RESUMO
Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Assuntos
Junções Comunicantes/fisiologia , Óxido Nítrico/metabolismo , Animais , Aterosclerose/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Conexinas/metabolismo , GMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Junções Comunicantes/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão/metabolismo , Camundongos , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/crescimento & desenvolvimento , Genes de Helmintos , Genes Supressores de Tumor , Proteínas de Helminto/fisiologia , Fosfatidilinositol 3-Quinases , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Animais , Caenorhabditis elegans/genética , Catálise , DNA Complementar/genética , Proteínas de Helminto/genética , Humanos , Larva/crescimento & desenvolvimento , Longevidade/genética , Lipídeos de Membrana/metabolismo , Família Multigênica , PTEN Fosfo-Hidrolase , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Receptor de Insulina/genética , Receptor de Insulina/fisiologiaRESUMO
Germline mutations in the RET proto-oncogene are responsible for two unrelated neural crest disorders: Hirschsprung disease, a congenital absence of the enteric nervous system in the hindgut, and multiple endocrine neoplasia type 2, a dominantly inherited cancer syndrome. Moreover, somatic rearrangements of RET are causally involved in the genesis of papillary thyroid carcinoma. The receptor tyrosine kinase encoded by the RET gene acts as the subunit of a multimolecular complex that binds four distinct ligands and activates a signalling network crucial for neural and kidney development. Over the past few years, a clearer picture of the mode of RET activation and of its multifaceted role during development has started to emerge. These findings, which provide new clues to the molecular mechanisms underlying RET signalling dysfunction in Hirschsprung disease, are summarized in this review.
Assuntos
Proteínas de Drosophila , Doença de Hirschsprung/genética , Fatores de Crescimento Neural , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Apoptose , Sistema Nervoso Entérico/anormalidades , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Doença de Hirschsprung/etiologia , Doença de Hirschsprung/fisiopatologia , Humanos , Ligantes , Microdomínios da Membrana/fisiologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/fisiologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Transdução de SinaisRESUMO
Hirschsprung's disease (HSCR) is a common congenital malformation characterized by the absence of intramural ganglion cells of the hindgut. Recently, mutations of the RET tyrosine kinase receptor have been identified in 50 and 15-20% of familial and sporadic HSCR, respectively. These mutations include deletion, insertion, frameshift, nonsense, and missense mutations dispersed throughout the RET coding sequence. To investigate their effects on RET function, seven HSCR missense mutations were introduced into either a 1114-amino acid wild-type RET isoform (RET51) or a constitutively activated form of RET51 (RET-MEN 2A). Here, we report that one mutation affecting the extracytoplasmic cadherin domain (R231H) and two mutations located in the tyrosine kinase domain (K907E, E921K) impaired the biological activity of RET-MEN 2A when tested in Rat1 fibroblasts and pheochromocytoma PC12 cells. However, the mechanisms resulting in RET inactivation differed since the receptor bearing R231H extracellular mutation resulted in an absent RET protein at the cell surface while the E921K mutation located within the catalytic domain abolished its enzymatic activity. In contrast, three mutations mapping into the intracytoplasmic domain neither modified the transforming capacity of RET-MEN 2A nor stimulated the catalytic activity of RET in our ligand-independent system (S767R, P1039L, M1064T). Finally, the C609W HSCR mutation exerts a dual effect on RET since it leads to a decrease of the receptor at the cell surface and converted RET51 into a constitutively activated kinase due to the formation of disulfide-linked homodimers. Taken together, our data show that allelic heterogeneity at the RET locus in HSCR is associated with various molecular mechanisms responsible for RET dysfunction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Drosophila , Doença de Hirschsprung/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Células 3T3 , Alelos , Animais , Biotinilação , Caderinas/genética , Fibroblastos , Expressão Gênica , Doença de Hirschsprung/metabolismo , Humanos , Técnicas Imunoenzimáticas , Proteínas de Membrana/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transformação Genética , Células Tumorais CultivadasRESUMO
