Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.     

Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells

Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes.     

A LCMT1-PME-1 methylation equilibrium controls mitotic spindle size

Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.     

Spatial control of protein phosphatase 2A (de)methylation

Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A(C)(1)) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A(C) antibodies revealed a good correlation with the methylation status of PP2A(C), demethylated PP2A(C) being substantially nuclear. Throughout mitosis, demethylated PP2A(C) is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A(C) in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.     

LCMT1 indicates poor prognosis and is essential for cell proliferation in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most malignant type of cancers. Leuci carboxyl methyltransferase 1 (LCMT1) is a protein methyltransferase that plays an improtant regulatory role in both normal and cancer cells. The aim of this study is to evaluate the expression pattern and clinical significance of LCMT1 in HCC.MethodsThe expression pattern and clinical relevance of LCMT1 were determined using the Gene Expression Omnibus (GEO) database, the Cancer Genome Atlas (TCGA) program, and our datasets. Gain-of-function and loss-of-function studies were employed to investigate the cellular functions of LCMT1 in vitro and in vivo. Quantitative real-time polymerase chain reaction (RT-PCR) analysis, western blotting, enzymatic assay, and high-performance liquid chromatography were applied to reveal the underlying molecular functions of LCMT1.ResultsLCMT1 was upregulated in human HCC tissues, which correlated with a “poor” prognosis. The siRNA-mediated knockdown of LCMT1 inhibited glycolysis, promoted mitochondrial dysfunction, and increased intracellular pyruvate levels by upregulating the expression of alani-neglyoxylate and serine-pyruvate aminotransferase (AGXT). The overexpression of LCMT1 showed the opposite results. Silencing LCMT1 inhibited the proliferation of HCC cells in vitro and reduced the growth of tumor xenografts in mice. Mechanistically, the effect of LCMT1 on the proliferation of HCC cells was partially dependent on PP2A.ConclusionsOur data revealed a novel role of LCMT1 in the proliferation of HCC cells. In addition, we provided novel insights into the effects of glycolysis-related pathways on the LCMT1regulated progression of HCC, suggesting LCMT1 as a novel therapeutic target for HCC therapy.     

Selection of protein phosphatase 2A regulatory subunits is mediated by the C terminus of the catalytic Subunit

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.     

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

Objectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.

Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells.

Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.

Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.     

Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice

Background

Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.

Methodology/Principal Findings

Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(−/−) tissues, which also displayed alterations in phosphoproteome content.

Conclusions

These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.     

Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast.

Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes. It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue. The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2). To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants. Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus. Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases. We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds. Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated. We did not detect any methylesterification of Pph3p under our conditions. Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast. The function of the PPM2 gene product remains unclear.     

Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.     

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.     

Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Here we show that methylation plays an essential role in promoting PP2A holoenzyme assembly and that demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

Methylation of protein phosphatase 2A—Influence of regulators and environmental stress factors

Protein phosphatase 2A catalytic subunit (PP2A‐C) has a terminal leucine subjected to methylation, a regulatory mechanism conserved from yeast to mammals and plants. Two enzymes, LCMT1 and PME1, methylate and demethylate PP2A‐C, respectively. The physiological importance of these posttranslational modifications is still enigmatic. We investigated these processes in Arabidopsis thaliana by mutant phenotyping, by global expression analysis, and by monitoring methylation status of PP2A‐C under different environmental conditions. The lcmt1 mutant, possessing essentially only unmethylated PP2A‐C, had less dense rosettes, and earlier flowering than wild type (WT). The pme1 mutant, with 30% reduction in unmethylated PP2A‐C, was phenotypically comparable with WT. Approximately 200 overlapping genes were twofold upregulated, and 200 overlapping genes were twofold downregulated in both lcmt1 and pme1 relative to WT. Differences between the 2 mutants were also striking; 97 genes were twofold upregulated in pme1 compared with lcmt1, indicating that PME1 acts as a negative regulator for these genes. Analysis of enriched GO terms revealed categories of both abiotic and biotic stress genes. Furthermore, methylation status of PP2A‐C was influenced by environmental stress, especially by hypoxia and salt stress, which led to increased levels of unmethylated PP2A‐C, and highlights the importance of PP2A‐C methylation/demethylation in environmental responses.     

Structure of protein phosphatase methyltransferase 1 (PPM1), a leucine carboxyl methyltransferase involved in the regulation of protein phosphatase 2A activity

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A. The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition. The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site. The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.     

NOM1 targets protein phosphatase I to the nucleolus

Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.     

Multifaceted roles of Arabidopsis PP6 phosphatase in regulating cellular signaling and plant development

Reversible protein phosphorylation catalyzed by kinases and phosphatases is a major form of posttranslational regulation that plays a central role in regulating many signaling pathways. While large families of both protein kinases and protein phosphatases have been identified in plants, kinases outnumber phosphatases. This raises the question of how a relatively limited number of protein phosphatases can maintain protein phosphorylation homeostasis in a cell. Recent studies have shown that Arabidopsis FyPP1 (Phytochrome-associated serine/threonine protein phosphatase 1) and FyPP3 encode the catalytic subunits of protein phosphatase 6 (PP6), and that they directly binds to the A subunits of protein phosphatase 2A (PP2AA proteins), and SAL (SAPS domain-like) proteins to form the heterotrimeric PP6 holoenzyme complex. Emerging evidence is suggesting that PP6, acts in opposition with multiple classes of kinases, to regulate the phosphorylation status of diverse substrates and subsequently numerous developmental processes and responses to environmental stimuli.     

Aralkyl selenoglycosides and related selenosugars in acetylated form activate protein phosphatase-1 and -2A

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ～2–4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT1^23–38) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (Br-BASG). MYPT1^23–38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1?h, and suppressed cell viability in 24?h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules.