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Influenza A viruses (IAVs) are respiratory pathogens that are able to hijack multiple cellular mechanisms to drive their replication. Consequently, several viral and cellular proteins undergo posttranslational modifications such as dynamic phosphorylation/dephosphorylation. In eukaryotic cells, dephosphorylation is mainly catalyzed by protein phosphatase 2A (PP2A). While the function of kinases in IAV infection is quite well studied, only little is known about the role of PP2A in IAV replication. Here, we show, by using knockdown and inhibition approaches of the catalytic subunit PP2Ac, that this phosphatase is important for efficient replication of several IAV subtypes. This could neither be attributed to alterations in the antiviral immune response nor to changes in transcription or translation of viral genes. Interestingly, decreased PP2Ac levels resulted in a significantly reduced cell viability after IAV infection. Comprehensive kinase activity profiling identified an enrichment of process networks related to apoptosis and indicated a synergistic action of hyper-activated PI3K/Akt, MAPK/JAK-STAT and NF-kB signaling pathways, collectively resulting in increased cell death. Taken together, while IAV seems to effectively tap leftover PP2A activity to ensure efficient viral replication, reduced PP2Ac levels fail to orchestrate cell survival mechanisms to protect infected cells from early cell death.

Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than nontransformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall phosphatase 2A (PP2A) activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate misregulated SET-Sgo1 pathway as an underlying mechanism.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that regulates many fundamental cellular processes. PP2A is involved in tumorigenesis because mutations in the scaffold subunit, PPP2R1B, were found in several types of cancers. However, the biological function of PPP2R1B remains largely unknown. We report here that homozygous deletion of Ppp2r1b in Mus musculus impairs meiotic recombination and causes meiotic arrest in spermatocytes. Consistently, male mice lacking Ppp2r1b are characterized with infertility. Furthermore, heterozygous missense mutations in the Homo sapiens PPP2R1B gene, which encodes PPP2R1B, are identified in azoospermia patients with meiotic arrest. We found that PPP2R1B mutants are susceptible to degradation by an E3 ligase CRL4A DCAF6 , and resistant to de-polyubiquitylation by ubiquitin-specific protease 5 (USP5). In addition, heterozygous mutations in PPP2R1B reduce stability of the wild-type PPP2R1B. Our results demonstrate an essential role of PPP2R1B in spermatogenesis and identify upstream regulators of PPP2R1B., (© 2021 Federation of American Societies for Experimental Biology.)

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP-GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.

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., Competing Interests: LB, FS, ST, CF, JB, PL, CS, JB, DC, FP No competing interests declared, (© 2020, Bel Borja et al.)

Insulin and insulin-like growth factors are longevity determinants that negatively regulate Forkhead box class O (FoxO) transcription factors. In C. elegans mutations that constitutively activate DAF-16, the ortholog of mammalian FoxO3a, extend lifespan by two-fold. While environmental insults induce DAF-16 activity in younger animals, it also becomes activated in an age-dependent manner in the absence of stress, modulating gene expression well into late adulthood. The mechanism by which DAF-16 activity is regulated during aging has not been defined. Since phosphorylation of DAF-16 generally leads to its inhibition, we asked whether phosphatases might be necessary for its increased transcriptional activity in adult C. elegans. We focused on the PP2A/4/6 subfamily of phosphoprotein phosphatases, members of which had been implicated to regulate DAF-16 under low insulin signaling conditions but had not been investigated during aging in wildtype animals. Using reverse genetics, we functionally characterized all C. elegans orthologs of human catalytic, regulatory, and scaffolding subunits of PP2A/4/6 holoenzymes in postreproductive adults. We found that PP2A complex constituents PAA-1 and PPTR-1 regulate DAF-16 transcriptional activity during aging and that they cooperate with the catalytic subunit LET-92 to protect adult animals from ultraviolet radiation. PP4 complex members PPH-4.1/4.2, and SMK-1 also appear to regulate DAF-16 in an age-dependent manner, and together with PPFR-2 they contribute to innate immunity. Interestingly, SUR-6 but no other subunit of the PP2A complex was necessary for the survival of pathogen-infected animals. Finally, we found that PP6 complex constituents PPH-6 and SAPS-1 contribute to host defense during aging, apparently without affecting DAF-16 transcriptional activity. Our studies indicate that a set of PP2A/4/6 complexes protect adult C. elegans from environmental stress, thus preserving healthspan. Therefore, along with their functions in cell division and development, the PP2A/4/6 phosphatases also appear to play critical roles later in life., Competing Interests: The authors have declared that no competing interests exist.

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The Icahn School of Medicine at Mount Sinai has filed patents covering composition of matter on the small molecules disclosed herein for the treatment of human cancer and other diseases (International Application Numbers: PCT/US15/19770, PCT/US15/19764; and US Patent: US 9,540,358 B2). Mount Sinai is actively seeking commercial partners for the further development of the technology. G.N. has a financial interest in the commercialization of the technology. The other authors have declared that no competing interests exist.

