Asunto(s)
Proteínas de Ciclo Celular , Proteínas Asociadas a Microtúbulos/fisiología , Transducción de Señal , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor , Animales , Caenorhabditis elegans , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Factores de Transcripción ForkheadRESUMEN
Transcription factors of the Myc/Max/Mad network affect multiple aspects of cellular behavior, including proliferation, differentiation, and apoptosis. Recent studies have shown that Mad proteins can inhibit cellular growth and transformation and thus antagonize the function of Myc proteins. To define further the contribution of these proteins to cellular growth control, we have studied the expression of the respective genes and proteins in 3T3-L1 cells, both upon serum stimulation of quiescent cells and during adipocytic differentiation in response to insulin, dexamethasone, and isobutylmethylxanthine. We found distinct expression patterns for the mad genes. Mad4 was induced when cells exit the cell cycle and, together with mad1, during the late phase of differentiation. In contrast, mad3 expression was associated with progression through S phase and the proliferative burst of differentiating preadipocytes, overlapping in part c-myc expression. DNA binding analyses revealed that the most prominent network complex both in cycling and in differentiating cells was Mnt/Max, whereas c-Myc/Max complexes were detectable only during peak c-Myc expression periods. Ectopic expression of Mad1 in preadipocytes resulted in the inhibition of S phase and the proliferation associated with the proliferative burst; as a consequence, adipocytic differentiation was significantly inhibited. Our findings suggest that the precise temporal regulation of Myc/Max/Mad network proteins is critical for determining cellular behavior.
Asunto(s)
Adipocitos/citología , Proteínas Portadoras , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células COS , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Dexametasona/farmacología , Humanos , Insulina/farmacología , Ratones , Fosfoproteínas/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , TransfecciónAsunto(s)
Proteínas Portadoras , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas I-kappa B , Proteínas Represoras/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Ciclo Celular , Proteínas de Ciclo Celular , División Celular , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de SecuenciaRESUMEN
Irradiation of mammalian cells with ultraviolet light (200-400 nm) results in the activation of a number of genes, the so-called "UV response" (Herrlich et al., Rev. Physiol. Biochem. Pharmacol., 119:187-223, 1992). Many of the UV responsive genes are also transcriptionally activated by growth factors and mitogens. Two transcription factors have been demonstrated to acutely respond to these stimuli, namely AP-1 and NF-kappa B. Whereas NF-kappa B proteins are primarily controlled via proteolysis of regulatory domains which prevent nuclear translocation, the AP-1 proteins are regulated at several levels including transcription and posttranslational modification. Here, we discuss progress in the identification of the components of pathways acutely regulating these transcription factors.
Asunto(s)
Proteínas Proto-Oncogénicas c-jun/fisiología , Transducción de Señal/fisiología , Activación Transcripcional , Animales , Línea Celular , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas c-jun/genética , Activación Transcripcional/efectos de la radiación , Rayos UltravioletaRESUMEN
c-Myc is a nuclear phosphoprotein which binds DNA as a heterodimer with Max. We have identified two in vivo phosphorylation sites, Thr58 and Ser62, within a domain highly conserved among all Myc family members. Thr58 is mutated in several viral forms of the protein and constitutes a mutational hot-spot in Burkitt's lymphoma. Members of the GSK-3 and MAP kinase families, but not CKII, specifically phosphorylated these sites in vitro. The effect of these phosphorylation sites on Myc function was assessed by cotransformation of primary rat embryo fibroblasts with Ras. Mutagenesis of Thr58 to alanine potentiated focus formation, whereas substitution of Ser62 severely inhibited transformation. Mutation of both residues restored wild-type activity. These data suggest acute, post-translational modulation of Myc via phosphorylation of a conserved region previously implicated in transactivation, transformation and autorepression.
Asunto(s)
Transformación Celular Neoplásica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Genes ras , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Ratas , Serina/metabolismo , Treonina/metabolismoAsunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Drosophila , Proteínas Asociadas a Microtúbulos/metabolismo , Transducción de Señal , Animales , Drosophila melanogaster/embriología , Drosophila melanogaster/fisiología , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Modelos Biológicos , Proteínas Quinasas/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Phorbol esters, such as phorbol myristate acetate (PMA), cause differentiation of U937 human monomyelocytic cells along the macrophage pathway. Within 15 min of PMA treatment DNA binding of the c-jun transcription factor is increased and is accompanied by rapid changes in the phosphate content of the c-jun protein. Phorbol esters stimulate phosphorylation of serines 63 and 73 located within the A1 transactivation domain of c-Jun that have previously been shown to positively regulate activity. A protein kinase activity is detectable in extracts of phorbol ester-treated U937 cells that specifically targets these two serines. Using novel assays, the protein kinase activity has been purified over 1000-fold. The major portion of protein kinase activity co-chromatographs over three columns with pp42/44 mitogen-activated protein kinases as judged by immunological methods. The significance of these results with respect to mitogen-induced transcription of AP-1-responsive genes is discussed.