The receptor tyrosine kinase RET functions as the signal transducing receptor for the GDNF (for "glial cell-derived neurotrophic factors") family of ligands. Mutations in the RET gene were implicated in Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN 2), and thyroid carcinomas. In this report we demonstrate that the docking protein FRS2 is tyrosine phosphorylated by ligand-stimulated and by constitutively activated oncogenic forms of RET. Complex formation between RET and FRS2 is mediated by binding of the phosphotyrosine-binding domain of FRS2 to pY1062, a residue in RET that also functions as a binding site for Shc. However, overexpression of FRS2 but not Shc potentiates mitogen-activated protein (MAP) kinase activation by RET oncoproteins. We demonstrate that oncogenic RET-PTC proteins are associated with FRS2 constitutively, leading to tyrosine phosphorylation of FRS2, MAP kinase stimulation, and cell proliferation. However, loss-of-function HSCR-associated RET mutants exhibit impaired FRS2 binding and reduced MAP kinase activation. These experiments demonstrate that FRS2 couples both ligand-regulated and oncogenic forms of RET, with the MAP kinase signaling cascade as part of the response of RET under normal biological conditions and pathological conditions, such as MEN 2 and papillary thyroid carcinomas.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Drosophila , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Genes Reporter/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Doença de Hirschsprung/genética , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
The RET tyrosine kinase is a functional receptor for neurotrophic ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Loss of function of RET is associated with congenital megacolon or Hirschsprung's disease, whereas germ-line point mutations causing RET activation are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and familial medullary thyroid carcinoma) syndromes. Here we show that the expression of a constitutively active RET-MEN2A oncogene promotes survival of rat pheochromocytoma PC12 cells upon growth factor withdrawal. Moreover, we show that the RET-MEN2A-mediated survival depends on signals transduced by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Thus, in PC12 cells, RET-MEN2A associates with the PI3K regulatory subunit p85 and promotes activation of Akt (also referred to as protein kinase B) in a PI3K-dependent fashion; in addition, RET-MEN2A promotes MAPK activation. PI3K recruitment and Akt activation as well as MAPK activation depend on RET-MEN2A tyrosine residue 1062. As a result, tyrosine 1062 of RET-MEN2A is essential for RET-MEN2A-mediated survival of PC12 cells cultured in growth factor-depleted media.
Assuntos
Proteínas de Drosophila , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Cromonas/farmacologia , Meios de Cultura Livres de Soro , Fragmentação do DNA , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Marcação In Situ das Extremidades Cortadas , Ligantes , Morfolinas/farmacologia , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Testes de Precipitina , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/química , Transdução de Sinais , TransfecçãoRESUMO
Distinct point mutations of RET, a tyrosine-kinase receptor encoding gene, are responsible for the inheritance of multiple endocrine neoplasia type 2 syndromes (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In particular, MEN2A is a more complex and aggressive disease than FMTC, being characterized by pheochromocytomas and parathyroid alterations, in addition to medullary thyroid carcinomas. The mutations associated with MEN2A and FMTC affect one of five cysteine residues mapping in the extracellular domain of the Ret protein. However, recent studies have indicated that MEN2A and FMTC disease phenotypes correlate with the position of mutations in RET. Mutations of Cys-634 are more frequent in families with MEN2A, whereas Cys-620 mutations are very rarely found in MEN2A patients and, in contrast, are frequently found in FMTC patients. We have reported previously that mutations of Cys-634 constitutively activate the RET transforming potential by causing a disulfide bridge-mediated homodimerization. Here, we report that the mutation Cys-620 --> Tyr is able to cause a constitutive dimerization of Ret, with consequent activation of its kinase and transforming activities, to a lower extent than mutation of Cys-634. We suggest that the difference in ability to activate RET shown by mutations associated with FMTC and MEN2A represents the molecular basis of the phenotypic diversity between the two syndromes.
Assuntos
Proteínas de Drosophila , Regulação Neoplásica da Expressão Gênica , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Células 3T3 , Animais , Camundongos , Peso Molecular , Células PC12 , Fenótipo , Proteínas Proto-Oncogênicas c-ret , RatosRESUMO
Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprung's disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.
Assuntos
Proteínas de Drosophila , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células 3T3 , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Proteínas Substratos do Receptor de Insulina , Camundongos , Mutação , Fosfoproteínas/genética , Fosforilação , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-ret , Proteínas de Ligação a RNA/metabolismo , Receptores Proteína Tirosina Quinases/genética , Ribonucleoproteínas/metabolismoRESUMO
The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.
