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Sample records for 2a pp2a activity

  1. Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

    SciTech Connect

    Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A.; Xing, Yongna

    2013-10-08

    Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.

  2. A subset of RAB proteins modulates PP2A phosphatase activity.

    PubMed

    Sacco, Francesca; Mattioni, Anna; Boldt, Karsten; Panni, Simona; Santonico, Elena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

    2016-09-09

    Protein phosphatase 2A (PP2A) is one of the most abundant serine-threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias.

  3. PP2A-activating drugs selectively eradicate TKI-resistant chronic myeloid leukemic stem cells

    PubMed Central

    Neviani, Paolo; Harb, Jason G.; Oaks, Joshua J.; Santhanam, Ramasamy; Walker, Christopher J.; Ellis, Justin J.; Ferenchak, Gregory; Dorrance, Adrienne M.; Paisie, Carolyn A.; Eiring, Anna M.; Ma, Yihui; Mao, Hsiaoyin C.; Zhang, Bin; Wunderlich, Mark; May, Philippa C.; Sun, Chaode; Saddoughi, Sahar A.; Bielawski, Jacek; Blum, William; Klisovic, Rebecca B.; Solt, Janelle A.; Byrd, John C.; Volinia, Stefano; Cortes, Jorge; Huettner, Claudia S.; Koschmieder, Steffen; Holyoake, Tessa L.; Devine, Steven; Caligiuri, Michael A.; Croce, Carlo M.; Garzon, Ramiro; Ogretmen, Besim; Arlinghaus, Ralph B.; Chen, Ching-Shih; Bittman, Robert; Hokland, Peter; Roy, Denis-Claude; Milojkovic, Dragana; Apperley, Jane; Goldman, John M.; Reid, Alistair; Mulloy, James C.; Bhatia, Ravi; Marcucci, Guido; Perrotti, Danilo

    2013-01-01

    The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs. PMID:23999433

  4. PP2A and GSK-3beta act antagonistically to regulate active zone development.

    PubMed

    Viquez, Natasha M; Füger, Petra; Valakh, Vera; Daniels, Richard W; Rasse, Tobias M; DiAntonio, Aaron

    2009-09-16

    The synapse is composed of an active zone apposed to a postsynaptic cluster of neurotransmitter receptors. Each Drosophila neuromuscular junction comprises hundreds of such individual release sites apposed to clusters of glutamate receptors. Here, we show that protein phosphatase 2A (PP2A) is required for the development of structurally normal active zones opposite glutamate receptors. When PP2A is inhibited presynaptically, many glutamate receptor clusters are unapposed to Bruchpilot (Brp), an active zone protein required for normal transmitter release. These unapposed receptors are not due to presynaptic retraction of synaptic boutons, since other presynaptic components are still apposed to the entire postsynaptic specialization. Instead, these data suggest that Brp localization is regulated at the level of individual release sites. Live imaging of glutamate receptors demonstrates that this disruption to active zone development is accompanied by abnormal postsynaptic development, with decreased formation of glutamate receptor clusters. Remarkably, inhibition of the serine-threonine kinase GSK-3beta completely suppresses the active zone defect, as well as other synaptic morphology phenotypes associated with inhibition of PP2A. These data suggest that PP2A and GSK-3beta function antagonistically to control active zone development, providing a potential mechanism for regulating synaptic efficacy at a single release site.

  5. IK-guided PP2A suppresses Aurora B activity in the interphase of tumor cells.

    PubMed

    Lee, Sunyi; Jeong, Ae Lee; Park, Jeong Su; Han, Sora; Jang, Chang-Young; Kim, Keun Il; Kim, Yonghwan; Park, Jong Hoon; Lim, Jong-Seok; Lee, Myung Sok; Yang, Young

    2016-09-01

    Aurora B activation is triggered at the mitotic entry and required for proper microtubule-kinetochore attachment at mitotic phase. Therefore, Aurora B should be in inactive form in interphase to prevent aberrant cell cycle progression. However, it is unclear how the inactivation of Aurora B is sustained during interphase. In this study, we find that IK depletion-induced mitotic arrest leads to G2 arrest by Aurora B inhibition, indicating that IK depletion enhances Aurora B activation before mitotic entry. IK binds to Aurora B, and colocalizes on the nuclear foci during interphase. Our data further show that IK inhibits Aurora B activation through recruiting PP2A into IK and Aurora B complex. It is thus believed that IK, as a scaffold protein, guides PP2A into Aurora B to suppress its activity in interphase until mitotic entry.

  6. Regulation of PP2A by Sphingolipid Metabolism and Signaling

    PubMed Central

    Oaks, Joshua; Ogretmen, Besim

    2014-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein. PMID:25642418

  7. Shugoshin-1 balances Aurora B kinase activity via PP2A to promote chromosome bi-orientation.

    PubMed

    Meppelink, Amanda; Kabeche, Lilian; Vromans, Martijn J M; Compton, Duane A; Lens, Susanne M A

    2015-04-28

    Correction of faulty kinetochore-microtubule attachments is essential for faithful chromosome segregation and dictated by the opposing activities of Aurora B kinase and PP1 and PP2A phosphatases. How kinase and phosphatase activities are appropriately balanced is less clear. Here, we show that a centromeric pool of PP2A-B56 counteracts Aurora B T-loop phosphorylation and is recruited to centromeres through Shugoshin-1 (Sgo1). In non-transformed RPE-1 cells, Aurora B, Sgo1, and PP2A-B56 are enriched on centromeres and levels diminish as chromosomes establish bi-oriented attachments. Elevating Sgo1 levels at centromeres recruits excess PP2A-B56, and this counteracts Aurora B kinase activity, undermining efficient correction of kinetochore-microtubule attachment errors. Conversely, Sgo1-depleted cells display reduced centromeric localization of Aurora B, whereas the remaining kinase is hyperactive due to concomitant reduction of centromeric PP2A-B56. Our data suggest that Sgo1 can tune the stability of kinetochore-microtubule attachments through recruitment of PP2A-B56 that balances Aurora B activity at the centromere.

  8. PP2A impaired activity is a common event in acute myeloid leukemia and its activation by forskolin has a potent anti-leukemic effect.

    PubMed

    Cristóbal, I; Garcia-Orti, L; Cirauqui, C; Alonso, M M; Calasanz, M J; Odero, M D

    2011-04-01

    Protein phosphatase 2A (PP2A) is a human tumor suppressor that inhibits cellular transformation by regulating the activity of several signaling proteins critical for malignant cell behavior. PP2A has been described as a potential therapeutic target in chronic myeloid leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia and B-cell chronic lymphocytic leukemia. Here, we show that PP2A inactivation is a recurrent event in acute myeloid leukemia (AML), and that restoration of PP2A phosphatase activity by treatment with forskolin in AML cells blocks proliferation, induces caspase-dependent apoptosis and affects AKT and ERK1/2 activity. Moreover, treatment with forskolin had an additive effect with Idarubicin and Ara-c, drugs used in standard induction therapy in AML patients. Analysis at protein level of the PP2A activation status in a series of patients with AML at diagnosis showed PP2A hyperphosphorylation in 78% of cases (29/37). In addition, we found that either deregulated expression of the endogenous PP2A inhibitors SET or CIP2A, overexpression of SETBP1, or downregulation of some PP2A subunits, might be contributing to PP2A inhibition in AML. In conclusion, our results show that PP2A inhibition is a common event in AML cells and that PP2A activators, such as forskolin or FTY720, could represent potential novel therapeutic targets in AML.

  9. PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects

    PubMed Central

    Zazo, Sandra; Arpí, Oriol; Menéndez, Silvia; Manso, Rebeca; Lluch, Ana; Eroles, Pilar; Rovira, Ana; Albanell, Joan; García-Foncillas, Jesús; Madoz-Gúrpide, Juan; Rojo, Federico

    2015-01-01

    The protein phosphatase 2A (PP2A) is a key tumor suppressor which has emerged as a novel molecular target in some human cancers. Here, we show that PP2A inhibition is a common event in breast cancer and identified PP2A phosphorylation and deregulation SET and CIP2A as molecular contributing mechanisms to inactivate PP2A. Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation. Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo. PP2A inhibition (CPscore: PP2A phosphorylation and/or CIP2A overexpression) was detected in 27% of cases (62/230), and associated with grade (p = 0.017), relapse (p < 0.001), negative estrogen (p < 0.001) and progesterone receptor expression (p < 0.001), HER2-positive tumors (p = 0.049), Ki-67 expression (p < 0.001), and higher AKT (p < 0.001) and ERK (p < 0.001) phosphorylation. Moreover, PP2A inhibition determined shorter overall (p = 0.006) and event-free survival (p = 0.003), and multivariate analysis confirmed its independent prognostic impact. Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease. PMID:25726524

  10. PP2A inhibition determines poor outcome and doxorubicin resistance in early breast cancer and its activation shows promising therapeutic effects.

    PubMed

    Rincón, Raúl; Cristóbal, Ion; Zazo, Sandra; Arpí, Oriol; Menéndez, Silvia; Manso, Rebeca; Lluch, Ana; Eroles, Pilar; Rovira, Ana; Albanell, Joan; García-Foncillas, Jesús; Madoz-Gúrpide, Juan; Rojo, Federico

    2015-02-28

    The protein phosphatase 2A (PP2A) is a key tumor suppressor which has emerged as a novel molecular target in some human cancers. Here, we show that PP2A inhibition is a common event in breast cancer and identified PP2A phosphorylation and deregulation SET and CIP2A as molecular contributing mechanisms to inactivate PP2A. Interestingly, restoration of PP2A activity after FTY720 treatment reduced cell growth, induced apoptosis and decreased AKT and ERK activation. Moreover, FTY720 led to PP2A activation then enhancing doxorubicin-induced antitumor effects both in vitro and in vivo. PP2A inhibition (CPscore: PP2A phosphorylation and/or CIP2A overexpression) was detected in 27% of cases (62/230), and associated with grade (p = 0.017), relapse (p < 0.001), negative estrogen (p < 0.001) and progesterone receptor expression (p < 0.001), HER2-positive tumors (p = 0.049), Ki-67 expression (p < 0.001), and higher AKT (p < 0.001) and ERK (p < 0.001) phosphorylation. Moreover, PP2A inhibition determined shorter overall (p = 0.006) and event-free survival (p = 0.003), and multivariate analysis confirmed its independent prognostic impact. Altogether, our results indicate that PP2A is frequently inactivated in breast cancer and determines worse outcome, and its restoration using PP2A activators represents an alternative therapeutic strategy in this disease.

  11. Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology.

    PubMed

    Sun, Lei; Ii, Adlai L Pappy; Pham, Tiffany T; Shanley, Thomas P

    2015-06-29

    Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces "immune tolerance" of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαl°xl°x;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαl°xl°x;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

  12. Opposing Activities of Aurora B Kinase and B56-PP2A Phosphatase on MKlp2 Determine Abscission Timing.

    PubMed

    Fung, Suet Yin Sarah; Kitagawa, Mayumi; Liao, Pei-Ju; Wong, Jasmine; Lee, Sang Hyun

    2017-01-09

    After cleavage furrow ingression during cytokinesis, nascent daughter cells remain connected by an intercellular bridge (ICB) and the midbody [1, 2]. The midbody becomes an assembly platform for ESCRT complexes that split apart the plasma membrane (PM) anchored to the ICB and complete abscission, which is the final step of cell division [3-5]. Aurora B governs abscission by regulating its timing as a checkpoint [6-10]. However, the underlying mechanisms for this process remain unknown. Here, we reveal the mechanism controlling abscission through integration of Aurora B kinase and B56-bound PP2A phosphatase activities on the kinesin motor protein MKlp2. We identify MKlp2 as an essential protein for promoting abscission, which may regulate tethering and stabilizing of the PM to the microtubule cytoskeleton at the ICB through its previously uncharacterized lipid association motif (LAM). MKlp2 recruits Aurora B to the ICB [11-15]. In turn, Aurora B phosphorylation of MKlp2 S878 in the LAM is a key inhibitory signal for abscission. Conversely, B56-PP2A promotes abscission by opposing Aurora B phosphorylation of MKlp2 S878. Strikingly, a phospho-resistant MKlp2 S878A mutant overcomes Aurora-B-mediated abscission blockade. Thus, abscission is determined by the balance of Aurora B and B56-PP2A activities on MKlp2 S878 within the LAM. Together, these findings establish a key mechanism for Aurora B regulation of abscission in mammalian cells.