Background: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Mice lacking the tumor-suppressive protein phosphatase 2A subunit B56δ (Ppp2r5d) spontaneously develop HCC, correlating with increased c-MYC oncogenicity., Materials and Methods: We used two-dimensional difference gel electrophoresis-coupled matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify differential proteomes of livers from wild-type, non-cancerous and HCC-affected B56δ knockout mice., Results: A total of 23 proteins were differentially expressed/regulated in liver between wild-type and non-cancerous knockout mice, and 119 between non-cancerous and HCC knockout mice ('cancer proteins'). Overlap with our reported differential transcriptome data was poor. Overall, 56% of cancer proteins were reported before in HCC proteomics studies; 44% were novel. Gene Ontology analysis revealed cancer proteins mainly associated with liver metabolism (18%) and mitochondria (15%). Ingenuity Pathway Analysis identified 'cancer' and 'gastrointestinal disease' as top hits., Conclusion: We identified several proteins for further exploration as novel potential HCC biomarkers, and independently underscored the relevance of Ppp2r5d knockout mice as a valuable hepatocarcinogenesis model., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2-enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell-specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the visual pigment melanopsin regulate non-image-forming visual tasks, such as circadian photoentrainment and pupil constriction, as well as contrast detection for image formation. Sustained ipRGC function throughout the day is, therefore, of great importance. Melanopsin is a bistable rhabdomeric-type (R-type) visual pigment, which is thought to use light to regenerate its chromophore from all-trans-retinal back to 11-cis-retinal and does not depend on constant chromophore supply to the extent required by visual pigment in rod and cone photoreceptors. Like the majority of photopigments and G-protein-coupled receptors (GPCRs), melanopsin deactivation requires C-terminal phosphorylation and subsequent β-arrestin binding. We hypothesize that melanopsin utilizes canonical GPCR resensitization mechanisms, including dephosphorylation and endocytosis, during the light, and together, they provide a mechanism for prolonged light responses., Methods: Here, we examined expression of protein phosphatases from a variety of subfamilies by RT-PCR and immunohistochemical analyses of the mouse retina. The expression of protein phosphatase 2A (PP2A) in ipRGCs was assessed. We also examine the role of phosphatase and endocytic activity in sustaining melanopsin signaling using transiently-transfected HEK293 cells., Results: Our analyses suggest that melanopsin-mediated light responses can be rapidly and extensively enhanced by PP2A activity. Light-activated melanopsin undergoes endocytosis in a clathrin-dependent manner. This endocytic activity enhances light responses upon repeated stimulation, implicating a role for endocytic activity in resensitization., Conclusions: Thus, we propose that melanopsin phototransduction is maintained by utilizing canonical GPCR resensitization mechanisms rather than reliance on chromophore replenishment from supporting cells.

Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.

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., Competing Interests: Declaration of Interests E.S.F. is a member of the scientific advisory board of C4 Therapeutics and a consultant to Novartis and Deerfield. He is a science advisory board member, founder, and shareholder for Civetta Therapeutics. E.S.F. received research funding from Novartis, Deerfield, and Astellas not related to this work. N.S.G. is a founder, science advisory board member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. A patent application related to this work has been submitted to the U.S. Patent and Trademark Office entitled “Compositions and Methods of Treating Cancers by Administering a Phenothiazine-Related Drug that Activates Protein Phosphatase 2A (PP2A) with Reduced Inhibitory Activity Targeted to the Dopamine D2 Receptor and Accompanying Toxicity” (application no. PCT/US19/67508)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is phytotoxic and detoxified primarily via phytochelatin (PC) complexation in Arabidopsis. Here, we explore Cd toxicity responses and defence mechanisms beyond the PC pathway using forward genetics approach. We isolated an Arabidopsis thaliana Cd-hypersensitive mutant, Cd-induced short root 1 (cdsr1) in the PC synthase mutant (cad1-3) background. Using genomic resequencing and complementation, we identified PP2A-4C as the causal gene for the mutant phenotype, which encodes a catalytic subunit of protein phosphatase 2A (PP2A). Root and shoot growth of cdsr1 cad1-3 and cdsr1 were more sensitive to Cd than their respective wild-type cad1-3 and Col-0. A mutant of the PP2A scaffolding subunit 1A was also more sensitive to Cd. PP2A-4C was localized in the cytoplasm and nucleus and PP2A-4C expression was downregulated by Cd in cad1-3. PP2A enzyme activity was decreased in cdsr1 and cdsr1 cad1-3 under Cd stress. The expression of 1-aminocyclopropane-1-carboxylic acid synthase genes ACS2 and ACS6 was upregulated by Cd more in cad1-3 and cdsr1 cad1-3 than in Col-0 and the double mutant had a higher ACS activity. cdsr1 cad1-3 and cdsr1 overproduced ethylene under Cd stress. The results suggest that PP2A containing 1A and 4C subunits alleviates Cd-induced growth inhibition by modulating ethylene production., (© 2020 John Wiley & Sons Ltd.)