Assuntos
Substituição de Aminoácidos , Cisteína/metabolismo , Proteínas de Drosophila , Neoplasia Endócrina Múltipla Tipo 2a/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células 3T3 , Animais , Transformação Celular Neoplásica , Cisteína/genética , Dimerização , Dissulfetos , Glicosilação , Doença de Hirschsprung/enzimologia , Doença de Hirschsprung/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Biológicos , Neoplasia Endócrina Múltipla Tipo 2a/enzimologia , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Células PC12 , Fosfotirosina/metabolismo , Testes de Precipitina , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Ativação TranscricionalRESUMO
Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two dominantly inherited disorders caused by germline mutations of the RET proto-oncogene. The RET gene codes for a receptor tyrosine kinase. The majority of MEN2A and FMTC mutations are clustered in the extra-cellular cysteine-rich domain and result in constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers. Recently, two novel point mutations have been identified in the germline of five distinct FMTC families. Both mutations occur within the catalytic domain of the RET kinase and lead to the substitution of either glutamic acid 768 or valine 804 by an aspartic acid and a leucine respectively. We have introduced each FMTC mutation in two RET isoforms: RET51 the long isoform (1114 aa) and RET9 the short isoform (1072 aa) which differ in the C-terminal region of the protein. The RET51 isoform carrying either E768D or V804L mutation was autophosphorylated, displayed a transforming activity upon expression in Rat1 fibroblasts and induced neuronal differentiation of PC12 cells. However, the transforming capacity of these RET51-FMTC mutants was found to be severalfold less potent compared to the same isoform carrying either the MEN2A mutation (C634R) or the MEN2B mutation (M918T). In contrast, RET9 containing mutations E768D or V804L was not autophosphorylated, exhibited a poor oncogenic potential in fibroblasts and did not promote neuritic outgrowth upon expression in PC12 cells. Overall, these findings demonstrate that mutations E768D and V804L are gain-of-function mutations that confer to the long RET isoform the capacity to exert a biological effect, although these mutations are more weakly activating than the MEN2A and MEN2B mutations. These results may provide a biochemical basis as to why the phenotypic consequences of these mutations are restricted to thyroid C-cells.
Assuntos
Carcinoma Medular/genética , Transformação Celular Neoplásica , Proteínas de Drosophila , Mutação Puntual , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias da Glândula Tireoide/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Diferenciação Celular , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Células PC12 , Proteínas Proto-Oncogênicas c-ret , RatosRESUMO
Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.
Assuntos
Proteínas de Drosophila , Mutação em Linhagem Germinativa/genética , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2b/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Diferenciação Celular , Transformação Celular Neoplásica , Ativação Enzimática , Vetores Genéticos/genética , Humanos , Neuritos/patologia , Feocromocitoma/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ret , Proto-Oncogenes/fisiologia , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Retroviridae/genética , TransfecçãoRESUMO
The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Processamento Alternativo , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Proteínas Serina-Treonina Quinases/químicaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) has potentially great clinical importance in the treatment of Parkinson's disease and several other neurodegenerative diseases, however its intracellular signaling mechanisms are poorly understood. Here we show that upon GDNF binding glycosyl-phosphatidylinositol (GPI)-linked GDNF receptor alpha1 (GFRalpha1) activates cytoplasmic Src family tyrosine kinase(s) in Ret tyrosine kinase-deficient cultured mouse dorsal root ganglion neurons and in two Ret-negative cell lines. GFRalpha1-mediated Src-type kinase activation subsequently triggers phosphorylation of mitogen-activated protein kinase, cAMP response element binding protein and phospholipase Cgamma. We therefore conclude that GDNF can activate intracellular signaling pathways Ret-independently via GPI-linked GFRalpha1.
Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Células 3T3 , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Gânglios Espinais/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Estabilidade Enzimática , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The RET proto-oncogene encodes a receptor tyrosine kinase which plays a crucial role during the embryonic development of the enteric nervous system and of the kidney. Cytogenetic analyses of papillary thyroid carcinoma (PTC), a neoplasm which originates from thyrocytes, have revealed that somatic rearrangements of the RET gene are involved in the etiology of a significant proportion of this tumour. Medullary thyroid carcinoma (MTC) which arises from neural-crest derived C-cells is the cardinal disease feature of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer syndrome. Recent studies have provided evidence that germline mutations of the RET gene are the underlying genetic events responsible for MEN 2. This review focuses on the role of RET mutations in the pathogenesis of PTC and MTC and summarizes our present knowledge on the consequences of these alterations on the RET tyrosine kinase function. We further describe a transgenic mouse model for hereditary MTC. Mice carrying a MEN 2A allele of RET under the control of the CGRP/calcitonin promoter develop bilateral and multifocal MTC, morphologically and biologically similar to human MTC.
Assuntos
Proteínas de Drosophila , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Carcinoma Medular/genética , Carcinoma Medular/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Humanos , Camundongos , Neoplasia Endócrina Múltipla Tipo 2a/enzimologia , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.