  13. Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    PubMed Central

    Shu, Guangwen; Zhang, Lang; Jiang, Shanqing; Cheng, Zhuo; Wang, Guan; Huang, Xu; Yang, Xinzhou

    2016-01-01

    Our previous study discovered that isoliensinine (isolie) triggers hepatocellular carcinoma (HCC) cell apoptosis via inducing p65 dephosphorylation at Ser536 and inhibition of NF-κB. Here, we showed that isolie promoted p65/PP2A interaction in vitro and in vivo. Repression of PP2A activity or knockdown of the expression of PP2A-C (the catalytic subunit of PP2A) abrogated isolie-provoked p65 dephosphorylation. I2PP2A is an endogenous PP2A inhibitor. Isolie directly impaired PP2A/I2PP2A interaction. Knockdown of I2PP2A boosted p65/PP2A association and p65 dephosphorylation. Overexpression of I2PP2A restrained isolie-induced p65 dephosphorylation. Untransformed hepatocytes were insensitive to isolie-induced NF-κB inhibition and cell apoptosis. In these cells, basal levels of I2PP2A and p65 phosphorylation at Ser536 were lower than in HCC cells. These findings collectively indicated that isolie suppresses NF-κB in HCC cells through impairing PP2A/I2PP2A interaction and stimulating PP2A-dependent p65 dephosphorylation at Ser536. PMID:27244888

  14. Isoliensinine induces dephosphorylation of NF-kB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction.

    PubMed

    Shu, Guangwen; Zhang, Lang; Jiang, Shanqing; Cheng, Zhuo; Wang, Guan; Huang, Xu; Yang, Xinzhou

    2016-06-28

    Our previous study discovered that isoliensinine (isolie) triggers hepatocellular carcinoma (HCC) cell apoptosis via inducing p65 dephosphorylation at Ser536 and inhibition of NF-κB. Here, we showed that isolie promoted p65/PP2A interaction in vitro and in vivo. Repression of PP2A activity or knockdown of the expression of PP2A-C (the catalytic subunit of PP2A) abrogated isolie-provoked p65 dephosphorylation. I2PP2A is an endogenous PP2A inhibitor. Isolie directly impaired PP2A/I2PP2A interaction. Knockdown of I2PP2A boosted p65/PP2A association and p65 dephosphorylation. Overexpression of I2PP2A restrained isolie-induced p65 dephosphorylation. Untransformed hepatocytes were insensitive to isolie-induced NF-κB inhibition and cell apoptosis. In these cells, basal levels of I2PP2A and p65 phosphorylation at Ser536 were lower than in HCC cells. These findings collectively indicated that isolie suppresses NF-κB in HCC cells through impairing PP2A/I2PP2A interaction and stimulating PP2A-dependent p65 dephosphorylation at Ser536.

  15. The PP2A inhibitor I2PP2A is essential for sister chromatid segregation in oocyte meiosis II.

    PubMed

    Chambon, Jean-Philippe; Touati, Sandra A; Berneau, Stéphane; Cladière, Damien; Hebras, Céline; Groeme, Rachel; McDougall, Alex; Wassmann, Katja

    2013-03-18

    Haploid gametes are generated through two consecutive meiotic divisions, with the segregation of chromosome pairs in meiosis I and sister chromatids in meiosis II. Separase-mediated stepwise removal of cohesion, first from chromosome arms and later from the centromere region, is a prerequisite for maintaining sister chromatids together until their separation in meiosis II [1]. In all model organisms, centromeric cohesin is protected from separase-dependent removal in meiosis I through the activity of PP2A-B56 phosphatase, which is recruited to centromeres by shugoshin/MEI-S332 (Sgo) [2-5]. How this protection of centromeric cohesin is removed in meiosis II is not entirely clear; we find that all the PP2A subunits remain colocalized with the cohesin subunit Rec8 at the centromere of metaphase II chromosomes. Here, we show that sister chromatid separation in oocytes depends on a PP2A inhibitor, namely I2PP2A. I2PP2A colocalizes with the PP2A enzyme at centromeres at metaphase II, independently of bipolar attachment. When I2PP2A is depleted, sister chromatids fail to segregate during meiosis II. Our findings demonstrate that in oocytes I2PP2A is essential for faithful sister chromatid segregation by mediating deprotection of centromeric cohesin in meiosis II.

  16. Structure-Activity Relationship Studies of Fostriecin, Cytostatin, and Key Analogs, with PP1, PP2A, PP5, and (β12–β13)-Chimeras (PP1/PP2A and PP5/PP2A), Provide Further Insight into the Inhibitory Actions of Fostriecin Family Inhibitors

    PubMed Central

    Swingle, Mark R.; Amable, Lauren; Lawhorn, Brian G.; Buck, Suzanne B.; Burke, Christopher P.; Ratti, Pukar; Fischer, Kimberly L.; Boger, Dale L.

    2009-01-01

    Fostriecin and cytostatin are structurally related natural inhibitors of serine/threonine phosphatases, with promising antitumor activity. The total synthesis of these antitumor agents has enabled the production of structural analogs, which are useful to explore the biological significance of features contained in the parent compounds. Here, the inhibitory activity of fostriecin, cytostatin, and 10 key structural analogs were tested in side-by-side phosphatase assays to further characterize their inhibitory activity against PP1c (Ser/Thr protein phosphatase 1 catalytic subunit), PP2Ac (Ser/Thr protein phosphatase 2A catalytic subunit), PP5c (Ser/Thr protein phosphatase 5 catalytic subunit), and chimeras of PP1 (Ser/Thr protein phosphatase 1) and PP5 (Ser/Thr protein phosphatase 5), in which key residues predicted for inhibitor contact with PP2A (Ser/Thr protein phosphatase 2A) were introduced into PP1 and PP5 using site-directed mutagenesis. The data confirm the importance of the C9-phosphate and C11-alcohol for general inhibition and further demonstrate the importance of a predicted C3 interaction with a unique cysteine (Cys269) in the β12–β13 loop of PP2A. The data also indicate that additional features beyond the unsaturated lactone contribute to inhibitory potency and selectivity. Notably, a derivative of fostriecin lacking the entire lactone subunit demonstrated marked potency and selectivity for PP2A, while having substantially reduced and similar activity against PP1 and PP1/PP2A- PP5/PP2A-chimeras that have greatly increased sensitivity to both fostriecin and cytostatin. This suggests that other features [e.g., the (Z,Z,E)-triene] also contribute to inhibitory selectivity. When considered together with previous data, these studies suggest that, despite the high structural conservation of the catalytic site in PP1, PP2A and PP5, the development of highly selective catalytic inhibitors should be feasible. PMID:19592665

  17. Methionine and S-Adenosylmethionine levels are critical regulators of PP2A activity modulating lipophagy during steatosis

    PubMed Central

    Zubiete-Franco, Imanol; García-Rodríguez, Juan Luis; Martínez-Uña, Maite; Martínez-Lopez, Nuria; Woodhoo, Ashwin; Juan, Virginia Gutiérrez-De; Beraza, Naiara; Lage-Medina, Sergio; Andrade, Fernando; Fernandez, Marta Llarena; Aldámiz-Echevarría, Luis; Fernández-Ramos, David; Falcon-Perez, Juan Manuel; Lopitz-Otsoa, Fernando; Fernandez-Tussy, Pablo; Barbier-Torres, Lucía; Luka, Zigmund; Wagner, Conrad; García-Monzón, Carmelo; Lu, Shelly C.; Aspichueta, Patricia; Mato, José María; Martínez-Chantar, María Luz; Varela-Rey, Marta

    2015-01-01

    Background & Aims Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe NAFLD. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism. Methods We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice. Results We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and Methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameloriated liver steatosis. Conclusions These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis. PMID:26394163

  18. PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

    PubMed

    Rodriguez Pino, Marbelys; Castillo, Boris; Kim, Bohye; Kim, Lou W

    2015-12-01

    We have previously shown that the Dictyostelium protein phosphatase 2A regulatory subunit B56, encoded by psrA, modulates Dictyostelium cell differentiation through negatively affecting glycogen synthase kinase 3 (GSK3) function. Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient. Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates. Expression of constitutively active Ras mutants or inhibition of GSK3 in psrA(-) cells increased activities of both PKBR1 and PKBA, but only the PKBR1 activity was increased in wild-type cells under the equivalent conditions, indicating that either B56- or GSK3-mediated suppressive mechanism is sufficient to maintain low PKBA activity, but both mechanisms are necessary for suppressing PKBR1. Finally, cells lacking RasD or RasC displayed normal PKBR1 regulation under GSK3-inhibiting conditions, indicating that RasC or RasD proteins are essential for GSK3-mediated PKBR1 inhibition. In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

  19. PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis

    PubMed Central

    Rodriguez Pino, Marbelys; Castillo, Boris; Kim, Bohye; Kim, Lou W.

    2015-01-01

    We have previously shown that the Dictyostelium protein phosphatase 2A regulatory subunit B56, encoded by psrA, modulates Dictyostelium cell differentiation through negatively affecting glycogen synthase kinase 3 (GSK3) function. Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA− cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient. Surprisingly, psrA− cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates. Expression of constitutively active Ras mutants or inhibition of GSK3 in psrA− cells increased activities of both PKBR1 and PKBA, but only the PKBR1 activity was increased in wild-type cells under the equivalent conditions, indicating that either B56- or GSK3-mediated suppressive mechanism is sufficient to maintain low PKBA activity, but both mechanisms are necessary for suppressing PKBR1. Finally, cells lacking RasD or RasC displayed normal PKBR1 regulation under GSK3-inhibiting conditions, indicating that RasC or RasD proteins are essential for GSK3-mediated PKBR1 inhibition. In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis. PMID:26424797

  20. PP2A Regulates HDAC4 Nuclear Import

    PubMed Central

    Paroni, Gabriela; Cernotta, Nadia; Dello Russo, Claudio; Gallinari, Paola; Pallaoro, Michele; Foti, Carmela; Talamo, Fabio; Orsatti, Laura; Steinkühler, Christian

    2008-01-01

    Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme Cα, Aα, B/PR55α. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import. PMID:18045992

  1. Silencing I2PP2A Rescues Tau Pathologies and Memory Deficits through Rescuing PP2A and Inhibiting GSK-3β Signaling in Human Tau Transgenic Mice

    PubMed Central

    Zhang, Yao; Ma, Rong-Hong; Li, Xia-Chun; Zhang, Jia-Yu; Shi, Hai-Rong; Wei, Wei; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2014-01-01

    Increase of inhibitor-2 of protein phosphatase-2A I2PP2A is associated with protein phosphatase-2A (PP2A) inhibition and tau hyperphosphorylation in Alzheimer’s disease (AD). Down-regulating I2PP2A attenuated amyloidogenesis and improved the cognitive functions in transgenic mice expressing amyloid precursor protein (tg2576). Here, we found that silencing I2PP2A by hippocampal infusion of Lenti - siI2PP2A down-regulated I2PP2A (~45%) with reduction of tau phosphorylation/accumulation, improvement of memory deficits, and dendritic plasticity in 12-month-old human tau transgenic mice. Silencing I2PP2A not only restored PP2A activity but also inhibited glycogen synthase kinase-3β (GSK-3β) with a significant activation of protein kinase A (PKA) and Akt. In HEK293/tau and N2a/tau cells, silencing I2PP2A by pSUPER - siI2PP2A also significantly reduced tau hyperphosphorylation with restoration of PP2A activity and inhibition of GSK-3β, demonstrated by the decreased GSK-3β total protein and mRNA levels, and the increased inhibitory phosphorylation of GSK-3β at serine-9. Furthermore, activation of PKA but not Akt mediated the inhibition of GSK-3β by I2PP2A silencing. We conclude that targeting I2PP2A can improve tau pathologies and memory deficits in human tau transgenic mice, and activation of PKA contributes to GSK-3β inhibition induced by silencing I2PP2A in vitro, suggesting that I2PP2A is a promising multiple target of AD. PMID:24987368

  2. The Brassinosteroid-Activated BRI1 Receptor Kinase Is Switched off by Dephosphorylation Mediated by Cytoplasm-Localized PP2A B' Subunits.

