BackgroundHairy 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.ResultsIn 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.ConclusionsThe 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.
Background and Purpose: Protein inhibitor of activated STAT1 (PIAS1) is phosphorylated by IKKα at Ser 90 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 β (Aβ) 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 Ser 503 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 Ser 503 determines PIAS1 E3 ligase activity. Moreover, PIAS1 mediates SUMOylation of Elk-1, which functions as an endogenous defence mechanism against Aβ toxicity in vivo. Targeting Elk-1 SUMOylation could be considered a novel therapeutic strategy against AD. 1 | INTRODUCTION One of the two pathological hallmarks of Alzheimer's disease (AD) in the brains of AD patients is the accumulation of senile plaques, composed largely of amyloid β (Aβ) peptides (Aβ 1-40 and Aβ 1-42). Aβ, generated by sequential proteolytic cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase (De Strooper & Annaert, 2000), initiates a detrimental cascade that increases lipid peroxidation, free radical production, caspase activation, and DNA damage,
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin E3 ligase that catalyzes lysine-63 linked polyubiquitination of various signaling proteins. The recruitment and formation of different protein complexes with TRAF6 leads to activation of TRAF6-associated signaling and regulates cell proliferation and apoptosis. In this study, we search for novel TRAF6-binding partners that may have implication in TRAF6 signaling and cancer progression. Bioinformatic analysis identified a putative TRAF6-binding protein (TRAF6BP) that contains the consensus TRAF6-binding motif X-X-P-X-E-X-X-Ar/Ac-X and thereby TRAF6BP was subjected to further characterization. In accord with the bioinformatics prediction, coimmunoprecipitation analysis demonstrated the binding between TRAF6BP and TRAF6. TRAF6BP also interacts with other TRAF proteins including TRAF-2, −3, −5 but the interaction was much weaker than TRAF6. Domain mapping with TRAF6 deletion mutants identified the TRAF-C domain of TRAF6 is important for its interaction with TRAF6BP. In vivo ubiquitination assay further unveiled that TRAF6BP is a substrate of TRAF6 E3 ligase and underwent ubiquitination in the presence of TRAF6. Only the expression of full-length TRAF6, but not the TRAF-C deletion mutants of TRAF6 resulted in the ubiquitination of TRAF6BP. In addition, TRAF6BP ubiquitiation was abrogated when the lysine-63 ubiquitin mutant (K63R) but not the lysine-48 mutant (K48R) was used as the ubiquitin substrate. Together, we demonstrated for the first time that TRAF6BP is a novel TRAF6-binding protein and is a potential lysine-63 ubiquitination substrate of TRAF6; the TRAF6BP-TRAF6 interaction is essential for TRAF6 to ubiquitinate TRAF6BP. The significances of TRAF6BP-TRAF6 interactions and TRAF6BP ubiquitination in cancer progression and TRAF6 signaling are worthy to further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1268.
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