During meiosis, the stepwise release of sister chromatid cohesion is crucial for the equal distribution of genetic material to daughter cells, enabling generation of fertile gametophytes. However, the molecular mechanism that protects centromeric cohesion from release at meiosis I is unclear in Arabidopsis ( Arabidopsis thaliana ). Here, we report that the protein phosphatase 2A regulatory subunits B'α and B'β participate in the control of sister chromatid separation. The double mutant b'αβ exhibited severe male and female sterility, caused by the lack of a nucleus or presence of an abnormal nucleus in mature microspores and embryo sacs. 4',6-Diamidino-2-phenylindole staining revealed unequal amounts of DNA in the mononuclear microspores. Transverse sections of the anthers revealed unevenly sized tetrads with or without a nucleus, suggesting a defect in meiocyte meiosis. An analysis of chromosome spreads showed that the sister chromatids separated prematurely at anaphase I in b'αβ Immunoblotting showed that AtRECOMBINATION DEFECTIVE8 (AtREC8), a key member of the cohesin complex, was hyperphosphorylated in b'αβ anthers and pistils during meiosis but hypophosphorylated in the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays showed that B'α and B'β interact specifically with AtREC8, AtSHUGOSHIN1 (AtSGO1), AtSGO2, and PATRONUS1. Given that B'α was reported to localize to the centromere in meiotic cells, we propose that protein phosphatase 2A B'α and B'β are recruited by AtSGO1/2 and PATRONUS1 to dephosphorylate AtREC8 at the site of centromere cohesion to shield it from cleavage until anaphase II, contributing to the balanced separation of sister chromatids at meiosis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope-based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6-regulated nuclear envelope-based dynein pulling forces and centrosome-nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

Cyclin-dependent kinase (CDK)-activating phosphatases, CDC25A and CDC25B, are labile proteins, and their levels vary in a cell cycle-dependent manner. Immediate-early response IER5 protein negatively regulates the cellular CDC25B levels, and stress-induced IER5 expression potentiates G2/M arrest. IER5 binds to protein phosphatase PP2A and regulates the PP2A substrate specificity. We show that IER5 binds to CDC25B and assists PP2A to convert CDC25B to hypophosphorylated forms. Hypophosphorylation at Ser323 results in the dissociation of CDC25B from 14-3-3 phospho-binding proteins. In IER5 expressing cells, CDC25B dissociated from 14-3-3 is unstable but slightly activated, because 14-3-3 inhibits CDC25B polyubiquitination and CDC25B binding to CDK1. The 14-3-3 binding to CDC25A also impedes CDC25A degradation and CDC25A-CDK2 interaction. We propose that 14-3-3 is an important regulator of CDC25A and CDC25B and that PP2A/IER5 controls the stability and activity of CDC25B through regulating the interaction of CDC25B and 14-3-3., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Normal functioning of the brain is dependent upon a complex web of communication between numerous cell types. Within neuronal networks, the faithful transmission of information between neurons relies on an equally complex organization of inter- and intra-cellular signaling systems that act to modulate protein activity. In particular, post-translational modifications (PTMs) are responsible for regulating protein activity in response to neurochemical signaling. The key second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), regulates one of the most ubiquitous and influential PTMs, phosphorylation. While cAMP is canonically viewed as regulating the addition of phosphate groups through its activation of cAMP-dependent protein kinases, it plays an equally critical role in regulating removal of phosphate through indirect control of protein phosphatase activity. This dichotomy of regulation by cAMP places it as one of the key regulators of protein activity in response to neuronal signal transduction throughout the brain. In this review we focus on the role of cAMP in regulation of the serine/threonine phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) and the relevance of control of PP1 and PP2A to regulation of brain function and behavior., (Copyright © 2018. Published by Elsevier B.V.)