    PubMed

    Wang, Ruiju; Liu, Mengmeng; Yuan, Min; Oses-Prieto, Juan A; Cai, Xingbo; Sun, Ying; Burlingame, Alma L; Wang, Zhi-Yong; Tang, Wenqiang

    2016-01-04

    Brassinosteroid (BR) binding activates the receptor kinase BRI1 by inducing heterodimerization with its co-receptor kinase BAK1; however, the mechanisms that reversibly inactivate BRI1 remain unclear. Here we show that cytoplasm-localized protein phosphatase 2A (PP2A) B' regulatory subunits interact with BRI1 to mediate its dephosphorylation and inactivation. Loss-of-function and overexpression experiments showed that a group of PP2A B' regulatory subunits, represented by B'η, negatively regulate BR signaling by decreasing BRI1 phosphorylation. BR increases the expression levels of these B' subunits, and B'η interacts preferentially with phosphorylated BRI1, suggesting that the dynamics of BR signaling are modulated by the PP2A-mediated feedback inactivation of BRI1. Compared with PP2A B'α and B'β, which promote BR responses by dephosphorylating the downstream transcription factor BZR1, the BRI1-inactivating B' subunits showed similar binding to BRI1 and BZR1 but distinct subcellular localization. Alteration of the nuclear/cytoplasmic localization of the B' subunits revealed that cytoplasmic PP2A dephosphorylates BRI1 and inhibits the BR response, whereas nuclear PP2A dephosphorylates BZR1 and activates the BR response. Our findings not only identify the PP2A regulatory B subunits that mediate the binding and dephosphorylation of BRI1, but also demonstrate that the subcellular localization of PP2A specifies its substrate selection and distinct effects on BR signaling.

  3. PhosphoTyrosyl phosphatase activator of Plasmodium falciparum: identification of its residues involved in binding to and activation of PP2A.

    PubMed

    Vandomme, Audrey; Fréville, Aline; Cailliau, Katia; Kalamou, Hadidjatou; Bodart, Jean-François; Khalife, Jamal; Pierrot, Christine

    2014-02-11

    In Plasmodium falciparum (Pf), the causative agent of the deadliest form of malaria, a tight regulation of phosphatase activity is crucial for the development of the parasite. In this study, we have identified and characterized PfPTPA homologous to PhosphoTyrosyl Phosphatase Activator, an activator of protein phosphatase 2A which is a major phosphatase involved in many biological processes in eukaryotic cells. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human are conserved in P. falciparum. Localization studies showed that PfPTPA and PfPP2A are present in the same compartment of blood stage parasites, suggesting a possible interaction of both proteins. In vitro binding and functional studies revealed that PfPTPA binds to and activates PP2A. Mutation studies showed that three residues (V(283), G(292) and M(296)) of PfPTPA are indispensable for the interaction and that the G(292) residue is essential for its activity. In P. falciparum, genetic studies suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Using Xenopus oocytes, we showed that PfPTPA blocked the G2/M transition. Taken together, our data suggest that PfPTPA could play a role in the regulation of the P. falciparum cell cycle through its PfPP2A regulatory activity.

  4. PhosphoTyrosyl Phosphatase Activator of Plasmodium falciparum: Identification of Its Residues Involved in Binding to and Activation of PP2A

    PubMed Central

    Vandomme, Audrey; Fréville, Aline; Cailliau, Katia; Kalamou, Hadidjatou; Bodart, Jean-François; Khalife, Jamal; Pierrot, Christine

    2014-01-01

    In Plasmodium falciparum (Pf), the causative agent of the deadliest form of malaria, a tight regulation of phosphatase activity is crucial for the development of the parasite. In this study, we have identified and characterized PfPTPA homologous to PhosphoTyrosyl Phosphatase Activator, an activator of protein phosphatase 2A which is a major phosphatase involved in many biological processes in eukaryotic cells. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human are conserved in P. falciparum. Localization studies showed that PfPTPA and PfPP2A are present in the same compartment of blood stage parasites, suggesting a possible interaction of both proteins. In vitro binding and functional studies revealed that PfPTPA binds to and activates PP2A. Mutation studies showed that three residues (V283, G292 and M296) of PfPTPA are indispensable for the interaction and that the G292 residue is essential for its activity. In P. falciparum, genetic studies suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Using Xenopus oocytes, we showed that PfPTPA blocked the G2/M transition. Taken together, our data suggest that PfPTPA could play a role in the regulation of the P. falciparum cell cycle through its PfPP2A regulatory activity. PMID:24521882

  5. Notch-1 signaling activates NF-κB in human breast carcinoma MDA-MB-231 cells via PP2A-dependent AKT pathway.

    PubMed

    Li, Li; Zhang, Jing; Xiong, Niya; Li, Shun; Chen, Yu; Yang, Hong; Wu, Chunhui; Zeng, Hongjuan; Liu, Yiyao

    2016-04-01

    Breast cancer has a high incidence in the world and is becoming a leading cause of death in female patients due to its high metastatic ability. High expression of Notch-1 and its ligand Jagged-1 correlates with poor prognosis in breast cancer. Our previous work has shown that Notch-1 signaling pathway upregulates NF-κB transcriptional activity and induces the adhesion, migration and invasion of human breast cancer cell line MDA-MB-231. However, the role of Notch-1 in NF-κB activation is still poorly understood. Here, we aim to understand the exact mechanism that Notch-1 regulates NF-κB activity. In MDA-MB-231 cells where Notch-1 is constitutively activated, the phosphorylation of p85 and AKT (Tyr308/Ser473) is upregulated, indicating PI3K/AKT pathway is activated. Notch-1 activation caused the increase of PP2A phosphorylation at Tyr307, indicating Notch-1 inhibits PP2A activity. NF-κB transcriptional activity was evaluated by dual-luciferase reporter assay, and the results showed that, while silencing of Notch-1, PP2A activity was upregulated and NF-κB activity was downregulated, whereas PP2A inhibitor okadaic acid (OA) restored NF-κB activity. Immunofluorescence and Western blots showed that OA treatment antagonized the decrease of p65 nuclear translocation caused by Notch-1 silencing. Moreover, OA treatment also upregulated MMP-2, MMP-9 and VEGF mRNA expression levels, indicating OA rescues Notch-1 silencing that caused low cell invasion. Taken together, our results suggest that Notch-1-activating PI3K/AKT/NF-κB pathway is PP2A dependent; PP2A may be a potential therapeutic target in breast cancer.

  6. The phosphorylated form of FTY720 activates PP2A, represses inflammation and is devoid of S1P agonism in A549 lung epithelial cells.

    PubMed

    Rahman, Md Mostafizur; Prünte, Laura; Lebender, Leonard F; Patel, Brijeshkumar S; Gelissen, Ingrid; Hansbro, Philip M; Morris, Jonathan C; Clark, Andrew R; Verrills, Nicole M; Ammit, Alaina J

    2016-11-16

    Protein phosphatase 2A (PP2A) activity can be enhanced pharmacologically by PP2A-activating drugs (PADs). The sphingosine analog FTY720 is the best known PAD and we have shown that FTY720 represses production of pro-inflammatory cytokines responsible for respiratory disease pathogenesis. Whether its phosphorylated form, FTY720-P, also enhances PP2A activity independently of the sphingosine 1-phosphate (S1P) pathway was unknown. Herein, we show that FTY720-P enhances TNF-induced PP2A phosphatase activity and significantly represses TNF-induced interleukin 6 (IL-6) and IL-8 mRNA expression and protein secretion from A549 lung epithelial cells. Comparing FTY720 and FTY720-P with S1P, we show that unlike S1P, the sphingosine analogs do not induce cytokine production on their own. In fact, FTY720 and FTY720-P significantly repress S1P-induced IL-6 and IL-8 production. We then examined their impact on expression of cyclooxygenase 2 (COX-2) and resultant prostaglandin E2 (PGE2) production. S1P did not increase production of this pro-inflammatory enzyme because COX-2 mRNA gene expression is NF-κB-dependent, and unlike TNF, S1P did not activate NF-κB. However, TNF-induced COX-2 mRNA expression and PGE2 secretion is repressed by FTY720 and FTY720-P. Hence, FTY720-P enhances PP2A activity and that PADs can repress production of pro-inflammatory cytokines and enzymes in A549 lung epithelial cells in a manner devoid of S1P agonism.

  7. B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation*

    PubMed Central

    Jayadeva, Girish; Kurimchak, Alison; Garriga, Judit; Sotillo, Elena; Davis, Anthony J.; Haines, Dale S.; Mumby, Marc; Graña, Xavier

    2010-01-01

    Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G0/G1 transition and the cell cycle restriction point in G1. Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G1 and G1/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation. PMID:20663872

  8. Mitotic exit: Determining the PP2A dephosphorylation program.

    PubMed

    Pereira, Gislene; Schiebel, Elmar

    2016-08-29

    In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55's dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis.

  9. Mitotic exit: Determining the PP2A dephosphorylation program

    PubMed Central

    2016-01-01

    In mitotic exit, proteins that were highly phosphorylated are sequentially targeted by the phosphatase PP2A-B55, but what underlies substrate selection is unclear. In this issue, Cundell et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201606033) identify the determinants of PP2A-B55’s dephosphorylation program, thereby influencing spindle disassembly, nuclear envelope reformation, and cytokinesis. PMID:27551057

  10. The Structural Basis for Tight Control of PP2A Methylation and Function by LCMT-1

    SciTech Connect

    V Stanevich; L Jiang; K Satyshur; Y Li; P Jeffrey; Z Li; P Menden; M Semmelhack; Y Xing

    2011-12-31

    Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, likely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.

  11. The Structural Basis for Tight Control of PP2A Methylation and Function by LCMT-1

    SciTech Connect

    Stanevich, Vitali; Jiang, Li; Satyshur, Kenneth A.; Li, Yongfeng; Jeffrey, Philip D.; Li, Zhu; Menden, Patrick; Semmelhack, Martin F.; Xing, Yongna

    2012-05-29

    Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, likely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.

  12. Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach.

    PubMed

    Kondo, Koyuki; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades.

  13. Suggested Involvement of PP1/PP2A Activity and De Novo Gene Expression in Anhydrobiotic Survival in a Tardigrade, Hypsibius dujardini, by Chemical Genetic Approach

    PubMed Central

    Kondo, Koyuki; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade, Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades. PMID:26690982

  14. From the Biology of PP2A to the PADs for Therapy of Hematologic Malignancies

    PubMed Central

    Ciccone, Maria; Calin, George A.; Perrotti, Danilo

    2015-01-01

    Over the past decades, an emerging role of phosphatases in the pathogenesis of hematologic malignancies and solid tumors has been established. The tumor-suppressor protein phosphatase 2A (PP2A) belongs to the serine–threonine phosphatases family and accounts for the majority of serine–threonine phosphatase activity in eukaryotic cells. Numerous studies have shown that inhibition of PP2A expression and/or function may contribute to leukemogenesis in several hematological malignancies. Likewise, overexpression or aberrant expression of physiologic PP2A inhibitory molecules (e.g., SET and its associated SETBP1 and CIP2A) may turn off PP2A function and participate to leukemic progression. The discovery of PP2A as tumor suppressor has prompted the evaluation of the safety and the efficacy of new compounds, which can restore PP2A activity in leukemic cells. Although further studies are needed to better understand how PP2A acts in the intricate phosphatases/kinases cancer network, the results reviewed herein strongly support the development on new PP2A-activating drugs and the immediate introduction of those available into clinical protocols for leukemia patients refractory or resistant to current available therapies. PMID:25763353

  15. Activation of CK1ε by PP2A/PR61ε is required for the initiation of Wnt signaling

    PubMed Central

    Curto, J; Padilla, M; Villarroel, A; Yang, J; de Herreros, AG; Duñach, M

    2016-01-01

    Canonical Wnt signaling induces the stabilization of β-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 coreceptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ε, γ and GSK3. Among these, activation of CK1ε, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ε is activated by the PP2A/PR61ε phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ε with Frizzled-bound PR61ε regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ε and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands. PMID:27321178

  16. Activation of CK1ɛ by PP2A/PR61ɛ is required for the initiation of Wnt signaling.

    PubMed

    Vinyoles, M; Del Valle-Pérez, B; Curto, J; Padilla, M; Villarroel, A; Yang, J; de Herreros, A G; Duñach, M

    2017-01-19

    Canonical Wnt signaling induces the stabilization of β-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 co-receptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ɛ, γ and GSK3. Among these, activation of CK1ɛ, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ɛ is activated by the PP2A/PR61ɛ phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ɛ with Frizzled-bound PR61ɛ regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ɛ and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.

