or solid tumors (lymphomas and carcinomas). In addition, transplantation of E14.5 fetal liver (FL) Prep1(i/i) cells into lethally irradiated mice induces lymphomas. In agreement with the above data, haploinsufficiency of a different Prep1-deficient (null) allele accelerates EmuMyc lymphoma growth. Therefore Prep1 has a tumor suppressor function in mice. Immunohistochemistry on tissue micrroarrays (TMA) generated from three distinct human cohorts comprising a total of some 1000 human tumors revealed that 70% of the tumors express no or extremely low levels of Prep1, unlike normal tissues. Our data in mice are thus potentially relevant to human cancer.
Assuntos
Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Neoplasias , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/patologiaRESUMO
The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Fenômenos Biomecânicos , Células , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Movimento/efeitos da radiação , MutaçãoRESUMO
Protein ubiquitination is critical for many cellular processes, through its ability to regulate protein degradation and various signaling mechanisms. In the ubiquitin (Ub) system, substrate specificity is achieved through the E3 family of Ub ligases. Because alterations of the ubiquitination machinery have been reported in human cancers, the selective interference with Ub ligases might represent a powerful therapeutic tool. Here, we report the first wide survey of misregulation of Ub ligases in cancer. We analysed 82 Ub ligases in nine types of cancer by in situ hybridization on tissue microarrays. We found 27 instances in which an Ub ligase was altered in a given type of tumor, when compared with normal tissues: 21 cases of overexpression and 6 cases of underexpression. We further analysed selected Ub ligases in large cohorts of breast and non-small-cell lung carcinomas. In five, of six, of these extended analyses (HUWE1, CCNB1IP1, SIAH1 and SIAH2 in breast cancer and CCNB1IP1 in lung cancer), we found that the levels of Ub ligases correlated significantly with relevant prognostic factors, and with clinical outcome. Our findings show that the alteration of Ub ligases is a frequent event in cancer and identify candidate targets for molecular therapies.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Humanos , Hibridização In Situ , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Neoplasias/mortalidade , Ubiquitina/químicaRESUMO
Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Metástase Linfática/genética , Adulto , Idoso , Algoritmos , Movimento Celular/genética , Análise por Conglomerados , Estudos de Coortes , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Análise por Pareamento , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Serpinas/fisiologiaRESUMO
Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.
Assuntos
Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Diferenciação Celular/genética , Proliferação de Células , Progressão da Doença , Seguimentos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Índice de Gravidade de Doença , Neoplasias Gástricas/classificação , Neoplasias Gástricas/metabolismoRESUMO
The gene numb encodes for a protein (Numb) involved in cell fate decisions in Drosophila, with proposed endocytic and developmental functions in mammalians. The distribution pattern of Numb in human tissues however, has not been fully characterized. We set out to explore the immunohistochemical expression of Numb in normal and neoplastic (28 adenoid cystic and 34 mucoepidermoid carcinomas) salivary glands, and correlated the results with the clinico-pathologic features of the neoplasms. Intense Numb immunoreactivity was detected in normal ductal cells and in a subset of acinar cells. In salivary carcinomas, we detected diffuse and intense Numb immunostaining in 5 adenoid cystic and 8 mucoepidermoid carcinomas. By contrast, the majority of adenoid cystic and mucoepidermoid cancers showed only moderate (14 and 5 cases) or focal staining (9 and 21 cases), respectively. The corresponding expression of Numb mRNA was documented in normal parotid gland and adenoid cystic carcinoma. Numb immunoreactivity was inversely correlated with the histological grade and Ki-67 immunoreactivity of both adenoid cystic and mucoepidermoid carcinomas. In addition, while tumor grade, stage, Ki-67 and Numb immunoreactivity were associated with disease-free survival in univariate analysis, only Numb and Ki-67 immunoreactivities retained independent prognostic significance in multivariate analysis. These data suggest that loss of Numb is implicated in aberrant differentiation programs of salivary gland carcinomas and may serve as a prognostic indicator in patients treated for these neoplasms.
