Hydrogen peroxide redistributes the localization of protein phosphatase methylesterase 1
Introduction
Protein phosphatase methylesterase-1 (PME-1), a 44 kDa intracellular protein encoded by the PPME1 gene, is a serine hydrolase that interacts with protein phosphatase 2A (PP2A) to catalyze its demethylation and that negatively regulates PP2A activity [1,2]. Recent studies have illustrated the cancer-associated functions of PME-1 and its potential clinical relevance to human glioma [3,4], gastric and lung cancer [5], colorectal cancer [6], and endometrial adenocarcinoma [7]. These emerging reports predict a great potential benefit to targeting PME-1 with cancer therapy treatment.
PP2A is a ubiquitously expressed serine/threonine phosphatase implicated in important cellular functions like cell cycle regulation, cell growth control development, the regulation of multiple signal transduction pathways, and so on [8,9]. The PP2A catalytic subunit (PP2Ac) can be methylated at the alpha carboxyl of Leu309 to regulate PP2A subunit composition, catalytic activity, and substrate specificity [10]. The methylation of PP2A is catalyzed by leucine carboxyl methyltransferase 1 (LCMT1) [11] while the removal of the carboxyl methyl group is catalyzed by PME-1 [12]. The knockdown of PME-1 or LCMT1 prevents the demethylation/methylation of PP2Ac [13,14]. The currently known functions of PME-1 are mostly related to PP2A. A crystal structure study showed that PME-1 exists in an inactive conformation when it is not bound to PP2A. Upon binding to PP2A, PME-1 catalytic triad residues rearrange into an active form, allowing PP2Ac C-terminal tail binding to the active site pocket of PME-1, which is thus catalyzed PP2Ac to be demethylated [2]. PME-1 methyl-esterase activity also protects PP2Ac from ubiquitin/proteasome degradation [15]. In addition to its role as PP2Ac methyl-esterase, PME-1 can directly bind to the active site of PP2Ac and block enzyme activity [2]. Furthermore, PME-1 is stably associated with the “inactive” form of PP2A complexes in tissues for stabilizing the inactive PP2A complex in the nucleus [16].
Oxidative stress is a common form of cellular stress that participates many physiological and pathological processes and refers to a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses. ROS act as a cellular messenger in redox signaling, regulating diverse important signaling pathways. PP2A is a signaling molecular target of ROS. Hydrogen peroxide (H2O2), a major form of ROS, has been reported to inhibit or activate PP2A activity [[17], [18], [19], [20]]. This contradiction may be attributed to the level of ROS exposed or to the particular methodological differences tested. Some studies have demonstrated that H2O2 induces PP2Ac demethylation at L309 [21,22]. However, no reports have yet explored the enzyme that regulates PP2Ac demethylation under oxidative stress.
In this study, we explore the effect of oxidative stress on PME-1 localization. We also address the distribution of LCMT1, as it is the pair partner of PME-1 in regulating PP2A methylation/demethylation. We hope to further understanding of the regulation of PME-1 and PP2A demethylation under oxidative stress.
Section snippets
Chemicals and reagents
AMZ30 was purchased from Merck (San Diego, CA). ABL-127, N-acetyl cysteine (NAC) and H2O2 were purchased from Sigma Aldrich (St. Louis, USA). Leptomycin B was purchased from the Beyond Technology Company (Shanghai, China).
Cell culture
Cells were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island, NY, USA), 2 mM l-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA, USA). Primary
PME-1 redistributed from the nucleus to the cytoplasm under oxidative stress
To study the effect of oxidative stress on PME-1, we examined the expression and cellular localization of PME-1 under H2O2 treatment. Western blot results show that the protein expression of PME-1 and LCMT1 did not change after H2O2 treatment of different concentrations (Fig. 1A). An immunofluorescence analysis shows that the PME-1 was ubiquitously distributed through the cytoplasm and nuclei of human primary fibroblasts under unstimulated conditions. H2O2-induced PME-1 redistributed from the
Discussion
In eukaryotic cells, the genetic material and transcriptional machinery of the nucleus are separated from translational machinery and metabolic systems of the cytoplasm. This segregation facilitates the precise regulation of gene expression, signal transduction, cell cycle progression and several other cellular processes through the selective regulation of bidirectional transport between the nucleus and cytoplasm. The proper control of nuclear–cytoplasmic shuttling and the localization of
Conclusion
In this study, we explored the effect of oxidative stress on PME-1 localization. We found that PME-1 is exported from the nucleus to the cytoplasm with H2O2 treatment and redistributes a species of dem-p-PP2Ac in subcellular compartments. These findings offer new insight into the regulation of PME-1 localization and PP2A demethylation under oxidative stress.
Declaration of interest
The authors have no conflicts of interest to declare.
CRediT authorship contribution statement
Shen Tang: Experimental designer, Experimental operator, Writing and editing, Analysis of datas. Cailing Lu: Experimental operator, Writing and editing, Analysis of datas. Laiming Mo: Experimental operator, Writing the first draft. Xinhang Wang: Experimental designer, Experimental operator. Ziwei Liang: Experimental designer, Experimental operator. Fu Qin: Experimental designer, Experimental operator. Yinpin Liu: Experimental designer, Experimental operator. Yuyang Liu: Experimental designer,
Acknowledgements
This research was supported by the National Science Foundation of China (NSFC) through grant 81460506 awarded to Shen Tang and grant NSFC 81860585 awarded to Xiyi Li and NSFC grant 21567006 and 81760576 awarded to Cailing Lu.
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Both authors contributed equally to this work.