Abstract
ATF5, a member of activating transcription factor (ATF)/cAMP-response element-binding protein (CREB) family of b-ZIP transcription factors, contributes to neural cell differentiation and is involved in cell apoptosis in response to cisplatin and a number of environment factors. However, the mechanisms governing the regulation of ATF5 protein during apoptosis are largely unknown. In this study we reported that ATF5 protein was a substrate of the ubiquitin-proteasome pathway. Interestingly, the ubiquitin-dependent degradation of exogenous ATF5 protein was independent of lysine residues. Instead, the addition of a large N-terminal enhanced green fluorescence protein tag increased the stability of ATF5 protein, and the free amino acid group of the N-terminal methionine of ATF5 protein was a site for ubiquitinylation, indicating that exogenous ATF5 was degraded via the ubiquitin-proteasome system through N-terminal ubiquitinylation. Furthermore, cisplatin increased ATF5 protein expression via preventing its ubiquitin-dependent degradation, which might be associated with its promoting the nucleus-to-cytoplasm translocation of E2 ubiquitin-conjugating enzyme Cdc34 and reducing the interaction between ATF5 and Cdc34. In summary, a down-regulation of proteasome-mediated degradation of ATF5 might contribute to cisplatin-induced apoptosis, providing a new mechanism of cisplatin-induced apoptosis.
Highlights
ATF53 transcription factor, a member of activating transcription factor (ATF)/cAMP-response element-binding protein (CREB) family of b-ZIP transcription factors [1], plays a critical role in regulating cAMP
We have showed that ATF5 was up-regulated by cisplatin, and its overexpression increased cisplatin-induced apoptosis in HeLa cells [5], indicating the pro-apoptotic role of ATF5 in DNA damageinduced apoptosis
When the same sample was immunoblotted with anti-ATF5 antibody, the Cisplatin Increases ATF5 Protein Expression by Inhibiting Its film demonstrated a similar ladder of ATF5 species (Fig. 1E, Ubiquitin-mediated Degradation—Our previous study showed lanes 7 and 8). These results strongly suggested that ATF5 that ATF5 protein was up-regulated during cisplatin-induced was poly-ubiquitinylated in vivo
Summary
Reagents—Cisplatin, cycloheximide, ubiquitin protein, FLAG-ubiquitin, ubiquitin-aldehyde, Cdc protein, phosphocreatine, phosphocreatine kinase, hexokinase, 2-deoxyglucose, anti--tubulin antibody, and anti-FLAG antibody were purchased from Sigma. To verify if cisplatin caused accumulation of ATF5 by preventing its proteasomemediated degradation, the level of ATF5 protein expression in HeLa cells pretreated with DMSO or proteasome inhibitor MG132 for 1 h followed by cisplatin treatment for 12 h was analyzed using Western blot assay. Reverse transcription-PCR analysis of ATF5 and P21 mRNA expression in HeLa cells treated with different concentrations of cisplatin (ϩ, 10 M; ϩϩ, 20 M) for 12 h. Ubiquitination assay of endogenous ATF5 was performed in HeLa cells treated (lanes 2 and 4) or untreated (lanes 1 and 3) with cisplatin in the absence (lanes 1 and 2) or in the presence (lanes 3 and 4) of MG132 (25 M). The same experiments were performed in HEK293 (Fig. 1C) and MDA231 (Fig. 1D) cells, and similar results were obtained, indicating that cisplatin might regulate ATF5 expression via a proteasome-dependent degradation. Nuclear and cytoplasmic protein was isolated We detected a high molecular weight of HA-marked moleaccording to the method of Schreiber et al [23]
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