The serine/threonine phosphatase PP2A regulates a vast portion of the phosphoproteome including pathways involved in apoptosis, proliferation and DNA damage response and PP2A inactivation is a vital step in malignant transformation. Many groups have explored the therapeutic venue of combining PP2A reactivation with kinase inhibition to counteract the very changes in tumor suppressors and oncogenes that lead to cancer development. Conversely, inhibition of PP2A to complement chemotherapy and radiation-induced cancer cell death is also an area of active investigation. Here we review the studies that utilize PP2A targeted agents as combination therapy in cancer. A potential role for PP2A in tumor immunity is also highlighted., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cancer cells depend on a supportive niche (the tumor microenvironment) that promotes tumor cell survival while protecting the malignant cells from therapeutic challenges and the host's defense systems. Cancer cells and the support cells in the tumor microenvironment communicate via cytokines/chemokines, cell:cell contact, or alterations in the metabolic state of the niche (e.g. hypoxia) that promote growth and survival of the tumor cell, influence metastasis, and defeat immune surveillance. These signaling pathways involve dysregulation of not only protein kinases but also protein phosphatases as normal signal transduction processes require both activation and deactivation. For instance, aberrant receptor signaling can result from constitutive activation of a tyrosine kinase such as FLT3 or inactivation of a tyrosine protein phosphatase such as SHP-2 (PTPN11). Activation of serine/threonine kinases such as AKT and ERK are often observed during the development of drug resistance while genomic and non-genomic suppression of serine/threonine protein phosphatases such as PP2A achieve similar results. It is fairly clear that the various protein phosphatases will impact processes that support drug resistance. Of growing interest is the emerging model whereby the support cells in the tumor microenvironment actually serve as drivers of tumorigenesis. This phenomenon has been most prominently observed in osteoblast cells in leukemic niches. At least one protein phosphatase, PTPN11, has emerged as a critical driver of this process in juvenile myelomonocytic leukemia. This review will cover the role of various serine/threonine and tyrosine protein phosphatases in processes that are central to tumor microenvironment function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein Phosphatase 2A (PP2A) encompasses a large family of Ser/Thr phosphatases, consisting of a catalytic C subunit and a structural A subunit that are, in most cases, further bound to a regulatory B-type subunit. The B-type subunits determine function and regulation of PP2A trimers, but despite their importance in PP2A biology, their roles in controlling dephosphorylation of a given substrate in a given cell or tissue remain poorly defined, particularly in the context of a complete organism. Besides twenty PP2A subunit encoding genes, some of which are tissue-specifically expressed, five additional genes encode major regulators of active PP2A trimer assembly, and at least seven genes encode cellular PP2A inhibitors, further adding to the complexity of the mammalian PP2A system. In this review, we summarize current knowledge on physiologic functions of PP2A in germ cell maturation, embryonic development, metabolic regulation, tumor suppression, and homeostasis of adult brain, heart, liver, immune system, lung, intestine, kidney, skin, bone and eye, all retrieved from in vivo studies using PP2A transgenic, knockout or knockin mice. Data from 63 mouse models, generated between 1998 and now, reveal the essentiality of PP2A in vivo, and shed light on tissue-specific functions of particular PP2A subunits on the one hand, and functional redundancies on the other hand. In future, it remains of utmost importance to further characterize the existing models, as well as to generate novel models, with the aim of deepening our insights in PP2A (patho)physiology and, particularly, in the therapeutic potential of PP2A targeting in human disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein synthesis is one of the most complex and energy-consuming processes in eukaryotic cells and therefore is tightly regulated. One of the main mechanisms of translational control is post-translational modifications of the components of translational apparatus. Phosphorylation status of translation factors depends on the balanced action of kinases and phosphatases. While many kinase-dependent events are well defined, phosphatases that counteract phosphorylation are rarely determined. This mini-review focuses on the regulation of activity of translational initiation factors by serine/threonine phosphatases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored., Methods: We studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1-SET-PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort., Results: We found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1-SET-PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study., Conclusions: Our results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1-SET-PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

Multiple interdependent mechanisms ensure faithful segregation of chromosomes during cell division. Among these, the spindle assembly checkpoint monitors attachment of spindle microtubules to the centromere of each chromosome, whereas the tension-sensing checkpoint monitors the opposing forces between sister chromatid centromeres for proper biorientation. We report here a new function for the deeply conserved Gcn5 acetyltransferase in the centromeric localization of Rts1, a key player in the tension-sensing checkpoint. Rts1 is a regulatory component of protein phopshatase 2A, a near universal phosphatase complex, which is recruited to centromeres by the Shugoshin (Sgo) checkpoint component under low-tension conditions to maintain sister chromatid cohesion. We report that loss of Gcn5 disrupts centromeric localization of Rts1. Increased RTS1 dosage robustly suppresses gcn5∆ cell cycle and chromosome segregation defects, including restoration of Rts1 to centromeres. Sgo1's Rts1-binding function also plays a key role in RTS1 dosage suppression of gcn5∆ phenotypes. Notably, we have identified residues of the centromere histone H3 variant Cse4 that function in these chromosome segregation-related roles of RTS1. Together, these findings expand the understanding of the mechanistic roles of Gcn5 and Cse4 in chromosome segregation.

