Protein Phosphatase 2A Complexes Research Articles

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.Graphical

Phosphorylation Dynamics in a flg22-Induced, G Protein–Dependent Network Reveals the AtRGS1 Phosphatase

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2–dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein–coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Regulation and role of the PP2A-B56 holoenzyme family in cancer.

Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a scaffolding A-subunit, a catalytic C-subunit, and a regulatory B-subunit. The functional complexity of PP2A holoenzymes arises mainly through the vast repertoire of regulatory B-subunits, which determine both their substrate specificity and their subcellular localization. Therefore, a major challenge for developing more effective therapeutic strategies for cancer is to identify the specific PP2A complexes to be targeted. Of note, the development of small molecules specifically directed at PP2A-B56α has opened new therapeutic avenues in both solid and hematological tumors. Here, we focus on the B56/PR61 family of PP2A regulatory subunits, which have a central role in directing PP2A tumor suppressor activity. We provide an overview of the mechanisms controlling the formation and regulation of these complexes, the pathways they control, and the mechanisms underlying their deregulation in cancer.

Distinct Clades of Protein Phosphatase 2A Regulatory B'/B56 Subunits Engage in Different Physiological Processes.

Protein phosphatase 2A (PP2A) is a strongly conserved and major protein phosphatase in all eukaryotes. The canonical PP2A complex consists of a catalytic (C), scaffolding (A), and regulatory (B) subunit. Plants have three groups of evolutionary distinct B subunits: B55, B' (B56), and B''. Here, the Arabidopsis B' group is reviewed and compared with other eukaryotes. Members of the B'α/B'β clade are especially important for chromatid cohesion, and dephosphorylation of transcription factors that mediate brassinosteroid (BR) signaling in the nucleus. Other B' subunits interact with proteins at the cell membrane to dampen BR signaling or harness immune responses. The transition from vegetative to reproductive phase is influenced differentially by distinct B' subunits; B'α and B'β being of little importance, whereas others (B'γ, B'ζ, B'η, B'θ, B'κ) promote transition to flowering. Interestingly, the latter B' subunits have three motifs in a conserved manner, i.e., two docking sites for protein phosphatase 1 (PP1), and a POLO consensus phosphorylation site between these motifs. This supports the view that a conserved PP1-PP2A dephosphorelay is important in a variety of signaling contexts throughout eukaryotes. A profound understanding of these regulators may help in designing future crops and understand environmental issues.

Protein-Protein Interactions and Quantitative Phosphoproteomic Analysis Reveal Potential Mitochondrial Substrates of Protein Phosphatase 2A-B'ζ Holoenzyme.

Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.

Activation of the PP2A-B56α heterocomplex synergizes with venetoclax therapies in AML through BCL2 and MCL1 modulation

AbstractVenetoclax combination therapies are becoming the standard of care in acute myeloid leukemia (AML). However, the therapeutic benefit of these drugs in older/unfit patients is limited to only a few months, highlighting the need for more effective therapies. Protein phosphatase 2A (PP2A) is a tumor suppressor phosphatase with pleiotropic functions that becomes inactivated in ∼70% of AML cases. PP2A promotes cancer cell death by modulating the phosphorylation state in a variety of proteins along the mitochondrial apoptotic pathway. We therefore hypothesized that pharmacological PP2A reactivation could increase BCL2 dependency in AML cells and, thus, potentiate venetoclax–induced cell death. Here, by using 3 structurally distinct PP2A-activating drugs, we show that PP2A reactivation synergistically enhances venetoclax activity in AML cell lines, primary cells, and xenograft models. Through the use of gene editing tools and pharmacological approaches, we demonstrate that the observed therapeutic synergy relies on PP2A complexes containing the B56α regulatory subunit, of which expression dictates response to the combination therapy. Mechanistically, PP2A reactivation enhances venetoclax-driven apoptosis through simultaneous inhibition of antiapoptotic BCL2 and extracellular signal-regulated kinase signaling, with the latter decreasing MCL1 protein stability. Finally, PP2A targeting increases the efficacy of the clinically approved venetoclax and azacitidine combination in vitro, in primary cells, and in an AML patient-derived xenograft model. These preclinical results provide a scientific rationale for testing PP2A-activating drugs with venetoclax combinations in AML.