  17. Pathologic significance of SET/I2PP2A-mediated PP2A and non-PP2A pathways in polycystic ovary syndrome (PCOS).

    PubMed

    Jiang, Shi-Wen; Xu, Siliang; Chen, Haibin; Liu, Xiaoqiang; Tang, Zuoqing; Cui, Yugui; Liu, Jiayin

    2017-01-01

    SET (SE translocation, SET), a constitutive inhibitor of protein phosphatase 2A (PP2A), is a multifunctional oncoprotein involved in DNA replication, histone modification, nucleosome assembly, gene transcription and cell proliferation. It is widely expressed in human tissues including the gonadal system and brain. Intensive studies have shown that overexpressed SET plays an important role in the development of Alzheimer's disease (AD), and may also contribute to the malignant transformation of breast and ovarian cancers. Recent studies indicated that through interaction with PP2A, SET may upregulate androgen biosynthesis and contribute to hyperandrogenism in polycystic ovary syndrome (PCOS) patients. This review article summarizes data concerning the SET expression in ovaries from PCOS and normal women, and analyzes the role/regulatory mechanism of SET for androgen biosynthesis in PCOS, as well as the significance of this action in the development of PCOS. The potential value of SET-triggered pathway as a therapeutic target and the application of anti-SET reagents for treating hyperandrogenism in PCOS patients are also discussed.

  18. Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia

    PubMed Central

    Gutierrez, Alejandro; Pan, Li; Groen, Richard W.J.; Baleydier, Frederic; Kentsis, Alex; Marineau, Jason; Grebliunaite, Ruta; Kozakewich, Elena; Reed, Casie; Pflumio, Francoise; Poglio, Sandrine; Uzan, Benjamin; Clemons, Paul; VerPlank, Lynn; An, Frank; Burbank, Jason; Norton, Stephanie; Tolliday, Nicola; Steen, Hanno; Weng, Andrew P.; Yuan, Huipin; Bradner, James E.; Mitsiades, Constantine; Look, A. Thomas; Aster, Jon C.

    2014-01-01

    T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug’s antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential. PMID:24401270

  19. AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway.

    PubMed

    Chen, Bo; Li, Jin; Zhu, Haibo

    2016-10-01

    The differentiation of macrophages into lipid-laden foam cells is a hallmark in early-stage atherosclerosis. The developmental role of adenosine monophosphate-activated protein kinase (AMPK) in a transformation of foam cells, especially in macrophage cholesterol uptake that remains undetermined. Here we demonstrate that AMPK activation in response to IMM-H007 or AICAR resulted in a decrease in macrophage cholesterol uptake and thus inhibited foam cell formation in macrophages mediated by oxidized low-density lipoprotein (oxLDL). This functional change was caused by a downregulation of mRNA and protein expression of LOX-1 but not other scavenger receptors, including scavenger receptor-A (SR-A), CD36 and scavenger receptor-BI (SR-BI). The expression of LOX-1 was regulated by AMPK activation induced decreased phosphorylation of nuclear transcription factor NF-κB, since siRNA interference or dominant negative AMPK overexpression significantly promotes Ser536 dephosphorylation of NF-κB p65 and thus increases LOX-1 expression. Moreover, pharmacological AMPK activation was shown to promote protein phosphatase 2A (PP2A) activity and the specific PP2A inhibitor, okadaic acid, could prevent the effects of IMM-H007 or AICAR on NF-κB and LOX-1. In vivo, pharmacological AMPK activation reduced the lesion size of atherosclerosis and the expression of LOX-1 in aortas in apolipoprotein E-deficient mice. Our current findings suggest a novel mechanism of LOX-1 regulation by AMPK to attenuate macrophage oxLDL uptake and atherosclerosis.

  20. Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.

    PubMed

    Kaur, Amanpreet; Westermarck, Jukka

    2016-12-15

    Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis by dephosphorylation of a variety of signaling proteins and acts as a tumor suppressor. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by highly complex mechanisms that are reviewed here. Importantly, recent studies have shown that PME-1 promotes oncogenic MAPK/ERK and AKT pathway activities in various cancer types. In human glioma, high PME-1 expression correlates with tumor progression and kinase inhibitor resistance. We discuss the emerging cancer-associated function of PME-1 and its potential clinical relevance.

  1. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    PubMed

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  2. Molecular cloning, expression and functional analysis of three subunits of protein phosphatase 2A (PP2A) from black tiger shrimps (Penaeus monodon).

    PubMed

    Zhao, Chao; Wang, Yan; Fu, Mingjun; Yang, Keng; Qiu, Lihua

    2017-02-01

    Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.

  3. Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A

    PubMed Central

    Persad, Amit; Venkateswaran, Geetha; Hao, Li; Garcia, Maria E.; Yoon, Jenny; Sidhu, Jaskiran; Persad, Sujata

    2016-01-01

    Dysregulation of Wnt/β-catenin signaling has been associated with the development and progression of many cancers. The stability and subcellular localization of β-catenin, a dual functional protein that plays a role in intracellular adhesion and in regulating gene expression, is tightly regulated. However, little is known about the transcriptionally active form of β-catenin, Active Beta Catenin (ABC), that is unphosphorylated at serine 37 (Ser37) and threonine 41 (Thr41). Elucidating the mechanism by which β-catenin is activated to generate ABC is vital to the development of therapeutic strategies to block β-catenin signaling for cancer treatment. Using melanoma, breast and prostate cancer cell lines, we show that while cellular β-catenin levels are regulated by the Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway and are dependent on the phosphatase activity of the protein phosphatase PP2A. Furthermore, we demonstrate that although the PI3K/PTEN pathway does not regulate total β-catenin protein levels within the cell, it plays a role in regulating the subcellular localization of β-catenin. Our results support a novel functional interaction/cross-talk between the PTEN/PI3K and Wnt pathways in the regulation of the subcellular/nuclear levels of ABC, which is crucially important for the protein's activity as a transcription factor and its biological effects in health and disease. PMID:28191283

  4. C-terminal truncation of GSK-3β enhances its dephosphorylation by PP2A.

    PubMed

    Jin, Nana; Wu, Yue; Xu, Wen; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2017-03-07

    Glycogen synthase kinase-3β (GSK-3β) is the major tau kinase. Its phosphorylation at Ser9 suppresses the activity. In Alzheimer's disease (AD) brain, GSK-3β is truncated at the C-terminus by over-activated calpain I, leading to an increase in its activity. However, the effect of truncation on its phosphorylation is unknown. We found here that in AD brain and in cultured cells, C-terminally truncated GSK-3β is less phosphorylated at Ser9 than the full-length enzyme. The truncation promotes GSK-3β nuclear translocation and enhances its interaction with protein phosphatase 2A (PP2A), leading to dephosphorylation. Thus, the truncation of GSK-3β may enhance its activity through Ser9 dephosphorylation by PP2A. Our findings shed new light onto the role of calpain - GSK-3β - PP2A in tau pathogenesis of AD. This article is protected by copyright. All rights reserved.

  5. Overexpression of the PP2A-C5 gene confers increased salt tolerance in Arabidopsis thaliana

    PubMed Central

    Hu, Rongbin; Zhu, Yinfeng; Shen, Guoxin; Zhang, Hong

    2017-01-01

    ABSTRACT Protein phosphatase 2A (PP2A) was shown to play important roles in biotic and abiotic stress signaling pathways in plants. PP2A is made of 3 subunits: a scaffolding subunit A, a regulatory subunit B, and a catalytic subunit C. It is believed that the B subunit recognizes specific substrates and the C subunit directly acts on the selected substrates, whereas the A subunit brings a B subunit and a C subunit together to form a specific PP2A holoenzyme. Because there are multiple isoforms for each PP2A subunit, there could be hundreds of novel PP2A holoenzymes in plants. For an example, there are 3 A subunits, 17 B subunits, and 5 C subunits in Arabidopsis, which could form 255 different PP2A holoenzymes. Understanding the roles of these PP2A holoenzymes in various signaling pathways is a challenging task. In a recent study,1 we discovered that PP2A-C5, the catalytic subunit 5 of PP2A, plays an important role in salt tolerance in Arabidopsis. We found that a knockout mutant of PP2A-C5 (i.e. pp2a-c5–1) was very sensitive to salt treatments, whereas PP2A-C5-overexpressing plants were more tolerant to salt stresses. Genetic analyses between pp2a-c5–1 and Salt-Overly-Sensitive (SOS) mutants indicated that PP2A-C5 does not function in the same pathway as SOS genes. Using yeast 2-hybrid analysis, we found that PP2A-C5 interacts with several vacuolar membrane bound chloride channel proteins. We hypothesize that these vacuolar chloride channel proteins might be PP2A-C5's substrates in vivo, and the action of PP2A-C5 on these channel proteins could increase or activate their activities, thereby result in accumulation of the chloride and sodium contents in vacuoles, leading to increased salt tolerance in plants. PMID:28045581

  6. The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma.

    PubMed

    Enjoji, Shuhei; Yabe, Ryotaro; Fujiwara, Nobuyuki; Tsuji, Shunya; Vitek, Michael P; Mizuno, Takuya; Nakagawa, Takayuki; Usui, Tatsuya; Ohama, Takashi; Sato, Koichi

    2015-11-01

    Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.

  7. Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

    PubMed

    Hales, Eric C; Orr, Steven M; Larson Gedman, Amanda; Taub, Jeffrey W; Matherly, Larry H

    2013-08-02

    Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

  8. Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56.

    PubMed

    Wang, Jiao; Okkeri, Juha; Pavic, Karolina; Wang, Zhizhi; Kauko, Otto; Halonen, Tuuli; Sarek, Grzegorz; Ojala, Päivi M; Rao, Zihe; Xu, Wenqing; Westermarck, Jukka

    2017-03-01

    Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two-hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N-terminal CIP2A fragment (amino acids 1-560) at 3.0 Å resolution, and by subsequent structure-based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N-terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure-function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy.

  9. A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi.

    PubMed Central

    González, Jorge; Cornejo, Alberto; Santos, Marcia R M; Cordero, Esteban M; Gutiérrez, Bessy; Porcile, Patricio; Mortara, Renato A; Sagua, Hernán; Da Silveira, José Franco; Araya, Jorge E

    2003-01-01

    Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite. PMID:12737627

  10. PP2A inhibition as a novel therapeutic target in castration-resistant prostate cancer.

    PubMed

    González-Alonso, Paula; Cristóbal, Ion; Manso, Rebeca; Madoz-Gúrpide, Juan; García-Foncillas, Jesús; Rojo, Federico

    2015-08-01

    Protein phosphatase 2A (PP2A) is a well-known tumor suppressor frequently inhibited in human cancer. Alterations affecting PP2A subunits together with the deregulation of endogenous PP2A inhibitors such as CIP2A and SET have been described as contributing mechanisms to inactivate PP2A in prostate cancer. Moreover, recent findings highlight that functional inactivation of PP2A could represent a key event in the acquisition of castration-resistant phenotype and a novel molecular target with high impact at both clinical and therapeutic levels in prostate cancer.

  11. Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau*

    PubMed Central

    Arif, Mohammad; Wei, Jianshe; Zhang, Qi; Liu, Fei; Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Iqbal, Khalid

    2014-01-01

    Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2PP2A). In AD brain, I2PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2PP2A containing a conserved basic motif, 179RKR181, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2PP2A in which the NLS was inactivated by 179RKR181 → AAA along with 168KR169 → AA mutations. In this model, the mutant NLS (mNLS)-I2PP2A (I2PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology. PMID:25128526

  12. Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases.

    PubMed

    Luo, J; Pato, M D; Riordan, J R; Hanrahan, J W

    1998-05-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity declines rapidly when excised from transfected Chinese hamster ovary (CHO) or human airway cells because of membrane-associated phosphatase activity. In the present study, we found that CFTR channels usually remained active in patches excised from baby hamster kidney (BHK) cells overexpressing CFTR. Those patches with stable channel activity were used to investigate the regulation of CFTR by exogenous protein phosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase to excised patches reduced CFTR channel activity by > 90% but did not abolish it completely. PP2B caused weak deactivation, whereas PP1 had no detectable effect on open probability (Po). Interestingly, the time course of deactivation by PP2C was identical to that of the spontaneous rundown observed in some patches after excision. PP2C and PP2A had distinct effects on channel gating Po declined during exposure to exogenous PP2C (and during spontaneous rundown, when it was observed) without any change in mean burst duration. By contrast, deactivation by exogenous PP2A was associated with a dramatic shortening of burst duration similar to that reported previously in patches from cardiac cells during deactivation of CFTR by endogenous phosphatases. Rundown of CFTR-mediated current across intact T84 epithelial cell monolayers was insensitive to toxic levels of the PP2A inhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and that multiple protein phosphatases may be required for complete deactivation of CFTR channels.

  13. PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1.

    PubMed

    Guergnon, Julien; Godet, Angélique N; Galioot, Amandine; Falanga, Pierre Barthélémy; Colle, Jean-Hervé; Cayla, Xavier; Garcia, Alphonse

    2011-11-01

    Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.

  14. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    PubMed Central

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  15. PP2A methylation controls sensitivity and resistance to β-amyloid-induced cognitive and electrophysiological impairments.