Background: Although MASTL (microtubule-associated serine/threonine kinase-like) is a key mitotic kinase that regulates mitotic progression through the inactivation of tumor suppressor protein phosphatase 2A (PP2A), the antitumor mechanism of MASTL targeting in cancer cells is still unclear., Methods: MASTL expression was evaluated by using breast cancer tissue microarrays and public cancer databases. The effects of MASTL depletion with siRNAs were evaluated in various breast cancer cells or normal cells. Various methods, including cell viability, cell cycle, soft agar, immunoblotting, immunofluorescence, PP2A activity, live image, and sphere forming assay, were used in this study., Results: This study showed the oncosuppressive mechanism of MASTL targeting that promotes mitotic catastrophe through PP2A activation selectively in breast cancer cells. MASTL expression was closely associated with tumor progression and poor prognosis in breast cancer. The depletion of MASTL reduced the oncogenic properties of breast cancer cells with high MASTL expression, but did not affect the viability of non-transformed normal cells with low MASTL expression. With regard to the underlying mechanism, we found that MASTL inhibition caused mitotic catastrophe through PP2A activation in breast cancer cells. Furthermore, MASTL depletion enhanced the radiosensitivity of breast cancer cells with increased PP2A activity. Notably, MASTL depletion dramatically reduced the formation of radioresistant breast cancer stem cells in response to irradiation., Conclusion: Our data suggested that MASTL inhibition promoted mitotic catastrophe through PP2A activation, which led to the inhibition of cancer cell growth and a reversal of radioresistance in breast cancer cells.

Background: Depression is characterized by a pathological injury to the hippocampal neurons. Senkyunolide A (SenA) is one of the major active components of Dan-zhi-xiao-yao-san, which is widely used in the treatment of depression-related disorders., Materials and Methods: In the present study, it was hypothesized that the antidepressant effect of Dan-zhi-xiao-yao-san depended on the function of SenA and the authors attempted to reveal the molecular mechanism associated with the treatment. An in vitro depression model was induced using corticosterone (Cort), and the effect of SenA on the cell viability, apoptosis, and protein phosphatase 2A/α-synuclein (PP2A/α-syn) signaling was detected. To validate the mechanism driving the therapeutic effect of SenA, activity of PP2A and α-syn was modulated and the effect on neural cells was evaluated., Results: The results showed that SenA protects Cort-induced cell apoptosis in PC12 cells. In addition, SenA increased Cort-induced reduction of PP2A activity, while it decreased the expression of p-PP2A, α-syn, and p-α-syn (Ser129). Further, modulation of PP2A activity with specific inhibitor okadaic acid (OA) increased Cort-induced cell apoptosis, while PP2A activator D-erythro-sphingosine (SPH) exhibited an opposite effect. The neuroprotective effects of SenA on neural cells also depended on inhibition of α-syn function, the regulation of which would influence the activity of PP2A in a negative loop., Conclusion: Collectively, the results suggested that the neuroprotective effects of SenA were exerted by modulating activities of PP2A activities and α-syn. The findings partially explained the mechanism associated with the neuroprotective effect of SenA., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The Niemann-Pick type C is a rare neurodegenerative disease that results from loss-of-function point mutations in NPC1 or NPC2, which affect the homeostasis of sphingolipids and sterols in human cells. We have previously shown that yeast lacking Ncr1, the orthologue of human NPC1 protein, display a premature ageing phenotype and higher sensitivity to oxidative stress associated with mitochondrial dysfunctions and accumulation of long chain bases. In this study, a lipidomic analysis revealed specific changes in the levels of ceramide species in ncr1Δ cells, including decreases in dihydroceramides and increases in phytoceramides. Moreover, the activation of Sit4, a ceramide-activated protein phosphatase, increased in ncr1Δ cells. Deletion of SIT4 or CDC55, its regulatory subunit, increased the chronological lifespan and hydrogen peroxide resistance of ncr1Δ cells and suppressed its mitochondrial defects. Notably, Sch9 and Pkh1-mediated phosphorylation of Sch9 decreased significantly in ncr1Δsit4Δ cells. These results suggest that phytoceramide accumulation and Sit4-dependent signaling mediate the mitochondrial dysfunction and shortened lifespan in the yeast model of Niemann-Pick type C1, in part through modulation of the Pkh1-Sch9 pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proper segregation of chromosomes in meiosis is essential to prevent miscarriages and birth defects. This requires that sister chromatids maintain cohesion at the centromere as cohesion is released on the chromatid arms when the homologs segregate at anaphase I. The Shugoshin proteins preserve centromere cohesion by protecting the cohesin complex from cleavage, and this has been shown in yeasts to be mediated by recruitment of the protein phosphatase 2A B' (PP2A B'). In metazoans, delineation of the role of PP2A B' in meiosis has been hindered by its myriad of other essential roles. The Drosophila Shugoshin MEI-S332 can bind directly to both of the B' regulatory subunits of PP2A, Wdb and Wrd, in yeast two-hybrid experiments. Exploiting experimental advantages of Drosophila spermatogenesis, we found that the Wdb subunit localizes first along chromosomes in meiosis I, becoming restricted to the centromere region as MEI-S332 binds. Wdb and MEI-S332 show colocalization at the centromere region until release of sister-chromatid cohesion at the metaphase II/anaphase II transition. MEI-S332 is necessary for Wdb localization, but, additionally, both Wdb and Wrd are required for MEI-S332 localization. Thus, rather than MEI-S332 being hierarchical to PP2A B', these proteins reciprocally ensure centromere localization of the complex. We analyzed functional relationships between MEI-S332 and the two forms of PP2A by quantifying meiotic chromosome segregation defects in double or triple mutants. These studies revealed that both Wdb and Wrd contribute to MEI-S332's ability to ensure accurate segregation of sister chromatids, but, as in centromere localization, they do not act solely downstream of MEI-S332., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

