RESUMO
The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten-/- ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Genes Supressores de Tumor/fisiologia , Neoplasias Experimentais/genética , Monoéster Fosfórico Hidrolases , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Supressoras de Tumor , Adenocarcinoma/patologia , Animais , Adesão Celular , Células Cultivadas , Neoplasias do Colo/patologia , Feminino , Genes Letais , Mutação em Linhagem Germinativa , Síndrome do Hamartoma Múltiplo/genética , Masculino , Camundongos , Camundongos Knockout , Neoplasias Experimentais/patologia , PTEN Fosfo-Hidrolase , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/análise , Células-Tronco/citologia , Teratocarcinoma/patologia , Neoplasias Testiculares/patologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27(KIP1) expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27(Kip1)) do not develop prostate cancer. PTEN activity leads to the induction of p27(KIP1) expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27(KIP1) may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten(+/-)/Cdkn1b(-/-) mice. Moreover, Pten(+/-)/Cdkn1b(-/-) mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27(Kip1) through the control of cell-cycle progression.
Assuntos
Proteínas de Ciclo Celular , Genes Supressores de Tumor , Proteínas Associadas aos Microtúbulos/genética , Monoéster Fosfórico Hidrolases/genética , Neoplasias da Próstata/genética , Proteínas Supressoras de Tumor , Animais , Inibidor de Quinase Dependente de Ciclina p27 , Masculino , Camundongos , Camundongos Mutantes , PTEN Fosfo-HidrolaseRESUMO
p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).
Assuntos
Proteínas de Ligação a DNA , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/etiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/prevenção & controle , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA , Proteínas ras/metabolismo , Animais , Divisão Celular , Células Cultivadas , Ativação Enzimática , Marcação de Genes , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Transdução de SinaisRESUMO
A major pathway by which growth factors, such as platelet-derived growth factor (PDGF), regulate cell proliferation is via the receptor tyrosine kinase/Ras/mitogen-activated protein kinase (MAPK) signaling cascade. The output of this pathway is subjected to tight regulation of both positive and negative regulators. One such regulator is p62(dok), the prototype of a newly identified family of adaptor proteins. We recently provided evidence, through the use of p62(dok)-deficient cells, that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation and the Ras/MAPK pathway. We show here that reintroduction of p62(dok) into p62(dok)-(/)- cells can suppress the increased cell proliferation and prolonged MAPK activity seen in these cells, and that plasma membrane recruitment of p62(dok) is essential for its function. We also show that the PDGF-triggered plasma membrane translocation of p62(dok) requires activation of phosphoinositide 3-kinase (PI3-kinase) and binding of its pleckstrin homology (PH) domain to 3'-phosphorylated phosphoinositides. Furthermore, we demonstrate that p62(dok) can exert its negative effect on the PDGFR/MAPK pathway independently of its ability to associate with RasGAP and Nck. We conclude that p62(dok) functions as a negative regulator of the PDGFR/Ras/MAPK signaling pathway through a mechanism involving PI3-kinase-dependent recruitment of p62(dok) to the plasma membrane.
Assuntos
Proteínas de Ligação a DNA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Transporte Biológico Ativo/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Proteínas Oncogênicas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/química , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Estrutura Terciária de Proteína , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas ras/metabolismoRESUMO
We generated purine nucleoside phosphorylase (PNP)-deficient mice to gain insight into the mechanism of immune deficiency disease associated with PNP deficiency in humans. Similar to the human disease, PNP deficiency in mice causes an immunodeficiency that affects T lymphocytes more severely than B lymphocytes. PNP knockout mice exhibit impaired thymocyte differentiation, reduced mitogenic and allogeneic responses, and decreased numbers of maturing thymocytes and peripheral T cells. T lymphocytes of PNP-deficient mice exhibit increased apoptosis in vivo and higher sensitivity to gamma irradiation in vitro. We propose that the immune deficiency in PNP deficiency is a result of inhibition of mitochondrial DNA repair due to the accumulation of dGTP in the mitochondria. The end result is increased sensitivity of T cells to spontaneous mitochondrial DNA damage, leading to T cell depletion by apoptosis.