    PubMed

    Nicholls, Russell E; Sontag, Jean-Marie; Zhang, Hong; Staniszewski, Agnieszka; Yan, Shijun; Kim, Carla Y; Yim, Michael; Woodruff, Caitlin M; Arning, Erland; Wasek, Brandi; Yin, Deqi; Bottiglieri, Teodoro; Sontag, Estelle; Kandel, Eric R; Arancio, Ottavio

    2016-03-22

    Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer's disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients.

  16. PP2A methylation controls sensitivity and resistance to β-amyloid–induced cognitive and electrophysiological impairments

    PubMed Central

    Nicholls, Russell E.; Sontag, Jean-Marie; Zhang, Hong; Staniszewski, Agnieszka; Yan, Shijun; Kim, Carla Y.; Yim, Michael; Woodruff, Caitlin M.; Arning, Erland; Wasek, Brandi; Yin, Deqi; Bottiglieri, Teodoro; Sontag, Estelle; Kandel, Eric R.; Arancio, Ottavio

    2016-01-01

    Elevated levels of the β-amyloid peptide (Aβ) are thought to contribute to cognitive and behavioral impairments observed in Alzheimer’s disease (AD). Protein phosphatase 2A (PP2A) participates in multiple molecular pathways implicated in AD, and its expression and activity are reduced in postmortem brains of AD patients. PP2A is regulated by protein methylation, and impaired PP2A methylation is thought to contribute to increased AD risk in hyperhomocysteinemic individuals. To examine further the link between PP2A and AD, we generated transgenic mice that overexpress the PP2A methylesterase, protein phosphatase methylesterase-1 (PME-1), or the PP2A methyltransferase, leucine carboxyl methyltransferase-1 (LCMT-1), and examined the sensitivity of these animals to behavioral and electrophysiological impairments caused by exogenous Aβ exposure. We found that PME-1 overexpression enhanced these impairments, whereas LCMT-1 overexpression protected against Aβ-induced impairments. Neither transgene affected Aβ production or the electrophysiological response to low concentrations of Aβ, suggesting that these manipulations selectively affect the pathological response to elevated Aβ levels. Together these data identify a molecular mechanism linking PP2A to the development of AD-related cognitive impairments that might be therapeutically exploited to target selectively the pathological effects caused by elevated Aβ levels in AD patients. PMID:26951658

  17. PR55α, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated β-Catenin Dephosphorylation

    PubMed Central

    Zhang, Wen; Yang, Jun; Liu, Yajuan; Chen, Xi; Yu, Tianxin; Jia, Jianhang; Liu, Chunming

    2009-01-01

    A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling. PMID:19556239

  18. PP2A Inhibitor PME-1 Drives Kinase Inhibitor Resistance in Glioma Cells.

    PubMed

    Kaur, Amanpreet; Denisova, Oxana V; Qiao, Xi; Jumppanen, Mikael; Peuhu, Emilia; Ahmed, Shafiq U; Raheem, Olayinka; Haapasalo, Hannu; Eriksson, John; Chalmers, Anthony J; Laakkonen, Pirjo; Westermarck, Jukka

    2016-12-01

    Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.

  19. Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

    PubMed Central

    Witt, Johannes; Barisic, Sandra; Schumann, Eva; Allgöwer, Frank; Sawodny, Oliver; Sauter, Thomas; Kulms, Dagmar

    2009-01-01

    Background Biological effects of nuclear factor-κB (NFκB) can differ tremendously depending on the cellular context. For example, NFκB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NFκB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NFκB, involving a lack of inhibitor of κB (IκBα) protein reappearance. Permanent activation of the upstream kinase IKKβ results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized IκBα. Results To investigate the mechanism underlying the general PP2A-mediated tuning of IKKβ phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKKβ and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKKβ phosphorylation thus allowing for IκBα resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKKβ phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of IκBα reappearance, is not a direct UVB action but requires instrumentality. Conclusion The model developed here can be used as a reliable building block of larger NFκB models and offers comprehensive simplification potential for future modeling of NFκB signaling. It gives more insight into the newly discovered

  20. Orchestrated Action of PP2A Antagonizes Atg13 Phosphorylation and Promotes Autophagy after the Inactivation of TORC1

    PubMed Central

    Kondo, Akihiro; Kaneko, Atsuki; Koike, Naoki; Ushimaru, Takashi

    2016-01-01

    Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1. PMID:27973551

  1. Imipramine blocks ethanol-induced ASMase activation, ceramide generation, and PP2A activation, and ameliorates hepatic steatosis in ethanol-fed mice.

    PubMed

    Liangpunsakul, Suthat; Rahmini, Yasmeen; Ross, Ruth A; Zhao, Zhenwen; Xu, Yan; Crabb, David W

    2012-03-01

    Our previous data showed the inhibitory effect of ethanol on AMP-activated protein kinase phosphorylation, which appears to be mediated, in part, through increased levels of hepatic ceramide and activation of protein phosphatase 2A (Liangpunsakul S, Sozio MS, Shin E, Zhao Z, Xu Y, Ross RA, Zeng Y, Crabb DW. Am J Physiol Gastrointest Liver Physiol 298: G1004-G1012, 2010). The effect of ethanol on AMP-activated protein kinase phosphorylation was reversed by imipramine, suggesting that the generation of ceramide via acid sphingomyelinase (ASMase) is stimulated by ethanol. In this study, we determined the effects of imipramine on the development of hepatic steatosis, the generation of ceramide, and downstream effects of ceramide on inflammatory, insulin, and apoptotic signaling pathways, in ethanol-fed mice. The effect of ethanol and imipramine (10 μg/g body wt ip) on ceramide levels, as well as inflammatory, insulin, and apoptotic signaling pathways, was studied in C57BL/6J mice fed the Lieber-DeCarli diet. Ethanol-fed mice developed the expected steatosis, and cotreatment with imipramine for the last 2 wk of ethanol feeding resulted in improvement in hepatic steatosis. Ethanol feeding for 4 wk induced impaired glucose tolerance compared with controls, and this was modestly improved with imipramine treatment. There was a significant decrease in total ceramide concentrations in response to imipramine in ethanol-fed mice treated with and without imipramine (287 ± 11 vs. 348 ± 12 pmol/mg tissue). The magnitude and specificity of inhibition on each ceramide species differed. A significant decrease was observed for C16 (28 ± 3 vs. 33 ± 2 pmol/mg tissue) and C24 (164 ± 9 vs. 201 ± 4 pmol/mg tissue) ceramide. Ethanol feeding increased the levels of the phosphorylated forms of ERK slightly and increased phospho-p38 and phospho-JNK substantially. The levels of phospho-p38 and phospho-JNK were reduced by treatment with imipramine. The activation of ASMase and generation

  2. All roads lead to PP2A: Exploiting the therapeutic potential of this phosphatase

    PubMed Central

    Sangodkar, Jaya; Farrington, Caroline; McClinch, Kimberly; Galsky, Matthew D.; Kastrinsky, David B.; Narla, Goutham

    2015-01-01

    Protein phosphatase 2A is a serine/threonine phosphatase involved in the regulation of many cellular processes. A confirmed tumor suppressor protein, PP2A is genetically altered or functionally inactivated in many cancers highlighting a need for its therapeutic reactivation. In this review we will discuss recent literature on PP2A: the elucidation of its structure and the functions of its subunits, and the identification of molecular lesions and post-translational modifications leading to its dysregulation in cancer. A final section will discuss the proteins and small molecules that modulate PP2A and how these might be used to target dysregulated forms of PP2A to treat cancers and other diseases. PMID:26507691

  3. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

  4. Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α

    PubMed Central

    Wang, Ling; Guo, Qingyuan; Fisher, Laura A; Liu, Dongxu; Peng, Aimin

    2015-01-01

    In addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55α, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55α and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery. PMID:25483054

  5. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A)

    PubMed Central

    Obanda, Diana N.; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T.

    2016-01-01

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation. PMID:26916435

  6. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A).

    PubMed

    Obanda, Diana N; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T

    2016-02-26

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

  7. Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits

    PubMed Central

    Sommer, Lauren M.; Cho, Hyuk; Choudhary, Madhusudan; Seeling, Joni M.

    2015-01-01

    Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αβε and B56γδ. Further duplications led to three B56αβε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core. PMID:25950761

  8. Psychosine induces the dephosphorylation of neurofilaments by deregulation of PP1 and PP2A phosphatases

    PubMed Central

    Cantuti-Castelvetri, Ludovico; Zhu, Hongling; Givogri, Maria I.; Chidavaenzi, Robstein L.; Lopez-Rosas, Aurora; Bongarzone, Ernesto R.

    2012-01-01

    Patients with Krabbe disease, a genetic demyelinating syndrome caused by deficiency of galactosyl-ceramidase and the resulting accumulation of galactosyl-sphingolipids, develop signs of a dying-back axonopathy compounded by a deficiency of large-caliber axons. Here, we show that axonal caliber in Twitcher mice, an animal model for Krabbe disease, is impaired in peripheral axons and is accompanied by a progressive reduction in the abundance and phosphorylation of the three neurofilament (NF) subunits. These changes correlate with an increase in the density of NFs per cross-sectional area in numerous mutant peripheral axons and abnormal increases in the activity of two serine/threonine phosphatases (PP1 and PP2A) in mutant tissue. Similarly, acutely isolated mutant cortical neurons show abnormal phosphorylation of NFs. Psychosine, the neurotoxin accumulated in Krabbe disease, was sufficient to induce abnormal dephosphorylation of NF subunits in a normal motor neuron cell line as well as in acutely isolated normal cortical neurons. This in vitro effect was mediated by PP1 and PP2A, which specifically dephosphorylated NFs. These results demonstrate that the reduced caliber observed in some axons in Krabbe disease involves abnormal dephosphorylation of NFs. We propose that a psychosine-driven pathogenic mechanism through deregulated phosphotransferase activities may be involved in this process. PMID:22326830

  9. Inhibition of Pten deficient Castration Resistant Prostate Cancer by Targeting of the SET - PP2A Signaling axis.

    PubMed

    Hu, Xiaoyong; Garcia, Consuelo; Fazli, Ladan; Gleave, Martin; Vitek, Michael P; Jansen, Marilyn; Christensen, Dale; Mulholland, David J

    2015-11-13

    The PP2A signaling axis regulates multiple oncogenic drivers of castration resistant prostate cancer (CRPC). We show that targeting the endogenous PP2A regulator, SET (I2PP2A), is a viable strategy to inhibit prostate cancers that are resistant to androgen deprivation therapy. Our data is corroborated by analysis of prostate cancer patient cohorts showing significant elevation of SET transcripts. Tissue microarray analysis reveals that elevated SET expression correlates with clinical cancer grading, duration of neoadjuvant hormone therapy (NHT) and time to biochemical recurrence. Using prostate regeneration assays, we show that in vivo SET overexpression is sufficient to induce hyperplasia and prostatic intraepithelial neoplasia. Knockdown of SET induced significant reductions in tumorgenesis both in murine and human xenograft models. To further validate SET as a therapeutic target, we conducted in vitro and in vivo treatments using OP449 - a recently characterized PP2A-activating drug (PAD). OP449 elicits robust anti-cancer effects inhibiting growth in a panel of enzalutamide resistant prostate cancer cell lines. Using the Pten conditional deletion mouse model of prostate cancer, OP449 potently inhibited PI3K-Akt signaling and impeded CRPC progression. Collectively, our data supports a critical role for the SET-PP2A signaling axis in CRPC progression and hormone resistant disease.