The functional inhibition of protein phosphatase 2A (PP2A) has emerged in the last years as a common alteration in breast cancer that determines poor outcome and contributes to disease progression and aggressiveness. Furthermore, expression of estrogen receptor (ER) is a high relevant molecular event with key therapeutic implications in breast cancer, and androgen receptor (AR) signaling is involved in the pathogenesis of breast cancer and represents a novel target with crescent importance in this disease. In this review, we summarize the role of the tumor suppressor PP2A in modulating ER and AR signaling in breast cancer, the molecular mechanisms involved, and its biological and therapeutic impact., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Renal dopamine receptor D1 played a critical role in the regulation of body blood pressure. Under hypertension, over-phosphorylation of D1 receptor impaired its function. G protein kinase 4 (GRK4) and protein phosphatase 2A (PP2A) exerted the effect to phosphorylate and de-phosphorylate D1 receptor. A current study revealed that the inhibition of GRK4 cannot normalize the phosphorylation level of D1 receptor. Meanwhile, the PP2A was activated under hypertension, indicating abnormal de-phosphorylation function of D1 receptor, the reason for which remains unknown. This study aimed to investigate the effect and mechanism of SUMO-1 modification on the regulation of dopamine receptor D1 to PP2A., Materials and Methods: Bioinformatics software predicted SUMO modification site in dopamine receptor D1. Cultured CHO cells were transfected with mutants of renal dopamine receptor D1. Co-immunoprecipitation and Western blot tested the interaction between over-phosphorylated D1 receptor and PP2A. Laser confocal microscopy examined their co-localization., Results: Bioinformatics predicted two SUMO modification sites K265 and K402 in dopamine receptor D1. Co-immunoprecipitation assay revealed weakened interaction between PP2A and phosphorylated D1 receptor, impeding the de-phosphorylation and normal function of D1 receptor., Conclusions: Two SUMO modification sites existed in dopamine receptor D1, the phosphorylation of which, due to SUMO modification, can interact with PP2A, leading to the inhibition of D1 de-phosphorylation and normal function, thus providing new insights for treatment and prevention of hypertension.

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

Our previous studies have described the toxic effects of microcystin-LR (MC-LR) in various normal cell lines and human hepatoma SMMC-7721 cells, but the specific effects of MC-LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion-transporting polypeptides (OATP) involved in cellular uptake of MC-LR, and thus probably make an appropriate in vitro model to assess MC-LR's cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC-LR for 24 h. The presence of MC-LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5-10 μM MC-LR inhibited PP2A activity significantly but 0.5-1 μM MC-LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC-LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen-activated protein kinase (MAPK) activation. However, exposure to MC-LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A-inhibition-induced hyperphosphorylation of Bcl-2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC-LR induced extensive molecular and cellular alterations in A549 cells through a PP2A-centered pathway, which differed in some respects from our previous study in SMMC-7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC-LR in A549 cells, and our findings provide insights into the mechanism of MC-LR toxicity in cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1065-1078, 2017., (© 2016 Wiley Periodicals, Inc.)

The complexity of the phosphatase, PP2A, is being unravelled and current research is increasingly providing information on the association of deregulated PP2A function with cancer initiation and progression. It has been reported that decreased activity of PP2A is a recurrent observation in many types of cancer, including colorectal and breast cancer (Baldacchino et al. EPMA J. 5:3, 2014; Cristobal et al. Mol Cancer Ther. 13:938-947, 2014). Since deregulation of PP2A and its regulatory subunits is a common event in cancer, PP2A is a potential target for therapy (Baldacchino et al. EPMA J. 5:3, 2014). In this review, the structural components of the PP2A complex are described, giving an in depth overview of the diversity of regulatory subunits. Regulation of the active PP2A trimeric complex, through phosphorylation and methylation, can be targeted using known compounds, to reactivate the complex. The endogenous inhibitors of the PP2A complex are highly deregulated in cancer, representing cases that are eligible to PP2A-activating drugs. Pharmacological opportunities to target low PP2A activity are available and preclinical data support the efficacy of these drugs, but clinical trials are lacking. We highlight the importance of PP2A deregulation in cancer and the current trends in targeting the phosphatase.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML., Competing Interests: The authors have no conflicts to declare.