Pleiotropy of PP2A Phosphatases in Cancer with a Focus on Glioblastoma IDH Wildtype.

Serine/Threonine protein phosphatase 2A (PP2A) is a heterotrimeric (or occasionally, heterodimeric) phosphatase with pleiotropic functions and ubiquitous expression. Despite the fact that they all contribute to protein dephosphorylation, multiple PP2A complexes exist which differ considerably by their subcellular localization and their substrate specificity, suggesting diverse PP2A functions. PP2A complex formation is tightly regulated by means of gene expression regulation by transcription factors, microRNAs, and post-translational modifications. Furthermore, a constant competition between PP2A regulatory subunits is taking place dynamically and depending on the spatiotemporal circumstance; many of the integral subunits can outcompete the rest, subjecting them to proteolysis. PP2A modulation is especially important in the context of brain tumors due to its ability to modulate distinct glioma-promoting signal transduction pathways, such as PI3K/Akt, Wnt, Ras, NF-κb, etc. Furthermore, PP2A is also implicated in DNA repair and survival pathways that are activated upon treatment of glioma cells with chemo-radiation. Depending on the cancer cell type, preclinical studies have shown some promise in utilising PP2A activator or PP2A inhibitors to overcome therapy resistance. This review has a special focus on "glioblastoma, IDH wild-type" (GBM) tumors, for which the therapy options have limited efficacy, and tumor relapse is inevitable.

PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability.

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

Coupling auxin‐inducible degradation with quantitative proteomics for in‐vivoprotein functional characterization

Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long‐term changes to the target protein expression/function. In contrast, the auxin‐inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long‐term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well‐suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10‐15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long‐term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms.

PP2A and cancer epigenetics: a therapeutic opportunity waiting to happen.

The epigenetic state of chromatin is altered by regulators which influence gene expression in response to environmental stimuli. While several post-translational modifications contribute to chromatin accessibility and transcriptional programs, our understanding of the role that specific phosphorylation sites play is limited. In cancer, kinases and phosphatases are commonly deregulated resulting in increased oncogenic signaling and loss of epigenetic regulation. Aberrant epigenetic states are known to promote cellular plasticity and the development of therapeutic resistance in many cancer types, highlighting the importance of these mechanisms to cancer cell phenotypes. Protein Phosphatase 2A (PP2A) is a heterotrimeric holoenzyme that targets a diverse array of cellular proteins. The composition of the PP2A complex influences its cellular targets and activity. For this reason, PP2A can be tumor suppressive or oncogenic depending on cellular context. Understanding the nuances of PP2A regulation and its effect on epigenetic alterations can lead to new therapeutic avenues that afford more specificity and contribute to the growth of personalized medicine in the oncology field. In this review, we summarize the known PP2A-regulated substrates and potential phosphorylation sites that contribute to cancer cell epigenetics and possible strategies to therapeutically leverage this phosphatase to suppress tumor growth.

Cryo-EM structure of the Hippo signaling integrator human STRIPAK.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a large, multisubunit protein phosphatase 2A (PP2A) assembly that integrates diverse cellular signals in the Hippo pathway to regulate cell proliferation and survival. The architecture and assembly mechanism of this critical complex are poorly understood. Using cryo-EM, we determine the structure of the human STRIPAK core comprising PP2AA, PP2AC, STRN3, STRIP1, and MOB4 at 3.2-Å resolution. Unlike the canonical trimeric PP2A holoenzyme, STRIPAK contains four copies of STRN3 and one copy of each the PP2AA-C heterodimer, STRIP1, and MOB4. The STRN3 coiled-coil domains form an elongated homotetrameric scaffold that links the complex together. An inositol hexakisphosphate (IP6) is identified as a structural cofactor of STRIP1. Mutations of key residues at subunit interfaces disrupt the integrity of STRIPAK, causing aberrant Hippo pathway activation. Thus, STRIPAK is established as a noncanonical PP2A complex with four copies of regulatory STRN3 for enhanced signal integration.

