ABSTRACT
BACKGROUND AND PURPOSE: Protein inhibitor of activated STAT1 (PIAS1) is phosphorylated by IKKα at Ser90 in a PIAS1 E3 ligase activity-dependent manner. Whether PIAS1 is also phosphorylated at other residues and the functional significance of these additional phosphorylation events are not known. The transcription factor Elk-1 remains SUMOylated under basal conditions, but the role of Elk-1 SUMOylation in brain is unknown. Here, we examined the functional significance of PIAS1-mediated Elk-1 SUMOylation in Alzheimer's disease (AD) using the APP/PS1 mouse model of AD and amyloid ß (Aß) microinjections in vivo. EXPERIMENTAL APPROACH: Novel phosphorylation site(s) on PIAS1 were identified by LC-MS/MS, and MAPK/ERK-mediated phosphorylation of Elk-1 demonstrated using in vitro kinase assays. Elk-1 SUMOylation by PIAS1 in brain was determined using in vitro SUMOylation assays. Apoptosis in hippocampus was assessed by measuring GADD45α expression by western blotting, and apoptosis of hippocampal neurons in APP/PS1 mice was assessed by TUNEL assay. KEY RESULTS: Using LC-MS/MS, we identified a novel MAPK/ERK-mediated phosphorylation site on PIAS1 at Ser503 and showed this phosphorylation determines PIAS1 E3 ligase activity. In rat brain, Elk-1 was SUMOylated by PIAS1, which decreased Elk-1 phosphorylation and down-regulated GADD45α expression. Moreover, lentiviral-mediated transduction of Elk-1-SUMO1 reduced the number of hippocampal apoptotic neurons in APP/PS1 mice. CONCLUSIONS AND IMPLICATIONS: MAPK/ERK-mediated phosphorylation of PIAS1 at Ser503 determines PIAS1 E3 ligase activity. Moreover, PIAS1 mediates SUMOylation of Elk-1, which functions as an endogenous defence mechanism against Aß toxicity in vivo. Targeting Elk-1 SUMOylation could be considered a novel therapeutic strategy against AD.
Subject(s)
Hippocampus/metabolism , Protein Inhibitors of Activated STAT/metabolism , ets-Domain Protein Elk-1/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Female , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Neurons/metabolism , Phosphorylation , Rats, Sprague-Dawley , Sumoylation , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: Hairy and Enhancer of split 1 (Hes-1) is a transcriptional repressor that plays an important role in neuronal differentiation and development, but post-translational modifications of Hes-1 are much less known. In the present study, we aimed to investigate whether Hes-1 could be SUMO-modified and identify the candidate SUMO acceptors on Hes-1. We also wished to examine the role of the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in SUMOylation of Hes-1 and the molecular mechanism of Hes-1 SUMOylation. Further, we aimed to identify the molecular target of Hes-1 and examine how Hes-1 SUMOylation affects its molecular target to affect cell survival. RESULTS: In this study, by using HEK293T cells, we have found that Hes-1 could be SUMO-modified and Hes-1 SUMOylation was greatly enhanced by the SUMO E3 ligase PIAS1 at Lys8, Lys27 and Lys39. Furthermore, Hes-1 SUMOylation stabilized the Hes-1 protein and increased the transcriptional suppressing activity of Hes-1 on growth arrest and DNA damage-inducible protein alpha (GADD45α) expression. Overexpression of GADD45α increased, whereas knockdown of GADD45αα expression decreased cell apoptosis. In addition, H2O2 treatment increased the association between PIAS1 and Hes-1 and enhanced the SUMOylation of Hes-1 for endogenous protection. Overexpression of Hes-1 decreased H2O2-induced cell death, but this effect was blocked by transfection of the Hes-1 triple sumo-mutant (Hes-1 3KR). Overexpression of PIAS1 further facilitated the anti-apoptotic effect of Hes-1. Moreover, Hes-1 SUMOylation was independent of Hes-1 phosphorylation and vice versa. CONCLUSIONS: The present results revealed, for the first time, that Hes-1 could be SUMO-modified by PIAS1 and GADD45α is a novel target of Hes-1. Further, Hes-1 SUMOylation mediates cell survival through enhanced suppression of GADD45α expression. These results revealed a novel role of Hes-1 in addition to its involvement in Notch signaling. They also implicate that SUMOylation could be an important posttranslational modification that regulates cell survival.
