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Título

Rapid, non-genomic actions of Retinoic Acid on Phosphatidyl-inositol-3-Kinase signalling pathway mediated by the Retinoic Acid Receptor

AutorMasiá, Susana CSIC ORCID; Laserna Mendieta, Emilio J.; Barettino, Domingo CSIC
Fecha de publicación2007
CitaciónEMBO Conference on Nuclear Receptors: Structure and Function in Health and Disease
ResumenRetinoic Acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signalling pathway, and this activation is required for RA-induced differentiation. RA activates PI3K and ERK1/2 MAP Kinase signalling pathways through a rapid, non-genomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor RAR, on the basis of the pharmacological profile of the activation, loss and gain of function experiments with MEF-RAR(αβγ)L-/L- null cells, and the physical association between liganded RAR and PI3K kinase activity. Ligand binding differentially regulated the association of RAR with the two subunits of PI3K. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARα and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR resulted to be relevant for PI3K activation. A chimerical RAR receptor fusing c-Src myristylation domain to the N-terminal of RARα (Myr-RARα) was targeted to plasma membrane. Transfection of Myr-RARα to MEF-RAR(αβγ)L-/L- null cells and COS-7 cells results in strong activation of PI3K signalling pathway, although both in the absence as well in the presence of RA. Our results suggest a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signalling complex involving RAR and the subunits of PI3K. Next we have investigated the consequences of the activation of signalling pathways by RA in neuroblastoma cells. Downstream targets of the PI3K/Akt signalling pathway, like mTOR or p70S6K, are activated upon RA addition. In addition we show that RA addition rapidly increased phosphorylation of several Akt kinase target proteins in the nucleus, identified by an antibody recognizing the phosphorylated Akt motif. We have analyzed possible nuclear protein targets for RA-induced phosphorylation, with the aim of finding functional links between non-genomic actions and classical transcriptional actions mediated by RAR. We have found that RA treatment rapidly results in phosphorylation of chromatin proteins (core histone H3), as well as proteins involved in transcriptional activation (transcription factor CREB). In a second approach, nuclear phospho-proteins from control and RA-treated neuroblastoma cells were purified by affinity chromatography and analysed by 2D-electrophoresis. The differentially expressed spots were identified by Mass Spectrometry. We have found that RA treatment induces phosphorylation of chromatin proteins (HMGB1 and Histone H1.5) and proteins involved in the processing and transport of mRNA (PABP, hnRNP-K, hnRNP-C1/C2, Nucleophosmin).
DescripciónPóster original presentado EMBO Conference on Nuclear Receptors: Structure and Function in Health and Disease, celebrado en Italia en 2007
URIhttp://hdl.handle.net/10261/123168
Aparece en las colecciones: (IBV) Comunicaciones congresos




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