MKP-3 SPLICE VARIANTS DIFFERENTIALLY REGULATE FOXO-1 TRANSCRIPTION FACTOR.

COHEN SABBAN JM; NUDLER S; MORI SEQUEIROS GARCÍA MM; MALOBERTI PM; CALVENTE N; PAZ C

Mar del Plata

Congreso; LXIV Annual Meeting of Argentine Society of Clinical Investigation; 2019

Sociedad Argentina de Investigaciones Clínicas

MAP kinase phosphatase-3 (MKP-3) is dual-specificity phosphatase up-regulated by proliferative stimuli and specific for the MAP kinases ERK1/2. However, it has been described that MKP-3 also dephosphorylates the transcription factor forkhead box protein 1 (FOXO1). The human MKP-3 gene generates the full length transcript, variant L, and an alternative splice product, or variant S. Our work focuses on the analysis of potential differences between isoforms and their effects on cellular functions. Given that FOXO1 dephosphorylation is required for its translocation to the nucleus, where it promotes the transcription of specific genes, we proposed that S and L variants of MKP-3 could differentially regulate its subcellular localization. Here, HEK293 cell lines stably expressing S or L variants (HEK-S and HEK-L respectively) and control HEK293 cells (HEK-C), were transfected for mVenus-tagged FOXO1 expression in order to analyze FOXO1 subcellular localization by fluorescence microscopy. The results showed that in serum-starved cells, mean FOXO1 fluorescence intensity ratio between nuclear and cytoplasmic regions in HEK-L cells is significantly higher than in HEK-S and HEK-C cells (L = 6.55 ± 0.8, S = 0.53 ± 0.5, C = 0.47 ± 0.5, p < 0.001). These results suggest that only the over-expression of L variant promotes FOXO1 translocation. Next, förster radius energy transfer (FRET) strategy was used to evaluate the interaction of each MKP-3 variant with FOXO1. These studies revealed a significant interaction between both mCerulean-tagged MKP-3 variants and mVenus-tagged FOXO-1 against not fused mVenus and mCerulean fluorophores (p < 0.001) , though the level of interaction was equivalent among variants (p > 0.05). Collectively, our results show that both MKP-3 variants are able to interact with FOXO1 but only L variant is able to regulate the localization of FOXO1.