Asunto(s)
Leucemia Mieloide/enzimología , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas c-jun/aislamiento & purificación , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Bases , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Fosforilación , Células Tumorales CultivadasAsunto(s)
Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Modelos Biológicos , Ésteres del Forbol/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Study of GSK-3 had an inauspicious beginning rooted in intermediary metabolism. However, owing to the fortuitous convergence of several disparate areas of biology, the enzyme now offers unique opportunities for study of the control of a variety cellular processes. While at first sight a role in transcriptional regulation appears unlikely for a protein first identified as acting on glycogen synthase, it is even more surprising that the same protein should be functionally interchangeable with a fruit fly homeotic gene. Such understandable scepticism, however, is based on teleological bias. Glycogen synthase is a critical enzyme regulating glucose storage. The c-Jun oncoprotein may have the potential to transform cells but this does not excuse it from similar mechanisms of control to glycogen synthase. Likewise, homeotic genes play a crucial role in setting up the body plan of an embryo but must also be subject to control. The main difference is that when such control is lost, the result is rather graphic. It is, therefore, only to be expected that regulatory protein kinases will surface in superficially quite unrelated areas and that many of their targets will be 'housekeeping' proteins. Perhaps the most difficult aspect of protein phosphorylation research is the linking of physiological substrates with particular protein kinases, hence reconstructing pathways. No matter how compelling in vitro data appear, there must be demonstration that the protein is targeted by the specific protein kinase in cells, an extremely difficult process. Most progress in this respect has been made using genetic analysis in lower organisms, especially yeast. Here another problem arises: demonstration of biochemical linkages underlying genetic interactions which requires function to be ascribed to genes identified by a gross effect. The challenge is to co-ordinate these two approaches, a strategy currently being employed to further unravel the biological role of GSK-3.
Asunto(s)
Proteínas Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Clonación Molecular , Drosophila melanogaster/embriología , Glucógeno Sintasa Quinasas , Mamíferos , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/metabolismo , Especificidad por SustratoRESUMEN
Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.
Asunto(s)
Baculoviridae/enzimología , Drosophila melanogaster/genética , Genes Homeobox , Isoenzimas/genética , Proteínas Quinasas/genética , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Cromatografía en Gel , ADN/genética , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glucógeno Sintasa Quinasas , Insectos/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfopéptidos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TransfecciónRESUMEN
The proto-oncogene c-jun is a component of the AP-1 transcription factor family involved in the mediation of nuclear events elicited by extracellular stimuli. The c-jun protein is negatively regulated by phosphorylation of residues near the carboxy terminus which are dephosphorylated in response to phorbol esters. Here we identify two serine residues in the amino terminal A1 transactivation domain which are phosphorylated in response to a variety of mitogens, phorbol esters and activated ras. We present evidence that mitogen-activated protein-serine (MAP) kinases (pp54 and pp42/44) specifically phosphorylate these sites and that their phosphorylation positively regulates the transacting activity of c-jun. The MAP kinase enzymes pp54 and pp42/44 are regulated by tyrosine as well as serine/threonine phosphorylation. MAP kinase activation of c-jun may underlie the common stimulation of this transcription factor by mitogens, growth factors and oncogenes.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/metabolismo , Ésteres del Forbol/farmacología , Fosforilación , Fosfoserina/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Activación Transcripcional , Transfección , TripsinaRESUMEN
Lytic infection with herpes simplex virus (HSV) results in the repression of most host cell protein synthesis but produces an increased activity of the cellular AP-1 transcription factor. This increase is paralleled by an increase in the transcription rate of the proto-oncogene encoding the AP-1 component, c-Jun resulting in an increase in c-Jun protein in infected cells. The increased AP-1 activity in infected cells is dependent upon the HSV immediate-early protein ICPO. Thus a mutant lacking the gene encoding this protein fails to increase AP-1 activity whilst an ICPO expression plasmid can specifically increase the activity of an AP-1 dependent promoter in co-transfection experiments. The implications of these effects in the interaction of HSV with cultured cells are discussed.