This study presents the effect of all-trans retinoic acid (ATRA) on cell growth and respiratory chain complex I in human keratinocyte cultures. Keratinocyte treatment results in increased level of GRIM-19 and other subunits of complex I, in particular of their carbonylated forms, associated with inhibition of its enzymatic activity. The results show that in keratinocytes ATRA-promoted phosphatase activity controls the proteostasis and activity of complex I.

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Administration of bone marrow stroma cells (BMSCs) has the potential to ameliorate degenerative disorders and to repair injured sites. The homing of transplanted BMSCs to damaged tissues is a critical property of engraftment. Therefore, it is important to understand signal molecules controlling migration of BMSCs. Here, we demonstrate that serine-threonine protein phosphatase 2A (PP2A) is responsive to migration of BMSCs. Pharmacological Inhibition of PP2A, using okadaic acid (OA), leads to attenuated cell migration in rat primary BMSCs both in the absence or presence of stromal cell-derived factor-1 (SDF-1). Consistent with the above findings, knockdown of the main catalytic subunit PP2Acα using small interfering RNA also attenuates chemotaxis of BMSCs. On the other hand, cell viability of BMSCs remains unchanged with OA treatment or knockdown of PP2Acα subunit. Moreover, we observed an upregulation of PP2A-B55β in transcription level after SDF-1 treatment, indicating their potential role as the functioning regulatory subunit of PP2A phosphatase in BMSCs migration model. Collectively, these data provide first insight into the modulation of BMSCs migration by PP2A phosphatase activity and lay a foundation for exploring PP2A signaling as a modulating target for BMSCs transplantation.

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

Objective: In this study, we investigated the role of Trefoil factor 1 (TFF1) in regulating cell proliferation and tumour development through β-catenin signalling using in vivo and in vitro models of gastric tumorigenesis., Design: Tff1-knockout (Tff1-KO) mice, immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot, and phosphatase assays were used to examine the role of TFF1 on β-catenin signalling pathway., Results: Nuclear localisation of β-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at an early age of 4-6 weeks and maintained during all stages of gastric tumorigenesis in the Tff1-KO mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the β-catenin/T-cell factor (TCF) transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear β-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signalling molecules upstream of β-catenin revealed a decrease in phosphorylated glycogen synthase kinase 3β (p-GSK3β) (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-β-catenin (Ser33/37/Thr41) and decrease of p-β-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3β, and AKT was dependent on protein phosphatase 2A (PP2A) activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localisation of β-catenin in stages of human gastric tumorigenesis., Conclusions: Our data indicate that loss of TFF1 promotes β-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3β signalling., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase involved in meiosis, mitosis, sperm capacitation, and apoptosis. Abberant activity of PP2A has been associated with a number of diseases. The homolog PPP2CA and PPP2CB can each function as the phosphatase catalytic subunit generally referred to as PP2AC. We generated a Ppp2ca conditional knockout (CKO) in C57BL/6J mice. Exon 2 of Ppp2ca was knocked out in a spatial or temporal-specific manner in primordial germ cells at E12.5. This Ppp2ca-null mutation caused infertility in male C57BL/6J mice. These CKO mice provide a powerful tool to study the mechanisms of Ppp2ca in development and disease., (© 2015 Society for Reproduction and Fertility.)

