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1.
EMBO J ; 20(16): 4349-59, 2001 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-11500362

RESUMEN

Eukaryotic initiation factor (eIF) 2B is a heteromeric guanine nucleotide exchange factor that plays an important role in regulating mRNA translation. Here we identify multiple phosphorylation sites in the largest, catalytic, subunit (epsilon) of mammalian eIF2B. These sites are phosphorylated by four different protein kinases. Two conserved sites (Ser712/713) are phosphorylated by casein kinase 2. They lie at the extreme C-terminus and are required for the interaction of eIF2Bepsilon with its substrate, eIF2, in vivo and for eIF2B activity in vitro. Glycogen synthase kinase 3 (GSK3) is responsible for phosphorylating Ser535. This regulatory phosphorylation event requires both the fourth site (Ser539) and a distal region, which acts to recruit GSK3 to eIF2Bepsilon in vivo. The fifth site, which lies outside the catalytic domain of eIF2Bepsilon, can be phosphorylated by casein kinase 1. All five sites are phosphorylated in the eIF2B complex in vivo.


Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa de la Caseína II , Línea Celular , Factor 2B Eucariótico de Iniciación/genética , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
2.
Curr Biol ; 11(1): 55-9, 2001 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-11166181

RESUMEN

GTP hydrolysis occurs at several specific stages during the initiation, elongation, and termination stages of mRNA translation. However, it is unclear how GTP hydrolysis occurs; it has previously been suggested to involve a GTPase active center in the ribosome, although proof for this is lacking. Alternatively, it could involve the translation factors themselves, e.g., be similar to the situation for small G in which the GTPase active site involves arginine residues contributed by a further protein termed a GTPase-activator protein (GAP). During translation initiation in eukaryotes, initiation factor eIF5 is required for hydrolysis of GTP bound to eIF2 (the protein which brings the initiator Met-tRNA(i) to the 40S subunit). Here we show that eIF5 displays the hallmarks of a classical GAP (e.g., RasGAP). Firstly, its interaction with eIF2 is enhanced by AlF(4)(-). Secondly, eIF5 possesses a conserved arginine (Arg15) which, like the "arginine fingers" of classical GAPs, is flanked by hydrophobic residues. Mutation of Arg15 to methionine abolishes the ability of eIF5 either to stimulate GTP hydrolysis or to support mRNA translation in vitro. Mutation studies suggest that a second conserved arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex. Our data thus show that eIF5 behaves as a classical GAP and that GTP hydrolysis during translation involves proteins extrinsic to the ribosome. Indeed, inspection of their sequences suggests that other translation factors may also act as GAPs.


Asunto(s)
GTP Fosfohidrolasas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Secuencia de Aminoácidos , Activación Enzimática , Factor 5 Eucariótico de Iniciación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/genética , Homología de Secuencia de Aminoácido
3.
Oncogene ; 19(38): 4437-40, 2000 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-10980620

RESUMEN

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) (Nanbru et al., 1997; Stoneley et al., 1998) and thus c-myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism (Stoneley et al., 2000). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c-myc and this correlates with a C-T mutation in the c-myc-IRES (Paulin et al., 1996). RNA derived from the mutant IRES displays enhanced binding of protein factors (Paulin et al., 1998). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c-myc-IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c-myc-IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.


Asunto(s)
Mieloma Múltiple/genética , Mutación Puntual , Proteínas Proto-Oncogénicas c-myc/genética , Secuencias Reguladoras de Ácidos Nucleicos , Ribosomas , Regiones no Traducidas 5' , Secuencia de Bases , Médula Ósea/fisiología , Línea Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Nucleic Acids Res ; 26(13): 3097-103, 1998 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-9628905

RESUMEN

A 340 nucleotide section of the c- myc 5' untranslated region (UTR) contains an internal ribosome entry segment. We have described previously a mutation in this region of RNA in cell lines derived from patients with multiple myeloma (MM) which exhibit increased expression of c- myc protein by an aberrant translational mechanism. In this study we show by electrophoretic mobility shift assays (EMSA), north-western blotting and UV cross-linking that radiolabelled c- myc 5' UTR RNA transcripts which harbour the mutation cause enhanced binding of cellular proteins. In addition, we also demonstrate that an MM derived cell line possesses an altered repertoire of RNA binding proteins. Our data suggest that the deregulated expression of c -myc in MM could result both from the effect of the mutation and the additional proteins which are present in these cell types.


Asunto(s)
Genes myc , Mutación Puntual , Ribosomas/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Citoplasma/metabolismo , Cartilla de ADN , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/efectos de la radiación , Rayos Ultravioleta
5.
Oncogene ; 16(3): 423-8, 1998 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-9467968

RESUMEN

Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5' end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure and in this case ribosomes are directed to the start codon by an internal ribosome entry segment (IRES). Picornaviruses represent the paradigm for this mechanism, but only a few examples exist which show that this mechanism is used by eukaryotic cells. In this report we show data which demonstrate that the 5' UTR of the proto-oncogene c-myc contains an IRES. When a dicistronic reporter vector, with c-myc 5' UTR inserted between the two cistrons, was transfected into both HepG2 and HeLa cells, the translation of the downstream cistron was increased by 50-fold, demonstrating that translational regulation of c-myc is mediated through cap-independent mechanisms. This is the first example of a proto-oncogene regulated in this manner and suggests that aberrant translational regulation of c-myc is likely to play a role in tumorigenesis.


Asunto(s)
Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Ribosomas/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Células Tumorales Cultivadas
6.
Curr Top Microbiol Immunol ; 224: 269-76, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9308251

RESUMEN

In cell lines derived from patients with multiple myeloma (MM) we have found an elevation in the amount of the c-myc protein which is not accompanied by an increase in the level of mRNA or a change in the half-life of the protein. There is a 3.4 fold enhancement in the degree of association of the c-myc message with polysomes. This is not accompanied by an alteration in polysome size or a change in the transit time of the c-myc mRNA on the polysomes thus suggesting that there is in increase in the degree of mobilisation of the c-myc message. Sequencing of the c-myc 5'UTR has revealed the presence of a mutation in all the MM cell lines studied and we demonstrate that this mutation causes altered binding of cellular proteins to this RNA species.


Asunto(s)
Genes myc , Mieloma Múltiple/genética , Secuencia de Bases , ADN de Neoplasias/genética , Humanos , Mutación Puntual , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Células Tumorales Cultivadas
7.
Oncogene ; 13(3): 505-13, 1996 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-8760292

RESUMEN

We demonstrate a 10- to 25-fold increase in the amount of c-myc protein in several independent cell lines derived from patients with multiple myeloma (MM). This is not accompanied by a corresponding increase in the overall level of the c-myc mRNA. There is, however, a 3.4-fold increase in the amount of c-myc mRNA associated with the polysomes in these cell lines without any detectable change in either the polysome size or the rate of translation elongation, thus suggesting that there is an increase in the extent of mobilisation of c-myc mRNA to the polysomes in MM. Analysis of the 5' untranslated region of c-myc has revealed the presence of a mutation, in all of the MM cell lines examined, in a region which has been implicated previously in the translational control of this mRNA species. These data suggest aberrant translational control of the c-myc gene in cell lines derived from patients with MM, which may contribute towards pathogenesis of the disease.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes myc , Mieloma Múltiple/metabolismo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Exones , Humanos , Mieloma Múltiple/genética , Mutación , Polirribosomas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas
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