Assuntos
Transformação Celular Neoplásica , Proteínas de Drosophila , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Neoplasia Endócrina Múltipla Tipo 2a/patologia , Mutação de Sentido Incorreto , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
In vitro infection of EBV-negative lymphoma cell lines with immortalizing strains of Epstein-Barr virus induces the cell-surface expression of B-cell markers, such as the EBV receptor/CR2 (CD21) and the CD23 antigen. The non-immortalizing EBV variant, P3HRI, which carries a deletion encompassing the EBV nuclear antigen 2 (EBNA2) gene, fails to induce any such expression. We show here that the EBV-mediated up-regulation of cell-surface expression of these molecules is associated with an increased level of the specific steady-state RNA corresponding to these 2 genes. These results suggest that the role of EBNA2 in B-cell growth and immortalization may be related to its role in transactivation of cellular genes. In order to identify other cellular genes whose expression may be modulated by EBV, we analyzed the level of transcription of a set of genes possibly involved in Burkitt's lymphoma pathogenesis. The level of the c-myc oncogene transcript was not significantly affected by in vitro EBV infection. The c-fgr oncogene, thought to be specifically activated in EBV-infected cells, was found to be expressed in some EBV-negative lymphoma cells and also to be activated by both non-immortalizing and immortalizing strains of EBV. The expression of vimentin, the major 56-kDa polypeptide of mesenchymal cell intermediate filaments, was altered by all EBV isolates, in either a negative or a positive way, depending on the cell line. Expression of lymphocyte-function-associated antigens, LFA-1 alpha/beta (CD11 a/18) and LFA-3 (CD58), involved in intercellular adhesion and the T-cytotoxic pathway, were differentially regulated by EBV; a crucial observation was the activation by immortalizing EBV isolates of LFA-1 beta chain (CD18) and of LFA-3 (CD58). The EBV-and possibly EBNA2-associated modulation of cellular genes, such as CR2 (CD21), CD23 and LFAs, probably represents key events for EBV-induced B-cell proliferation, and also for in vivo immune control of EBV-infected B cells.
Assuntos
Antígenos de Superfície/biossíntese , Linfócitos B/metabolismo , Linfoma de Burkitt/metabolismo , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/patogenicidade , Antígenos CD/biossíntese , Biomarcadores , Northern Blotting , Linfoma de Burkitt/genética , Antígenos CD58 , Moléculas de Adesão Celular/biossíntese , Genes myc , Humanos , Técnicas In Vitro , Molécula 1 de Adesão Intercelular , Antígeno-1 Associado à Função Linfocitária/biossíntese , Glicoproteínas de Membrana/biossíntese , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA/biossíntese , Transcrição Gênica , Regulação para Cima , Vimentina/biossíntese , Quinases da Família srcRESUMO
Myc proteins are basic helix-loop-helix/leucine-zipper proteins that bind to specific DNA sequences. In vivo, Myc proteins have been found associated with Max, another basic helix-loop-helix/leucine-zipper protein. However, it is not known to what extent the dimerization of Myc with Max is required for the manifestation of the Myc-induced phenotype. To investigate this, we constructed a dominant-negative mutant of Max, named dMax, that inhibits sequence-specific DNA binding of Myc proteins. Using a rat neuroblastoma model system, we show that dMax reverts N-Myc-induced changes in cellular gene expression. A control mutant of dMax that contains a proline residue in the leucine-zipper region was unable to bind to N-Myc and did not revert the N-Myc-induced changes in cellular gene expression. These data support the hypothesis that N-Myc affects neuroblastoma gene expression through the formation of a DNA-binding heterodimeric complex with Max in vivo.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes myc , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Clonagem Molecular , Genes Dominantes , Dados de Sequência Molecular , Mutagênese , Neuroblastoma , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Deleção de Sequência , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Multiple endocrine neoplasia type 2 (MEN2) and Hirschsprung's disease (HSCR) are two dominantly inherited neurocristopathies ascribed to mutations in the RET gene [Chakravarti, 1996; Pasini et al., 1996; Eng and Mulligan, 1997]. MEN2 is a cancer syndrome comprising three related clinical subtypes: (1) MEN type 2A (MEN2A; MIM# 171400) characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo), and hyperparathyroidism; (2) MEN type 2B (MEN2B; MIM# 162300), which includes MTC, Pheo, mucosal neuromas, ganglioneuromatosis of the digestive tract, and skeletal abnormalities; and (3) familial MTC (FMTC; MIM# 155240), defined by the sole occurrence of MTC. HSCR (MIM# 142623) is a congenital malformation caused by the absence of enteric plexuses in the hindgut, leading to bowel obstruction in neonates. The RET gene (MIM# 164761) codes for a transmembrane tyrosine kinase, a component of a multimeric complex that also comprises one of four members of a novel family of glycosylphosphatidylinositol (GPI)-anchored receptor, GFRalpha((1-4) (e.g., GFRA1, MIM# 601496; references are detailed in Baloh et al. [1998]. Four structurally related soluble factors-glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin-are the ligands of these multimolecular receptors in which the nature of the GFRalpha determines the ligand specificity of the complex [see Baloh et al., 1998, for references]. It is well documented that RET/GFRalpha-1/GDNF delivers a signal critical for the survival of the early neural crest-derived precursors that colonize the intestine below the rostral foregut and give rise to the enteric nervous plexuses [Gershon, 1997; Cacalano et al., 1998; Enomoto et al., 1998].