Non-genomic actions of retinoic acid induce pi3k signaling pathway and phosphorylation of nuclear proteins

Abstract

Retinoic acid (RA), the active form of vitamin A, induces neuroblastoma cells SH-SY5Y to differentiate. In addition to its classical transcriptional actions regulating the expression of specific genes, RA acts in an extra-genomic way, modulating the activity of relevant signalling cascades. In particular, RA treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation (Lopez-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002). RA activates PI3K and also ERK1/2 MAP Kinase signaling 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. 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. 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( Masiá S. et al., Mol. Endrocinol., in press, 2007). Next, we have investigated the consequences of the activation of signalling pathways by RA in neuroblastoma cells. Western blots using an antibody which specifically recognizes the phosphorylation motif of Akt have demonstrated that Akt activation in response to RA treatment produced a rapid phosphorylation of downstream substrates. Moreover, RA treatment resulted in rapid phosphorylation of well-known targets of Akt, such as mTOR and the ribosomal kinase p70S6, and other proteins, as CREB and histone H3, which are likely targets of RA non-genomic actions according to the scientific literature. 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). Finally, the observed RA-induced phosphorylation changes in these proteins were confirmed in 2-D western blots and immunoprecipitation experiments.