Efecto de la sobreexpresión de la proteína ANKK1 en la organización del citoesqueleto en neuroblastoma

Abstract

[ES] The protein Ankyrin Repeat and Kinase Domain containing I (ANKK1) is a member of the RIP (Receptor-Interacting Protein) family of kinases. ANKK1 has a kinase domain in the N-terminal end, an interdomain region and a C-terminal end containing multiple ankyrin repeats. Based on preliminary lab work, it was observed that ANKK1 is expressed in the central nervous system and it could interact with the cytosqueleton. The aim of this study is to provide evidences about ANKK1 relationship with some specific cytosqueleton proteins involved in axonal dynamics. Specifically, we will study the effect of ANKK1 overexpression in two neuroblastoma cell lines, SH-SY-5Y and SK-N-SH. The first step will be to perform transfections with the plasmids pcDNA3.1-FLcH2 and pEGFP-N1A2, which contain the long and short isoforms of ANKK1 fused with a GFP tag in the carboxyl terminal, respectively. Also, in some cases, the cells will be treated with the dopaminergic agonist apomorphine (APO). The functional analysis will comprise western blots and immunofluorescence (IFI). Se estudiará de parámetros relacionados con la dinámica axonal: 1. Longitud y número de neuritas: IFIs con α-TUBB3 y α -Ankk1; 2.Dinámica de microtúbulos: IFIs con α -TUBa acetilada, α -TUBa tiroxinada y α -Ankk1.

[EN] The gene ANKK1 that codes for the Receptor Interacting Protein 5 (RIP5) has been widely associated to cognitive traits, psychiatric disorders and the dopaminergic functioning per se. Besides, there are some evidences that ANKK1 protein participates in neurodevelopment and plasticity in the brain, processes for which cytoskeleton dynamics is crucial. However, the function of ANKK1 is still unknown, and there is a great interest in studying this protein and the signal pathways in which it is involved. Our objective was to study a possible relation between ANKK1 function and cytoskeleton dynamics, specifically the -tubulin post translational modifications (PTMs) that tune microtubule dynamic functions. To this purpose, we investigated the effect of ANKK1-K and ANKK1-FL isoforms overexpression upon microtubules organization in neuroblastoma cell lines. After the overexpression, we found a morphological change in neuroblastoma cells towards a more neuronal-like phenotype, which shifted into a more astrocyte-like phenotype after apomorphine treatment. Besides, we found an increase of -tubulin tyrosination levels in SK-N-SH neuroblastoma cell line after ANKK1-FL isoform overexpression, and in SH-SY5Y neuroblastoma cell lines after the overexpression of the two ANKK1 isoforms. Finally, a -tubulin acetylation signal change was observed in SH-SY5Y neuroblastoma cell line after the overexpression of ANKK1-FL isoform, specifically an expanded pattern is observed throughout the soma both at proximal and distal areas. Despite futher studies and replicas should be done, we think these results suggest that ANKK1 could be involved in the radial glia differentiation of neural lineages and that dopaminergic stimulation might be a modulator of the protein function or the pathways where it is involved. Regarding the study of PTMs changes, our results suggest that ANKK1 function might be implicated in neural processes which function require microtubule dynamics.