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2.
Biochim Biophys Acta ; 1783(12): 2241-54, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18809440

RESUMEN

The ribosomal protein S6 kinase 1 (S6K1) is emerging as a common downstream target of signalling by hormones and nutrients such as insulin and amino acids. Here, we have investigated how amino acids signal through the S6K1 pathway. First, we found that a commercial anti-phospho-Thr389-S6K1 antibody detects an 80-90 kDa protein that is rapidly phosphorylated in response to amino acids. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI-3 kinase inhibitors, and knockdown experiments showed that this protein was not S6K1. Looking for candidate targets of this phosphorylation, we found that amino acids stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. In turn, these phosphorylations required the activity of either p38 or ERK MAP kinases, which could compensate for each other. Moreover, we show that these MAP kinases are also needed for the amino acid-induced phosphorylation of S6K1 at Thr421/Ser424, as well as for that of S6K1 substrate, the S6 ribosomal protein. Consistent with these results, concomitant inhibition of p38 and ERK pathways also antagonised the well-known effects of amino acids on the process of autophagy. Altogether, these findings demonstrate a previously unknown role for MAP kinases in amino acid signalling.


Asunto(s)
Aminoácidos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Androstadienos/farmacología , Antibióticos Antineoplásicos/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Immunoblotting , Inmunoprecipitación , Inmunosupresores/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Plásmidos , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Wortmanina , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores
3.
Biochim Biophys Acta ; 1783(8): 1467-79, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18442486

RESUMEN

The Regulator of Chromosome Condensation 1 (RCC1) was identified over 20 years ago as a critical cell cycle regulator. By analyzing its amino acid sequence, RCC1 was found to consist of seven homologous repeats of 51-68 amino acid residues, which were later shown to adopt a seven-bladed beta-propeller fold. Since the initial identification of RCC1, a number of proteins have been discovered that contain one or more RCC1-like domains (RLDs). As we show here, these RCC1 superfamily proteins can be subdivided in five subgroups based on structural criteria. In recent years, a number of studies have been published regarding the functions of RCC1 superfamily proteins. From these studies, the emerging picture is that the RLD is a versatile domain which may perform many different functions, including guanine nucleotide exchange on small GTP-binding proteins, enzyme inhibition or interaction with proteins and lipids. Here, we review the available structural and functional data on RCC1 superfamily members, paying special attention to the human proteins and their involvement in disease.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/clasificación , Enfermedades Genéticas Congénitas/genética , Factores de Intercambio de Guanina Nucleótido/clasificación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/clasificación , Filogenia , Alineación de Secuencia , Distribución Tisular
4.
Electrophoresis ; 27(20): 3935-8, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17054096

RESUMEN

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.


Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/aislamiento & purificación , Acrilamidas/química , Proteínas Fluorescentes Verdes/aislamiento & purificación , Factores de Intercambio de Guanina Nucleótido/aislamiento & purificación , Peso Molecular , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ubiquitina-Proteína Ligasas
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