Assuntos
Nucleotídeos de Desoxiguanina/metabolismo , Mitocôndrias/metabolismo , Purina-Núcleosídeo Fosforilase/deficiência , Purina-Núcleosídeo Fosforilase/genética , Imunodeficiência Combinada Severa/etiologia , Linfócitos T/metabolismo , Animais , Apoptose , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Citotoxicidade Imunológica , Camundongos , Camundongos Knockout , Subpopulações de Linfócitos T/metabolismo , Timo/citologiaRESUMO
Inactivating mutations in the PTEN tumor suppressor gene, encoding a phosphatase, occur in three related human autosomal dominant disorders characterized by tumor susceptibility. Here it is shown that Pten heterozygous (Pten+/-) mutants develop a lethal polyclonal autoimmune disorder with features reminiscent of those observed in Fas-deficient mutants. Fas-mediated apoptosis was impaired in Pten+/- mice, and T lymphocytes from these mice show reduced activation-induced cell death and increased proliferation upon activation. Phosphatidylinositol (PI) 3-kinase inhibitors restored Fas responsiveness in Pten+/- cells. These results indicate that Pten is an essential mediator of the Fas response and a repressor of autoimmunity and thus implicate the PI 3-kinase/Akt pathway in Fas-mediated apoptosis.
Assuntos
Apoptose , Doenças Autoimunes/imunologia , Nefropatias/imunologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor , Receptor fas/fisiologia , Animais , Anticorpos Antinucleares/sangue , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Heterozigoto , Imunoglobulina G/sangue , Nefropatias/patologia , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.
Assuntos
Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas/genética , Supressão Genética , Proteínas ras/genética , Células 3T3 , Animais , Gentamicinas/farmacologia , Humanos , Camundongos , Proteína Supressora de Tumor p14ARFRESUMO
ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We performed Northern blot experiments using sequences from ERV9-LTR, and we observed a different pattern of expression in several different hemopoietic tumor cell lines. It is possible that by the result of a somatic integration event, or by virtue of their original dispersal in the genome, ERV9-LTRs may specifically induce the expression of different cellular sequences in different cell lineages. Here, we describe the identification and analysis of four chimeric cDNA clones isolated from the T-lymphoma Peer cell line, having a structure consistent with transcription initiation from an ERV9-LTR. All the cDNA clones represent transcripts derived from unique cellular sequences. We also report the genomic localization of these cDNA clones.
Assuntos
Genes Reguladores/genética , Genes Virais , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Sequência de Bases , Quimera , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
It is well known that thyroid disease is more frequent in women than in men; however, the molecular basis for this gender-based difference is still poorly understood. The activation of phosphoinositide 3-kinase (PI3K), through different mechanisms including loss of the PTEN tumor suppressor, is being increasingly recognized as a major player in the development of thyroid neoplastic lesions. Loss of Pten in the mouse thyroid results in a significant increase in the thyrocyte proliferative index, which is more prominent in the female mice. In this study, we show that 52% of the Pten(-/-) female mice, but only 12% of the males, develop follicular adenomas by 1 year of age. In addition, 50% of female mutants, but only 35% of males older than 1 year of age develop invasive, and often metastatic, follicular carcinomas. Mutant females have a significantly shorter overall survival compared with male mutants. Hormonal manipulation experiments established a direct role of estrogens in controlling the increased thyrocyte proliferation index in mutant females. Furthermore, while genetic ablation of one Cdkn1b allele accelerated the development of neoplastic lesions, it also abolished the gender differences in survival and reduced the difference in neoplastic lesion development rate, underlining a key role of p27 in mediating estrogen action in the thyroid follicular cells. These data, based on a clinically relevant model of thyroid follicular carcinoma, provide, to the best of our knowledge, for the first time in vivo evidence that circulating estrogens are directly responsible for the increased female susceptibility to thyroid disease, at least on activation of the PI3K pathway, and provide new insights into the gender-based differences characterizing thyroid neoplastic disorders.
Assuntos
Adenocarcinoma Folicular/metabolismo , Estrogênios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor Cross-Talk/fisiologia , Caracteres Sexuais , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Folicular/genética , Adenocarcinoma Folicular/patologia , Animais , Western Blotting , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Incidência , Masculino , Camundongos , Camundongos Mutantes , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
The human genome contains a variety of genetic elements similar in structure to retroviruses and retrotransposons. We report here the structural and functional organization of a novel human endogenous retroviral family (ERV9). Three polyadenylated RNAs, 8, 2, and 1.5 kb long, are detected by Northern blot in undifferentiated embryonal carcinoma NT2/D1 cells. Upon genomic cloning of an expressed ERV9 locus, we demonstrated that the three polyadenylated RNAs are originated by a single ERV9 locus by alternative usage of splicing and polyadenylation signals. DNA sequence analysis of different ERV9 LTRs have revealed that they are heterogeneous in length and that the length variability is due to the number of tandemly repeated subelements present in both U3 and U5 regions; moreover, the ERV9 LTRs are capable to drive expression of a reporter gene in transient expression assays. Finally, analysis of the ERV9 5' transcription start site has allowed us to define the U3-R-U5 organization of the ERV9 LTR.