  10. Inhibition of Pten deficient Castration Resistant Prostate Cancer by Targeting of the SET - PP2A Signaling axis

    PubMed Central

    Hu, Xiaoyong; Garcia, Consuelo; Fazli, Ladan; Gleave, Martin; Vitek, Michael P.; Jansen, Marilyn; Christensen, Dale; Mulholland, David J

    2015-01-01

    The PP2A signaling axis regulates multiple oncogenic drivers of castration resistant prostate cancer (CRPC). We show that targeting the endogenous PP2A regulator, SET (I2PP2A), is a viable strategy to inhibit prostate cancers that are resistant to androgen deprivation therapy. Our data is corroborated by analysis of prostate cancer patient cohorts showing significant elevation of SET transcripts. Tissue microarray analysis reveals that elevated SET expression correlates with clinical cancer grading, duration of neoadjuvant hormone therapy (NHT) and time to biochemical recurrence. Using prostate regeneration assays, we show that in vivo SET overexpression is sufficient to induce hyperplasia and prostatic intraepithelial neoplasia. Knockdown of SET induced significant reductions in tumorgenesis both in murine and human xenograft models. To further validate SET as a therapeutic target, we conducted in vitro and in vivo treatments using OP449 - a recently characterized PP2A-activating drug (PAD). OP449 elicits robust anti-cancer effects inhibiting growth in a panel of enzalutamide resistant prostate cancer cell lines. Using the Pten conditional deletion mouse model of prostate cancer, OP449 potently inhibited PI3K-Akt signaling and impeded CRPC progression. Collectively, our data supports a critical role for the SET-PP2A signaling axis in CRPC progression and hormone resistant disease. PMID:26563471

  11. PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos.

    PubMed

    Kitagawa, Daiju; Flückiger, Isabelle; Polanowska, Jolanta; Keller, Debora; Reboul, Jérôme; Gönczy, Pierre

    2011-04-19

    Centrosome duplication occurs once per cell cycle and ensures that the two resulting centrosomes assemble a bipolar mitotic spindle. Centriole formation is fundamental for centrosome duplication. In Caenorhabditis elegans, the evolutionarily conserved proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but how they function is not fully understood. Here, we demonstrate that Protein Phosphatase 2A (PP2A) is also critical for centriole formation in C. elegans embryos. We find that PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex. Furthermore, we show that PP2A-mediated dephosphorylation promotes centriolar targeting of SAS-5 and ensures SAS-6 delivery to the site of centriole assembly. We find that PP2A is similarly needed for the presence of HsSAS-6 at centrioles and for centriole formation in human cells. These findings lead us to propose that PP2A-mediated loading of SAS-6 proteins is critical at the onset of centriole formation.

  12. Protein Phosphatase 2A (PP2A) Regulates Low Density Lipoprotein Uptake through Regulating Sterol Response Element-binding Protein-2 (SREBP-2) DNA Binding*

    PubMed Central

    Rice, Lyndi M.; Donigan, Melissa; Yang, Muhua; Liu, Weidong; Pandya, Devanshi; Joseph, Biny K.; Sodi, Valerie; Gearhart, Tricia L.; Yip, Jenny; Bouchard, Michael; Nickels, Joseph T.

    2014-01-01

    LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake. PMID:24770487

  13. Targeting A Tumor Suppressor To Suppress Tumor Growth: News and Views on Protein Phosphatase 2A (PP2A) as a Target for Anti-cancer Therapy

    PubMed Central

    Perrotti, Danilo; Neviani, Paolo

    2013-01-01

    Protein phosphatase 2A (PP2A), one of the major serine-threonine phosphatases in mammalian cells, maintains cell homeostasis by counteracting most of the kinase-driven intracellular signaling pathways. Unrestrained activation of oncogenic kinases together with inhibition of tumor suppressors is frequently required for the development of cancer. Because it has been found genetically altered or functionally inactivated in many solid cancers and leukemias, PP2A is indeed a bona fide tumor suppressor. For example, the phosphatase activity of PP2A is suppressed in chronic myelogenous leukemia and other malignancies characterized by the aberrant activity of oncogenic kinases. Notably, preclinical studies indicate that pharmacologic restoration of PP2A tumor suppressor activity by PP2A activating drugs (PADs, e.g. FTY720) effectively antagonizes cancer development and progression. Herein, we systematically discuss the importance of PP2A as a druggable tumor suppressor in light of the possible introduction of PADs into anti-cancer therapeutic protocols. PMID:23639323

  14. PP2A: The Achilles Heal in MDS with 5q Deletion.

    PubMed

    Sallman, David A; Wei, Sheng; List, Alan

    2014-01-01

    Myelodysplastic syndromes (MDS) represent a hematologically diverse group of myeloid neoplasms, however, one subtype characterized by an isolated deletion of chromosome 5q [del(5q)] is pathologically and clinically distinct. Patients with del(5q) MDS share biological features that account for the profound hypoplastic anemia and unique sensitivity to treatment with lenalidomide. Ineffective erythropoiesis in del(5q) MDS arises from allelic deletion of the ribosomal processing S-14 (RPS14) gene, which leads to MDM2 sequestration with consequent p53 activation and erythroid cell death. Since its approval in 2005, lenalidomide has changed the natural course of the disease. Patients who achieve transfusion independence and/or a cytogenetic response with lenalidomide have a decreased risk of progression to acute myeloid leukemia and an improved overall survival compared to non-responders. Elucidation of the mechanisms of action of lenalidomide in del(5q) MDS has advanced therapeutic strategies for this disease. The selective cytotoxicity of lenalidomide in del(5q) clones derives from inhibition of a haplodeficient phosphatase whose catalytic domain is encoded within the common deleted region on chromosome 5q, i.e., protein phosphatase 2A (PP2Acα). PP2A is a highly conserved, dual specificity phosphatase that plays an essential role in regulation of the G2/M checkpoint. Inhibition of PP2Acα results in cell-cycle arrest and apoptosis in del(5q) cells. Targeted knockdown of PP2Acα using siRNA is sufficient to sensitize non-del(5q) clones to lenalidomide. Through its inhibitory effect on PP2A, lenalidomide stabilizes MDM2 to restore p53 degradation in erythroid precursors, with subsequent arrest in G2/M. Unfortunately, the majority of patients with del(5q) MDS develop resistance to lenalidomide over time associated with PP2Acα over-expression. Targeted inhibition of PP2A with a more potent inhibitor has emerged as an attractive therapeutic approach for patients with del

  15. The mitotic PP2A regulator ENSA/ARPP-19 is remarkably conserved across plants and most eukaryotes.

    PubMed

    Labandera, Anne-Marie; Vahab, Ahmad R; Chaudhuri, Sibapriya; Kerk, David; Moorhead, Greg B G

    2015-03-20

    Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase of eukaryotes. PP2A containing the B55 subunit is a key regulator of mitosis and must be inhibited by phosphorylated α-endosulfine (ENSA) or cyclic AMP-regulated 19 kDa phosphoprotein (ARPP-19) to allow passage through mitosis. Exit from mitosis then requires dephosphorylation of ENSA/ARPP-19 to relieve inhibition of PP2A/B55. ENSA/ARPP-19 has been characterized in several vertebrates and budding yeast, but little is known about its presence in plants and the majority of other eukaryotes. Here we show that three isoforms of ENSA/ARPP-19 are present in the Arabidopsis thaliana genome with distinct expression profiles across various plant tissues. The ENSA/ARPP-19 proteins, and in particular their key inhibitory sequence FDSGDY (FDSADW in plants), is remarkably conserved across plants and most eukaryotes suggesting an ancient origin and conserved function to control PP2A activity.

  16. PP2A-twins is antagonized by greatwall and collaborates with polo for cell cycle progression and centrosome attachment to nuclei in drosophila embryos.

    PubMed

    Wang, Peng; Pinson, Xavier; Archambault, Vincent

    2011-08-01

    Cell division and development are regulated by networks of kinases and phosphatases. In early Drosophila embryogenesis, 13 rapid nuclear divisions take place in a syncytium, requiring fine coordination between cell cycle regulators. The Polo kinase is a conserved, crucial regulator of M-phase. We have recently reported an antagonism between Polo and Greatwall (Gwl), another mitotic kinase, in Drosophila embryos. However, the nature of the pathways linking them remained elusive. We have conducted a comprehensive screen for additional genes functioning with polo and gwl. We uncovered a strong interdependence between Polo and Protein Phosphatase 2A (PP2A) with its B-type subunit Twins (Tws). Reducing the maternal contribution of Polo and PP2A-Tws together is embryonic lethal. We found that Polo and PP2A-Tws collaborate to ensure centrosome attachment to nuclei. While a reduction in Polo activity leads to centrosome detachments observable mostly around prophase, a reduction in PP2A-Tws activity leads to centrosome detachments at mitotic exit, and a reduction in both Polo and PP2A-Tws enhances the frequency of detachments at all stages. Moreover, we show that Gwl antagonizes PP2A-Tws function in both meiosis and mitosis. Our study highlights how proper coordination of mitotic entry and exit is required during embryonic cell cycles and defines important roles for Polo and the Gwl-PP2A-Tws pathway in this process.

  17. Carnosic acid stimulates glucose uptake in skeletal muscle cells via a PME-1/PP2A/PKB signalling axis.

    PubMed

    Lipina, Christopher; Hundal, Harinder S

    2014-11-01

    Carnosic acid (CA) is a major constituent of the labiate herbal plant Rosemary (Rosmarinus officinalis), which has been shown to exhibit a number of beneficial health properties. In particular, recently there has been growing interest into the anti-obesity effects conveyed by CA, including its ability to counteract obesity-associated hyperglycaemia and insulin resistance. However, the mechanisms underlying its anti-diabetic responses are not fully understood. In this study, we hypothesized that CA may act to improve glycaemic status through enhancing peripheral glucose clearance. Herein, we demonstrate that CA acts to mimic the metabolic actions of insulin by directly stimulating glucose uptake in rat skeletal L6 myotubes, concomitant with increased translocation of the GLUT4 glucose transporter to the plasma membrane. Mechanistically, CA-induced glucose transport was found to be dependent on protein kinase B (PKB/Akt) but not AMPK, despite both kinases being activated by CA. Crucially, in accordance with its ability to activate PKB and stimulate glucose uptake, we show that CA conveys these effects through a pathway involving PME-1 (protein phosphatase methylesterase-1), a key negative regulator of the serine/threonine phosphatase PP2A (protein phosphatase 2A). Herein, we demonstrate that CA promotes PME-1 mediated demethylation of the PP2A catalytic subunit leading to its suppressed activity, and in doing so, alleviates the repressive action of PP2A towards PKB. Collectively, our findings provide new insight into how CA may improve glucose homeostasis through enhancing peripheral glucose clearance in tissues such as skeletal muscle through a PME-1/PP2A/PKB signalling axis, thereby mitigating pathological effects associated with the hyperglycaemic state.

  18. Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest

    PubMed Central

    Shouse, Geoffrey; de Necochea-Campion, Rosalia; Mirshahidi, Saied; Liu, Xuan; Chen, Chien-Shing

    2016-01-01

    Activation of the Protein Kinase B (PKB), or AKT pathway has been shown to correlate with acute myeloid leukemia (AML) prognosis. B55α-Protein Phosphatase 2A (PP2A) has been shown to dephosphorylate AKT at Thr-308 rendering it inactive. In fact, low expression of the PP2A regulatory subunit B55α was associated with activated phospho-AKT and correlated with inferior outcomes in AML. Despite this fact, no studies have specifically demonstrated a mechanism whereby B55α expression is regulated in AML. In this study, we demonstrate novel loss of function mutations in the PPP2R2A gene identified in leukemic blasts from three AML patients. These mutations eliminate B55α protein expression thereby allowing constitutive AKT activation. In addition, leukemic blasts with PPP2R2A gene mutation were more sensitive to treatment with the AKT inhibitor MK2206, but less responsive to the PP2A activator FTY720. Using leukemia cell lines, we further demonstrate that B55α expression correlates with AKT Thr-308 phosphorylation and predicts responsiveness to AKT inhibition and PP2A activation. Together our data illustrate the importance of the B55α-PP2A-AKT pathway in leukemogenesis. Screening for disruptions in this pathway at initial AML diagnosis may predict response to targeted therapies against AKT and PP2A. PMID:27531894

  19. Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers

    PubMed Central

    Williams, Byron C; Filter, Joshua J; Blake-Hodek, Kristina A; Wadzinski, Brian E; Fuda, Nicholas J; Shalloway, David; Goldberg, Michael L

    2014-01-01

    During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55’s action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55’s attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55. DOI: http://dx.doi.org/10.7554/eLife.01695.001 PMID:24618897

  20. A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit.

    PubMed

    Cundell, Michael J; Hutter, Lukas H; Nunes Bastos, Ricardo; Poser, Elena; Holder, James; Mohammed, Shabaz; Novak, Bela; Barr, Francis A

    2016-08-29

    PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.

  1. A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit

    PubMed Central

    Cundell, Michael J.; Holder, James

    2016-01-01

    PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis. PMID:27551054

  2. Synthesis of Highly Selective Submicromolar Microcystin‐Based Inhibitors of Protein Phosphatase (PP)2A over PP1

    PubMed Central

    Fontanillo, Miriam; Zemskov, Ivan; Häfner, Maximilian; Uhrig, Ulrike; Salvi, Francesca; Simon, Bernd; Wittmann, Valentin

    2016-01-01

    Abstract Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure–activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid‐ and solution‐phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues. PMID:27723199

  3. Standardised extract of Bacopa monniera (CDRI-08) improves contextual fear memory by differentially regulating the activity of histone acetylation and protein phosphatases (PP1α, PP2A) in hippocampus.

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Venkataraman, Jois Shreyas; Rajan, Koilmani Emmanuvel

    2014-05-01

    Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15-29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.