Assuntos
Carcinoma Adenoide Cístico/genética , Carcinoma Mucoepidermoide/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Neoplasias das Glândulas Salivares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Carcinoma Adenoide Cístico/epidemiologia , Carcinoma Adenoide Cístico/patologia , Carcinoma Mucoepidermoide/epidemiologia , Carcinoma Mucoepidermoide/patologia , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/genética , Masculino , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/imunologia , Glândula Parótida/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias das Glândulas Salivares/epidemiologia , Neoplasias das Glândulas Salivares/patologia , Glândula Submandibular/metabolismo , Análise de SobrevidaRESUMO
Ubiquitination is a post-translational modification in which a small conserved peptide, ubiquitin, is appended to target proteins in the cell, through a series of complex enzymatic reactions. Recently, a particular form of ubiquitination, monoubiquitination, has emerged as a nonproteolytic reversible modification that controls protein function. In this review, we highlight recent findings on monoubiquitination as a signaling-induced modification, controlled, among others, by pathways originating from active receptor tyrosine kinases. Furthermore, we review the major cellular processes controlled by ubiquitin modification, including membrane trafficking, histone function, transcription regulation, DNA repair, and DNA replication.
Assuntos
Membrana Celular/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Animais , Reparo do DNA , Replicação do DNA , Endocitose , Anemia de Fanconi/metabolismo , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Receptores Proteína Tirosina Quinases/metabolismo , Saccharomyces cerevisiae , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Clatrina/metabolismo , Vírus JC/patogenicidade , Neuroglia/virologia , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação ao Cálcio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoflavonas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosfoproteínas/genéticaRESUMO
Tyrosine phosphorylation is one of the major mechanisms involved in the intracellular propagation of external signals. Strategies aimed at interfering with this process might allow the control of several cellular phenotypes. SH2 domains mediate protein-protein interactions by recognizing phosphotyrosine (pY) residues in the context of specific phosphopeptides. We created an SH2-scaffolded repertoire library by randomly mutagenizing five critical amino acid positions in the specificity-determining region of the PLCgamma C-terminal SH2 domain. Synthetic SH2 domains were selected from the library using biotinylated phosphopeptides derived from a natural PLCgamma-SH2 ligand as well as unrelated SH2 ligands. The isolated SH2s displayed high binding affinity constants for the selecting peptides and were capable of interacting with the corresponding proteins.
Assuntos
Biblioteca de Peptídeos , Engenharia de Proteínas , Domínios de Homologia de src , Animais , Sítios de Ligação , Bovinos , Cinética , Ligantes , Modelos Moleculares , Mutagênese , Peptídeos/química , Fenótipo , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Tirosina/metabolismoRESUMO
Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Tecido Nervoso/isolamento & purificação , Sistema Nervoso/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia , Vesículas Sinápticas/metabolismo , Aldicarb/farmacologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Caenorhabditis elegans/citologia , Proteínas de Ligação ao Cálcio/genética , Dinaminas , Imunofluorescência , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Deleção de Genes , Genes Reporter/fisiologia , Inseticidas/farmacologia , Microscopia Eletrônica , Dados de Sequência Molecular , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/fisiopatologia , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/ultraestrutura , Fenótipo , Fosfoproteínas/genética , Transporte Proteico/efeitos dos fármacos , Homologia de Sequência do Ácido Nucleico , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , TemperaturaRESUMO
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Proteína SOS1/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Proteínas de Transporte/genética , Fracionamento Celular , Extensões da Superfície Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Citocalasina D/farmacologia , Proteínas do Citoesqueleto , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genes Reporter , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Microscopia de Fluorescência , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SOS1/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas ras/genéticaRESUMO
Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and alpha-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Endocitose/fisiologia , Proteínas Monoméricas de Montagem de Clatrina , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Transporte Ativo do Núcleo Celular/fisiologia , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Ativação Transcricional/fisiologiaAssuntos
Receptores ErbB/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfotirosina/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Anticorpos , Anticorpos Monoclonais , Bactérias , Técnicas de Cultura de Células/métodos , Linhagem Celular , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Clonagem Molecular/métodos , DNA Complementar , Receptores ErbB/genética , Biblioteca Gênica , Humanos , Indicadores e Reagentes , Fosfoproteínas/metabolismo , Coelhos , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.