Assuntos
Retroviridae/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Viral , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. In this report we have analyzed the minimal promoter region located within the ERV9 LTR. Using the transient CAT expression assay we have identified the minimal promoter region, which includes sequences spanning from -70 to +6 relative to the major transcription start site. Deletion analysis, primer extension mapping of the transcription start sites and DNA-protein interactions assays have allowed us to define two important regions within the ERV9 minimal promoter. One region located between -70 to -39 acts as a transcriptional activating sequence and contains an Sp 1 binding site. The second region from -7 to +6, which resembles an initiator element (Inr), was necessary for the correct transcription start site utilization, and binds to a regulatory protein. Cross-competition experiments using various Inr elements have indicated that the protein that binds to the ERV9 Inr element can be competed by the HIV-1 and TdT Inr sequences.
Assuntos
Regulação Viral da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Provírus/genética , Retroviridae/genética , Transcrição Gênica/genética , Sequência de Bases , Sítios de Ligação/genética , Células-Tronco de Carcinoma Embrionário , Humanos , Dados de Sequência Molecular , Células-Tronco Neoplásicas , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We report here the structural organization in different primate species of a zinc-finger coding gene whose expression is driven in humans by a solitary ERV9-LTR promoter. Using a PCR strategy and library screening, we were able to trace the origin of the insertion event in the primate lineage and to evaluate the impact of this event on gene structure. Our findings indicate that the integration of the ERV9 element occurred after the split of orangutang from the great apes, but before the divergence of the gorilla lineage. These results suggest that ERV9 elements have been mobile within the primate lineages and may still be active in humans.
Assuntos
Evolução Biológica , Genes Reguladores/genética , Primatas/genética , Retroviridae/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , DNA Viral , Gorilla gorilla , Humanos , Macaca mulatta , Dados de Sequência Molecular , Pan troglodytes , Reação em Cadeia da Polimerase , Pongo pygmaeus , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We describe here the structure of the ZNF80 cDNA clone putatively coding for a zinc-finger protein, whose 5' terminus starts from within an ERV9-LTR. Characterization of the single copy genomic locus indicates that a complete ERV9-LTR element is present upstream of the ZNF80 coding region and that this element acts as a functional promoter in both in vivo and in vitro experiments. A 2.6 kb long transcript is selectively expressed only in some hematopoietic cell lineages. Interestingly we mapped the ZNF80 locus to the 3q13.3 band, a region involved in karyotype rearrangements associated with myelocytic disorders. We have also analyzed the ZNF80 genomic organization in African green monkey and we show that this lower primate does not harbour an ERV9 element at this locus. Our findings strongly suggest that the expression of a zinc finger gene, which is highly conserved during evolution of primates, is regulated in humans by an LTR element of the ERV9 family.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Retroviridae/genética , Dedos de Zinco/genética , Animais , Sequência de Bases , Células Cultivadas , Chlorocebus aethiops , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , Clonagem Molecular , Proteínas de Ligação a DNA/química , Genes/genética , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Retroviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , DNA de Neoplasias/análise , DNA de Neoplasias/genética , DNA Viral/análise , DNA Viral/genética , Células-Tronco de Carcinoma Embrionário , Genes Virais , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Regiões Promotoras Genéticas , RNA Neoplásico/análise , RNA Neoplásico/genética , RNA Viral/análise , RNA Viral/genética , Mapeamento por Restrição , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag, pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3-->q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages.
Assuntos
Cromossomos Humanos/genética , Retrovirus Endógenos/genética , Regulação Viral da Expressão Gênica , Mapeamento de Híbridos Radioativos , Animais , Southern Blotting , Cromossomos Humanos Par 11/genética , Cricetinae , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Integração Viral/genéticaRESUMO
The PTEN tumor suppressor is mutated in diverse human cancers and in hereditary cancer predisposition syndromes. PTEN is a phosphatase that can act on both polypeptide and phosphoinositide substrates in vitro. The PTEN structure reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTEN's membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane.
Assuntos
Genes Supressores de Tumor , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans , Gráficos por Computador , Cristalografia por Raios X/métodos , Drosophila , Humanos , Modelos Moleculares , Dados de Sequência Molecular , PTEN Fosfo-Hidrolase , Fosfatidilinositóis/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210(bcr-abl), a chimeric protein with tyrosine kinase activity. Substrates for p210(bcr-abl) are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56(dok-2) (Dok-2), from p210(bcr-abl) expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120(RasGAP). Dok-2 was shown to be 35% identical to p62(dok-1), a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210(bcr-abl) in CML.