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes.

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.

Selective Activation of Protein Phosphatase 2A Complexes

Selective Activation of Protein Phosphatase 2A Complexes

Abstract 2981: Biased activation of PP2A through selective heterotrimer binding and stabilization

Abstract Development of molecularly targeted therapies have shifted our clinical approach to cancer allowing for an integrated treatment approach where tumor genotype and molecular profiles are paired in a clinically actionable manner with specific targeted therapies. This approach maximizes efficacy and minimizes toxicity for each patient. While the vast majority of molecularly targeted therapies have focused on the development of kinase inhibition, an equally effective yet underexplored strategy exists in the activation or regulation of phosphatases. The tumor suppressor Protein Phosphatase 2A (PP2A) negatively regulates multiple oncogenic drivers common to a broad range of human malignancies including MYC, AKT, and ERK; and has been demonstrated as a critical tumor suppressor. In cancer, PP2A is inactivated through several mechanisms including increased expression of endogenous inhibitors, post-translational modifications of the catalytic subunit, and somatic mutation in its catalytic, regulatory or scaffold subunits. The vast mechanisms employed to dysregulate and inhibit PP2A in cancer, highlights it's potential as an endogenous molecular target to combat malignant growth. Our laboratory has been involved in the development of a novel series of small molecule activators of PP2A (SMAPs). Using structural, biochemical, cellular, and in vivo model systems we reveal how the lead SMAP, DT-061, specifically stabilizes a B56 containing PP2A holoenzyme, in its fully assembled, active state, to direct enzymatic activity towards specific oncogenic targets, including its well-known substrate c-MYC. Our structure identifies a unique interfacial small molecule binding pocket that involves all three PP2A subunits to prevent dissociation of select B56 subunit containing holoenzymes. Elucidating the mechanism of SMAPs allowed us to explore appropriate clinical placement for this targeting strategy and investigate potential mechanisms of resistance. For example, in vivo xenograft studies of cancer-associated Aα mutants, most notably R183W, demonstrates that these mutations render malignant cells resistant to these SMAPs and further support the mechanism of SMAPs described. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets. Citation Format: Daniel Leonard, Wei Huang, Sudeh Izadmehr, Caitlin M. O'Connor, Danica D. Wiredja, Zhizhi Wang, Nilesh Zaware, Daniela M. Schlatzer, Stefan Schuchner, Janna Kiselar, Yinghua Chen, Nikhil Vasireddi, Abbey L. Perl, Matthew D. Glasky, Wenqing Xu, David L. Brautigan, Egon Ogris, Derek J. Taylor, Goutham Narla. Biased activation of PP2A through selective heterotrimer binding and stabilization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2981.

RETRACTED: Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2

Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium.

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified protein phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hippo kinase, and consequently its substrate Warts, to release the transcriptional co-activator Yorkie into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.

PS1000 POST‐TRANSLATIONAL MODIFICATIONS OF PROTEIN PHOSPHATASE 2A (PP2A) IN MIXED LINEAGE LEUKEMIA (MLL)