  4. PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration

    PubMed Central

    Janssens, Veerle; Zwaenepoel, Karen; Rossé, Carine; Petit, Marleen M. R.; Goris, Jozef; Parker, Peter J.

    2017-01-01

    Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn2+-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP–PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130–LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics. PMID:26945059

  5. PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

    PubMed Central

    Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min

    2014-01-01

    Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602

  6. Compression regulates gene expression of chondrocytes through HDAC4 nuclear relocation via PP2A-dependent HDAC4 dephosphorylation.

    PubMed

    Chen, Chongwei; Wei, Xiaochun; Wang, Shaowei; Jiao, Qiang; Zhang, Yang; Du, Guoqing; Wang, Xiaohu; Wei, Fangyuan; Zhang, Jianzhong; Wei, Lei

    2016-07-01

    Biomechanics plays a critical role in the modulation of chondrocyte function. The mechanisms by which mechanical loading is transduced into intracellular signals that regulate chondrocyte gene expression remain largely unknown. Histone deacetylase 4 (HDAC4) is specifically expressed in chondrocytes. Mice lacking HDAC4 display chondrocyte hypertrophy, ectopic and premature ossification, and die early during the perinatal period. HDAC4 has a remarkable ability to translocate between the cell's cytoplasm and nucleus. It has been established that subcellular relocation of HDAC4 plays a critical role in chondrocyte differentiation and proliferation. However, it remains unclear whether subcellular relocation of HDAC4 in chondrocytes can be induced by mechanical loading. In this study, we first report that compressive loading induces HDAC4 relocation from the cytoplasm to the nucleus of chondrocytes via stimulation of Ser/Thr-phosphoprotein phosphatases 2A (PP2A) activity, which results in dephosphorylation of HDAC4. Dephosphorylated HDAC4 relocates to the nucleus to achieve transcriptional repression of Runx2 and regulates chondrocyte gene expression in response to compression. Our results elucidate the mechanism by which mechanical compression regulates chondrocyte gene expression through HDAC4 relocation from the cell's cytoplasm to the nucleus via PP2A-dependent HDAC4 dephosphorylation.

  7. The role of PP2A-associated proteins and signal pathways in microcystin-LR toxicity.

    PubMed

    Liu, Jing; Sun, Yu

    2015-07-02

    Microcystins are a family of monocyclic heptapeptides produced by cyanobacteria during water blooms. Microcystin-LR (MC-LR) is the most common member of this family. Microcystins induce a variety of toxic cellular effects, including oxidative damage, apoptosis, cytoskeletal destabilization, and cancer cell invasion. Recent studies have examined the molecular mechanism of their toxicity. Protein phosphatase 2A (PP2A) is emerging as a critical regulator of the microcystin-induced molecular network. Furthermore, it has been shown that several molecules or signal pathways associated with PP2A play important roles in microcystin-induced toxic effects. This review summarizes the recent research progress of the molecular mechanism and focuses on the role of PP2A in MC-LR toxicity, which will contribute to a better understanding of the mechanism of microcystin toxicity, and will provide biomarkers for toxicity assessment and control.

  8. Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.

    PubMed

    Ribeiro, Paulo S; Josué, Filipe; Wepf, Alexander; Wehr, Michael C; Rinner, Oliver; Kelly, Gavin; Tapon, Nicolas; Gstaiger, Matthias

    2010-08-27

    The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, we identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development.

  9. Protein phosphatase 2A family members (PP2A and PP6) associate with U1 snRNP and the spliceosome during pre-mRNA splicing

    PubMed Central

    Kamoun, Malek; Filali, Mohammed; Murray, Michael V.; Awasthi, Sita; Wadzinski, Brian E.

    2013-01-01

    Protein phosphorylation and dephosphorylation are both important for multiple steps in the splicing pathway. Members of the PP1 and PP2A subfamilies of phospho-serine/threonine phosphatases play essential but redundant roles in the second step of the splicing reaction. PP6, a member of the PP2A subfamily, is the mammalian homologue of yeast Sit4p and ppe1, which are involved in cell cycle regulation; however, the involvement of PP6 in the splicing pathway remains unclear. Here we show that PP2A family members physically associate with the spliceosome throughout the splicing reaction. PP2A holoenzyme and PP6 were found stably associated with U1 snRNP. Together our findings indicate that these phosphatases regulate splicing catalysis involving U1 snRNP and suggest an important evolutionary conserved role of PP2A family phosphatases in pre-mRNA splicing. PMID:24064353

  10. PP2A inhibitors arrest G2/M transition through JNK/Sp1- dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21.

    PubMed

    Gong, Fei-Ran; Wu, Meng-Yao; Shen, Meng; Zhi, Qiaoming; Xu, Ze-Kuan; Wang, Rong; Wang, Wen-Jie; Zong, Yang; Li, Zeng-Liang; Wu, Yadi; Zhou, Binhua P; Chen, Kai; Tao, Min; Li, Wei

    2015-07-30

    Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.

  11. Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer.

    PubMed

    Liu, Hao; Gu, Yixue; Wang, Hongsheng; Yin, Jiang; Zheng, Guopei; Zhang, Zhijie; Lu, Minyin; Wang, Chenkun; He, Zhimin

    2015-06-20

    SET oncoprotein is an endogenous inhibitor of protein phosphatase 2A (PP2A), and SET-mediated PP2A inhibition is an important regulatory mechanism for promoting cancer initiation and progression of several types of human leukemia disease. However, its potential relevance in solid tumors as non-small cell lung cancer (NSCLC) remains mostly unknown. In this study, we showed that SET was evidently overexpressed in human NSCLC cell lines and NSCLC tissues. Clinicopathologic analysis showed that SET expression was significantly correlated with clinical stage (p < 0.001), and lymph node metastasis (p < 0.05). Kaplan-Meier analysis revealed that patients with high SET expression had poorer overall survival rates than those with low SET expression. Moreover, knockdown of SET in NSCLC cells resulted in attenuated proliferative and invasive abilities. The biological effect of SET on proliferation and invasion was mediated by the inhibition of the PP2A, which in turn, activation of AKT and ERK, increased the expression of cyclin D1 and MMP9, and decreased the expression of p27. Furthermore, we observed that restoration of PP2A using SET antagonist FTY720 impaired proliferative and invasive potential in vitro, as well as inhibited tumor growth in vivo of NSCLC cells. Taken together, SET oncoprotein plays an important role in NSCLC progression, which could serve as a potential prognosis marker and a novel therapeutic target for NSCLC patients.

  12. MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia.

    PubMed

    Pippa, Raffaella; Dominguez, Ana; Malumbres, Raquel; Endo, Akinori; Arriazu, Elena; Marcotegui, Nerea; Guruceaga, Elizabeth; Odero, María D

    2016-06-06

    The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.

  13. A triangular connection between Cyclin G, PP2A and Akt1 in the regulation of growth and metabolism in Drosophila

    PubMed Central

    Fischer, Patrick; Preiss, Anette; Nagel, Anja C.

    2016-01-01

    ABSTRACT Size and weight control is a tightly regulated process, involving the highly conserved Insulin receptor/target of rapamycin (InR/TOR) signaling cascade. We recently identified Cyclin G (CycG) as an important modulator of InR/TOR signaling activity in Drosophila. cycG mutant flies are underweight and show a disturbed fat metabolism resembling TOR mutants. In fact, InR/TOR signaling activity is disturbed in cycG mutants at the level of Akt1, the central kinase linking InR and TORC1. Akt1 is negatively regulated by protein phosphatase PP2A. Notably the binding of the PP2A B′-regulatory subunit Widerborst (Wdb) to Akt1 is differentially regulated in cycG mutants, presumably by a direct interaction of CycG and Wdb. Since the metabolic defects of cycG mutant animals are abrogated by a concomitant loss of Wdb, CycG presumably influences Akt1 activity at the PP2A nexus. Here we show that Well rounded (Wrd), another B' subunit of PP2A in Drosophila, binds CycG similar to Wdb, and that its loss ameliorates some, but not all, of the metabolic defects of cycG mutants. We propose a model, whereby the binding of CycG to a particular B′-regulatory subunit influences the tissue specific activity of PP2A, required for the fine tuning of the InR/TOR signaling cascade in Drosophila. PMID:26980713

  14. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation

    SciTech Connect

    LeNoue-Newton, Michele; Watkins, Guy R.; Zou, Ping; Germane, Katherine L.; McCorvey, Lisa R.; Wadzinski, Brian E.; Spiller, Benjamin W.

    2012-04-30

    Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: (1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and (2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

  15. Identification of the Adenovirus E4orf4 Protein Binding Site on the B55α and Cdc55 Regulatory Subunits of PP2A: Implications for PP2A Function, Tumor Cell Killing and Viral Replication

    PubMed Central

    Mui, Melissa Z.; Kucharski, Michael; Miron, Marie-Joëlle; Hur, Woosuk Steve; Berghuis, Albert M.; Blanchette, Paola; Branton, Philip E.

    2013-01-01

    Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) is required, and such binding inhibits PP2AB55 activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2AB55α. We propose that E4orf4 inhibits PP2AB55 activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2AB55 to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product. PMID:24244166

  16. PP2A-3 interacts with ACR4 and regulates formative cell division in the Arabidopsis root.

    PubMed

    Yue, Kun; Sandal, Priyanka; Williams, Elisabeth L; Murphy, Evan; Stes, Elisabeth; Nikonorova, Natalia; Ramakrishna, Priya; Czyzewicz, Nathan; Montero-Morales, Laura; Kumpf, Robert; Lin, Zhefeng; van de Cotte, Brigitte; Iqbal, Mudassar; Van Bel, Michiel; Van De Slijke, Eveline; Meyer, Matthew R; Gadeyne, Astrid; Zipfel, Cyril; De Jaeger, Geert; Van Montagu, Marc; Van Damme, Daniël; Gevaert, Kris; Rao, A Gururaj; Beeckman, Tom; De Smet, Ive

    2016-02-02

    In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

  17. URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells.

    PubMed

    Mita, Paolo; Savas, Jeffrey N; Briggs, Erica M; Ha, Susan; Gnanakkan, Veena; Yates, John R; Robins, Diane M; David, Gregory; Boeke, Jef D; Garabedian, Michael J; Logan, Susan K

    2016-12-02

    URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

  18. The PP2A-B56 phosphatase opposes cyclin E autocatalytic degradation via site-specific dephosphorylation.

    PubMed

    Davis, Ryan J; Swanger, Jherek; Hughes, Bridget T; Clurman, Bruce E

    2017-01-30

    Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCF(Fbw7) ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells, and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity, while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.

  19. The Role of PP2A Methylation in Susceptibility and Resistance to TBI and AD-Induced Neurodegeneration

    DTIC Science & Technology

    2013-09-01

    73 Tanzi,  R.E.  (2012).  The   Genetics  of   Alzheimer  Disease.  Cold  Spring  Harbor   Perspectives  in  Medicine  2...ABSTRACT The focus of the current study is to test the effect of genetic manipulations that target the tau phosphatase, PP2A, on behavioral...impairments resulting from shockwave exposure in a mouse model, and to compare those results with the effects of the same genetic manipulations on the

  20. Silencing [Formula: see text] Rescues Tau Pathologies and Memory Deficits through Rescuing PP2A and Inhibiting GSK-3β Signaling in Human Tau Transgenic Mice.

    PubMed

    Zhang, Yao; Ma, Rong-Hong; Li, Xia-Chun; Zhang, Jia-Yu; Shi, Hai-Rong; Wei, Wei; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2014-01-01

    Increase of inhibitor-2 of protein phosphatase-2A [Formula: see text] is associated with protein phosphatase-2A (PP2A) inhibition and tau hyperphosphorylation in Alzheimer's disease (AD). Down-regulating [Formula: see text] attenuated amyloidogenesis and improved the cognitive functions in transgenic mice expressing amyloid precursor protein (tg2576). Here, we found that silencing [Formula: see text] by hippocampal infusion of [Formula: see text] down-regulated [Formula: see text] (~45%) with reduction of tau phosphorylation/accumulation, improvement of memory deficits, and dendritic plasticity in 12-month-old human tau transgenic mice. Silencing [Formula: see text] not only restored PP2A activity but also inhibited glycogen synthase kinase-3β (GSK-3β) with a significant activation of protein kinase A (PKA) and Akt. In HEK293/tau and N2a/tau cells, silencing [Formula: see text] by [Formula: see text] also significantly reduced tau hyperphosphorylation with restoration of PP2A activity and inhibition of GSK-3β, demonstrated by the decreased GSK-3β total protein and mRNA levels, and the increased inhibitory phosphorylation of GSK-3β at serine-9. Furthermore, activation of PKA but not Akt mediated the inhibition of GSK-3β by [Formula: see text] silencing. We conclude that targeting [Formula: see text] can improve tau pathologies and memory deficits in human tau transgenic mice, and activation of PKA contributes to GSK-3β inhibition induced by silencing [Formula: see text]in vitro, suggesting that [Formula: see text] is a promising multiple target of AD.