Background: Impairment of endothelium-dependent vasorelaxation has been suggested to play a principle role of endothelial dysfunction in the development of cardiovascular complications of diabetes. Recent studies have demonstrated a protective effect of Tanshinone IIA (Tan) on endothelial nitric oxide synthase (eNOS)-NO pathway. However, its role in endothelium-dependent vasorelaxation in diabetes and precise mechanisms remain elusive., Methods: Sprague-Dawley rats were injected intraperitoneally with streptozotocin (STZ) to induce diabetes and then administered orally with Tan for 2 weeks. For the in vitro study, human umbilical vein endothelial cells (HUVECs) were co-incubated with Tan and high glucose for 48 h., Results: eNOS expression and NO generation were significantly decreased in diabetic rats. These decreases were accompanied by an impairment of endothelium-dependent relaxation. Administration of Tan ameliorated the aberrant changes in eNOS expression, NO generation and endothelium-dependent relaxation in diabetic rats. Expectedly, Tan also inhibited high glucose-induced decrease of eNOS expression and NO generation in a concentration-dependent manner in HUVECs. Mechanistically, high glucose attenuated eNOS transcriptional activity through inhibiting the binding activity and nuclear translocation of Sp1 and AP-1. However, Tan did not prevent these effects. At post-transcriptional level, Tan increased eNOS expression and activity through multiple mechanisms including regulation of mRNA and protein half-life, degradation, coupling and serine 1177 phosphorylation. Rather than affecting protein phosphatase 2A (PP2A) expression and activity, Tan markedly inhibited the translocation of PP2A-A from cytosol to membrane and subsequently impaired PP2A-A/eNOS interaction, leading to prevent eNOS dephosphorylation. All these alterations underlie the protective role of Tan on eNOS expression following high glucose stimulation., Conclusions: Our data demonstrate that high glucose decreases eNOS expression initiating at a transcriptional level, whereas Tan prevents such effect through multiple ways of post-transcriptional mechanism., General Significance: Our work provided novel mechanisms for Tan in regulating vasorelaxation and may help to better understand the cardiovascular protective action of Tan., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To facilitate rapid accumulation of Cdk1-phosphorylated substrate proteins, the Cdk1 counter-acting phosphatase, PP2A-B55 is inhibited during M phase by stoichiometric inhibitors (ENSA and Arpp19). These inhibitors are activated when phosphorylated by Cdk1-activated Greatwall-kinase. Recent experiments show that ENSA is dephosphorylated and inactivated by the PP2A-B55 itself, and acts as an unfair substrate inhibiting PP2A-B55 activity towards other Cdk1 substrates. Mathematical modelling shows that this mutual antagonism between the phosphatase and its inhibitor is insufficient to explain the switch-like characteristics of mitotic entry and exit. We show that the feedback regulation of Greatwall activating kinase and/or inactivating phosphatase can explain the abruptness of these cell cycle transitions., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Glycogen synthase kinase-3β (GSK-3β) and protein phosphatase 2A (PP2A) are the important enzymes controlling tau hyperphosphorylation. The relationship between these two enzymes and its impact on tau hyperphosphorylation are not well understood. In the present study, we determined the cross talk between PI3K-AKT-GSK-3β and PP2A pathways and found that the former regulated the methylation of PP2Ac via GSK-3β. Upregulation of GSK-3β led to an increase in the methylation and activity of PP2Ac through suppression of protein phosphatase methylesterase-1 expression and phosphorylation of leucine carboxyl methyltransferase 1. PP2A also regulated GSK-3β phosphorylation. Downregulation of PP2A enhanced Ser9 phosphorylation of GSK-3β and inhibited its kinase activity. Thus, GSK-3β and PP2A regulate each other and control tau phosphorylation both directly and indirectly through each other. Reduction of tau phosphorylation by inhibition of GSK-3β may be more than offset by inhibition of PP2A through a shift in phosphatase methylesterase-1/leucine carboxyl methyltransferase 1 balance; PP2A regulates phosphorylation of tau at Ser262/356, a required site for tau pathology. These findings suggest targeting PP2A rather than GSK-3β to inhibit tau pathology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2-7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2-7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Candida albicans, a major human fungal pathogen, is the primary cause of invasive candidiasis in a wide array of immunocompromised patients. C. albicans virulence requires the ability to undergo a reversible morphological transition from yeast to filaments in response to a variety of host environmental cues. These cues are sensed by the pathogen and activate multiple signal transduction pathways to induce filamentation. Reversible phosphorylation events are critical for regulation of many of these pathways. While a variety of protein kinases are known to function as components of C. albicans filamentous growth signal transduction pathways, considerably little is known about the role of phosphatases. Here we demonstrate that PPG1, encoding a putative type 2A-related protein phosphatase, is important for C. albicans filament extension, invasion, and virulence in a mouse model of systemic candidiasis. PPG1 is also important for downregulation of NRG1, a key transcriptional repressor of C. albicans filamentous growth, and is shown to affect the expression of several filament-specific target genes. An epistasis analysis suggests that PPG1 controls C. albicans filamentation via the cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway. We demonstrate that Ppg1 possesses phosphatase activity and that a ppg1 catalytic mutant shows nearly equivalent filamentation, invasion, and virulence defects compared to those of a ppg1Δ/Δ strain. Overall, our results suggest that phosphatases, such as Ppg1, play critical roles in controlling and fine-tuning C. albicans filament extension and virulence as well as signal transduction pathways, transcriptional regulators, and target genes associated with these processes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In yeast, the initiation of telomere replication at the late S phase involves in combined actions of kinases on Cdc13, the telomere binding protein. Cdc13 recruits telomerase to telomeres through its interaction with Est1, a component of telomerase. However, how cells terminate the function of telomerase at G2/M is still elusive. Here we show that the protein phosphatase 2A (PP2A) subunit Pph22 and the yeast Aurora kinase homologue Ipl1 coordinately inhibit telomerase at G2/M by dephosphorylating and phosphorylating the telomerase recruitment domain of Cdc13, respectively. While Pph22 removes Tel1/Mec1-mediated Cdc13 phosphorylation to reduce Cdc13-Est1 interaction, Ipl1-dependent Cdc13 phosphorylation elicits dissociation of Est1-TLC1, the template RNA component of telomerase. Failure of these regulations prevents telomerase from departing telomeres, causing perturbed telomere lengthening and prolonged M phase. Together our results demonstrate that differential and additive actions of PP2A and Aurora on Cdc13 limit telomerase action by removing active telomerase from telomeres at G2/M phase.

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC., (© 2014 Espert et al.)

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites., (© 2014 International Society for Neurochemistry.)