Background:Mixed lineage leukemia (MLL) is the most common leukemia in infant patients. This leukemia is due to chromosomal translocations at locus 11q23 and is associated with poor clinical outcome. Novel, targeted therapeutic approaches are needed. Protein phosphatase 2A (PP2A) is a serine threonine phosphatase which regulates the phosphorylation of several kinases, including Erk, Akt, GSK3 which are fundamental for MLL cells’ survival. PP2A is a trimeric protein complex in which a core dimer formed between the scaffold subunit and the catalytic subunit is associated with one of the many regulatory subunits that facilitate and direct the interaction of the trimer with substrate proteins. PP2A activity is regulated by interaction with PP2A inhibitor proteins (PIPs), as well as by post‐translational modifications of PP2A complex components.Aims:In this context, our aim is to understand how PP2A post‐translational modifications affect the activity of PP2A on its downstream targets and to investigate whether PP2A re‐activation, by preventing phosphorylation and activation of collateral pathways, might represent a valid therapeutic strategy for MLL.Methods:12 different leukemic cell lines and 8 primary samples from MLL patients were included in this study. We measured the PP2A phosphatase activity by a colorimetric assay using a synthetic phospho‐peptide and malachite green reagent. The protein levels of PP2A, phospho‐PP2A, demethyl‐PP2A, LCMT‐1, PME‐1, Akt, phospho‐Akt, Erk, and phospho‐Erk were determined by western blot using specific antibodies.Results:Compared to healthy bone marrow, we found an increase in the phosphorylation at Tyrosine 307 (Tyr307) and a decrease in the methylation at Leucine 309 (Leu309) of the catalytic subunit of PP2A in MLL samples. These changes in the post‐translational modifications of the catalytic subunit correlated with a decrease in the phosphatase activity of PP2A. Methylation at Leu309 has been described as an absolute requirement for the binding of the regulatory subunit B55α to PP2A core. B55α was found up‐regulated in MLL cell lines and primary samples compared to healthy bone marrow. The methylation of the catalytic subunit of PP2A is catalyzed by leucine carboxyl methyltransferase (LCMT‐1), which we found down‐regulated in MLL samples, whereas demethylation is catalyzed by the phosphatase methylesterase (PME‐1) that was found up‐regulated in MLL. In line with the inactivation of PP2A in the MLL samples, PP2A downstream targets such as Akt and Erk were found hyper‐phosphorylated in MLL samples.Summary/Conclusion:Our results suggest that PP2A phosphatase activity inhibition might sustain in MLL samples a persistent serine/threonine phosphorylation of PP2A substrates, such as Akt and Erk that mediate pro‐survival and anti‐apoptotic signals. This inhibition might be explained, at least in part, by phosphorylation and demethylation of PP2A catalytic subunit. A better understanding of the mechanisms that regulate PP2A activity could provide new strategies to rescue PP2A phosphatase activity and target fundamental mechanisms of leukemic survival and chemotherapy resistance.

NDRG2 Sensitizes Myeloid Leukemia to Arsenic Trioxide via GSK3β-NDRG2-PP2A Complex Formation.

N-Myc downstream-regulated gene 2 (NDRG2) was characterized as a tumor suppressor, inducing anti-metastatic and anti-proliferative effects in several tumor cells. However, NDRG2 functions on anticancer drug sensitivity, and its molecular mechanisms are yet to be fully investigated. In this study, we investigated the mechanism of NDRG2-induced sensitization to As2O3 in the U937 cell line, which is one of the most frequently used cells in the field of resistance to As2O3. NDRG2-overexpressing U937 cells (U937-NDRG2) showed a higher sensitivity to As2O3 than mock control U937 cell (U937-Mock). The higher sensitivity to As2O3 in U937-NDRG2 was associated with Mcl-1 degradation through glycogen synthase kinase 3β (GSK3β) activation. Inhibitory phosphorylation of GSK3β was significantly reduced in U937-NDRG2, and the reduction was diminished by okadaic acid, a protein phosphatase inhibitor. NDRG2 mediated the interaction between GSK3β and protein phosphatase 2A (PP2A), inducing dephosphorylation of GSK3β at S9 by PP2A. Although the C-terminal deletion mutant of NDRG2 (ΔC NDRG2), which could not interact with PP2A, interacted with GSK3β, the mutant failed to dephosphorylate GSK3β at S9 and increased sensitivity to As2O3. Our findings suggest that NDRG2 is a kind of adaptor protein mediating the interaction between GSK3β and PP2A, inducing GSK3β activation through dephosphorylation at S9 by PP2A, which increases sensitivity to As2O3 in U937 cells.

Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent β-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of β-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, β-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.