  1. PP2A Ligand ITH12246 Protects against Memory Impairment and Focal Cerebral Ischemia in Mice

    PubMed Central

    2013-01-01

    ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimer’s disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca2+ overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimer’s disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant. PMID:23763493

  2. Dual Regulation of the Mitotic Exit Network (MEN) by PP2A-Cdc55 Phosphatase

    PubMed Central

    Baro, Barbara; Hernáez, María Luisa; Gil, Concha; Queralt, Ethel

    2013-01-01

    Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2ACdc55 phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2ACdc55 phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2ACdc55 regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2ACdc55 initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2ACdc55 functions affect the regulation of various MEN components, contributing to mitotic exit. PMID:24339788

  3. An integrated workflow for charting the human interaction proteome: insights into the PP2A system

    PubMed Central

    Glatter, Timo; Wepf, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

    2009-01-01

    Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes. PMID:19156129

  4. An integrated workflow for charting the human interaction proteome: insights into the PP2A system.

    PubMed

    Glatter, Timo; Wepf, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

    2009-01-01

    Protein complexes represent major functional units for the execution of biological processes. Systematic affinity purification coupled with mass spectrometry (AP-MS) yielded a wealth of information on the compendium of protein complexes expressed in Saccharomyces cerevisiae. However, global AP-MS analysis of human protein complexes is hampered by the low throughput, sensitivity and data robustness of existing procedures, which limit its application for systems biology research. Here, we address these limitations by a novel integrated method, which we applied and benchmarked for the human protein phosphatase 2A system. We identified a total of 197 protein interactions with high reproducibility, showing the coexistence of distinct classes of phosphatase complexes that are linked to proteins implicated in mitosis, cell signalling, DNA damage control and more. These results show that the presented analytical process will substantially advance throughput and reproducibility in future systematic AP-MS studies on human protein complexes.

  5. Rapamycin inhibits BAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells.

    PubMed

    Zeng, Qingyu; Zhang, Hai; Qin, Jiamin; Xu, Zhigang; Gui, Lin; Liu, Beibei; Liu, Chunxiao; Xu, Chong; Liu, Wen; Zhang, Shuangquan; Huang, Shile; Chen, Long

    2015-12-01

    B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin's suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin's effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

  6. Suspension survival mediated by PP2A-STAT3-Col XVII determines tumour initiation and metastasis in cancer stem cells

    PubMed Central

    Liu, Chen-Chi; Lin, Shih-Pei; Hsu, Han-Shui; Yang, Shung-Haur; Lin, Chiu-Hua; Yang, Muh-Hwa; Hung, Mien-Chie; Hung, Shih-Chieh

    2016-01-01

    Targeting tumour-initiating cells (TICs) would lead to new therapies to cure cancer. We previously demonstrated that TICs have the capacity to survive under suspension conditions, while other cells undergo anoikis. Here we show that TICs exhibit increased phosphorylation levels of S727STAT3 because of PP2A inactivation. Collagen 17 gene expression is upregulated in a STAT3-dependent manner, which also stabilizes laminin 5 and engages cells to form hemidesmosome-like junctions in response. Blocking the PP2A-S727STAT3-collagen 17 pathway inhibits the suspension survival of TICs and their ability to form tumours in mice, while activation of the same pathway increases the suspension survival and tumour-initiation capacities of bulk cancer cells. The S727STAT3 phosphorylation levels correlate with collagen 17 expression in colon tumour samples, and correlate inversely with survival. Finally, this signalling axis enhances the ability of TIC to form tumours in mouse models of malignant lung cancer pleural effusion and spontaneous colon cancer metastasis. PMID:27306323

  7. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

    PubMed

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-02-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

  8. Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle.

    PubMed

    Lorca, Thierry; Bernis, Cyril; Vigneron, Suzanne; Burgess, Andrew; Brioudes, Estelle; Labbé, Jean-Claude; Castro, Anna

    2010-07-01

    Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.

  9. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

    PubMed Central

    2011-01-01

    Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of M. persicae as a result

  10. Altered regulation of the Spry2/Dyrk1A/PP2A triad by homocysteine impairs neural progenitor cell proliferation.

    PubMed

    Rabaneda, Luis G; Geribaldi-Doldán, Noelia; Murillo-Carretero, Maribel; Carrasco, Manuel; Martínez-Salas, José M; Verástegui, Cristina; Castro, Carmen

    2016-12-01

    Hyperhomocysteinemia reduces neurogenesis in the adult mouse brain. Homocysteine (Hcy) inhibits postnatal neural progenitor cell (NPC) proliferation by specifically impairing the fibroblast growth factor receptor (FGFR)-Erk1/2-cyclin E signaling pathway. We demonstrate herein that the inhibition of FGFR-dependent NPC proliferation induced by Hcy is mediated by its capacity to alter the cellular methylation potential. Our results show that this alteration modified the expression pattern and activity of Sprouty2 (Spry2), a negative regulator of the above mentioned pathway. Both elevated concentrations of Hcy and methyltransferase activity inhibition induced Spry2 promoter demethylation in NPC cultures leading to a sustained upregulation of the expression of Spry2 mRNA and protein. In addition, protein levels of two kinases responsible for Spry2 activation/deactivation were altered by Hcy: Spry2 kinase Dyrk1A levels diminished while Spry2 phosphatase PP2A increased, leading to changes in the phosphorylation pattern, activity and stability of Spry2. In conclusion, Hcy inhibits NPC proliferation by indirect mechanisms involving alterations in DNA methylation, gene expression, and Spry2 function, causing FGFR signaling impairment.

  11. Docosahexaenoic acid inhibits vascular endothelial growth factor (VEGF)-induced cell migration via the GPR120/PP2A/ERK1/2/eNOS signaling pathway in human umbilical vein endothelial cells.

    PubMed

    Chao, Che-Yi; Lii, Chong-Kuei; Ye, Siou-Yu; Li, Chien-Chun; Lu, Chia-Yang; Lin, Ai-Hsuan; Liu, Kai-Li; Chen, Haw-Wen

    2014-05-07

    Cell migration plays an important role in angiogenesis and wound repair. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is essential for endothelial cell survival, proliferation, and migration. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, shows both anti-inflammatory and antioxidant activities in vitro and in vivo. This study investigated the molecular mechanism by which DHA down-regulates VEGF-induced cell migration. HUVECs were used as the study model, and the MTT assay, Western blot, wound-healing assay, and phosphatase activity assay were used to explore the effects of DHA on cell migration. GPR120 is the putative receptor for DHA action. The results showed that DHA, PD98059 (an ERK1/2 inhibitor), and GW9508 (a GPR120 agonist) inhibited VEGF-induced cell migration. In contrast, pretreatment with okadaic acid (OA, a PP2A inhibitor) and S-nitroso-N-acetyl-DL-penicillamine (an NO donor) reversed the inhibition of cell migration by DHA. VEGF-induced cell migration was accompanied by phosphorylation of ERK1/2 and eNOS. Treatment of HUVECs with DHA increased PP2A enzyme activity and decreased VEGF-induced phosphorylation of ERK1/2 and eNOS. However, pretreatment with OA significantly decreased DHA-induced PP2A enzyme activity and reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS phosphorylation. These results suggest that stimulation of PP2A activity and inhibition of the VEGF-induced ERK1/2/eNOS signaling pathway may be involved in the DHA suppression of VEGF-induced cell migration. Thus, the effect of DHA on angiogenesis and wound repair is at least partly by virtue of its attenuation of cell migration.

  12. A PP2A regulatory subunit PPTR-1 regulates the C. elegans Insulin/IGF-1 signaling pathway by modulating AKT-1 phosphorylation

    PubMed Central

    Padmanabhan, Srivatsan; Mukhopadhyay, Arnab; Narasimhan, Sri Devi; Tesz, Gregory; Czech, Michael P.; Tissenbaum, Heidi A.

    2009-01-01

    Summary The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in the regulation of lifespan, dauer diapause, metabolism and stress response. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. In a RNAi screen for serine/threonine protein phosphatases that counter-balance the effect of the kinases in the IIS pathway, we identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects phenotypes associated with the IIS pathway including lifespan, dauer, stress resistance and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56β regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this modulation ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in modulating insulin signaling through AKT dephosphorylation and thereby has widespread implications in cancer and diabetes research. PMID:19249087

  13. par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans

    PubMed Central

    Jiu, Yaming; Hasygar, Kiran; Tang, Lois; Liu, Yanbo; Holmberg, Carina I.; Bürglin, Thomas R.; Hietakangas, Ville; Jäntti, Jussi

    2013-01-01

    The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process. PMID:24192838

  14. Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage

    PubMed Central

    Tong, Y; Ying, H; Liu, R; Li, L; Bergholz, J; Xiao, Z-X

    2015-01-01

    Inactivation of the retinoblastoma protein (Rb) has a key role in tumorigenesis. It is well established that Rb function is largely regulated by a dynamic balance of phosphorylation and dephosphorylation. Although much research has been done to understand the mechanisms and function of RB phosphorylation, the regulation of Rb dephosphorylation is still not well understood. In this study, we demonstrate that Pin1 has an important role in the regulation of Rb function in cell cycle progression and S-phase checkpoint upon DNA damage. We show that the Rb C-pocket directly binds to the Pin1 WW domain in vitro and in vivo, and that the phosphorylation of Rb C-pocket by G1/S Cyclin/Cyclin-dependent kinase complexes is critical for mediating this interaction. We further show that Rb-mediated cell cycle arrest and Rb-induced premature cellular senescence are effectively inhibited by Pin1 expression. In addition, DNA damage induces Rb dephosphorylation in a PP2A-dependent manner, and this process is inhibited by Pin1. Furthermore, the overexpression of Pin1 promotes Rb hyperphosphorylation upon S-phase DNA damage. Importantly, both the Pin1 WW domain and isomerase activity are required for its effect on S-phase checkpoint. Moreover, the overexpression of Pin1 is correlated with Rb hyperphosphorylation in breast cancer biopsies. These results indicate that Pin1 has a critical role in the modulation of Rb function by the regulation of Rb dephosphorylation, which may have an important pathological role in cancer development. PMID:25675300

  15. Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage.

    PubMed

    Tong, Y; Ying, H; Liu, R; Li, L; Bergholz, J; Xiao, Z-X

    2015-02-12

    Inactivation of the retinoblastoma protein (Rb) has a key role in tumorigenesis. It is well established that Rb function is largely regulated by a dynamic balance of phosphorylation and dephosphorylation. Although much research has been done to understand the mechanisms and function of RB phosphorylation, the regulation of Rb dephosphorylation is still not well understood. In this study, we demonstrate that Pin1 has an important role in the regulation of Rb function in cell cycle progression and S-phase checkpoint upon DNA damage. We show that the Rb C-pocket directly binds to the Pin1 WW domain in vitro and in vivo, and that the phosphorylation of Rb C-pocket by G1/S Cyclin/Cyclin-dependent kinase complexes is critical for mediating this interaction. We further show that Rb-mediated cell cycle arrest and Rb-induced premature cellular senescence are effectively inhibited by Pin1 expression. In addition, DNA damage induces Rb dephosphorylation in a PP2A-dependent manner, and this process is inhibited by Pin1. Furthermore, the overexpression of Pin1 promotes Rb hyperphosphorylation upon S-phase DNA damage. Importantly, both the Pin1 WW domain and isomerase activity are required for its effect on S-phase checkpoint. Moreover, the overexpression of Pin1 is correlated with Rb hyperphosphorylation in breast cancer biopsies. These results indicate that Pin1 has a critical role in the modulation of Rb function by the regulation of Rb dephosphorylation, which may have an important pathological role in cancer development.

  16. MiR-1 Overexpression Enhances Ca2+ release and Promotes Cardiac Arrhythmogenesis by Targeting PP2A Regulatory Subunit B56α and Causing CaMKII-Dependent Hyperphosphorylation of RyR2

    PubMed Central

    Terentyev, Dmitry; Belevych, Andriy E.; Terentyeva, Radmila; Martin, Mickey M.; Malana, Geraldine E.; Kuhn, Donald E.; Abdellatif, Maha; Feldman, David S; Elton, Terry S.; Gyorke, Sandor

    2015-01-01

    MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca2+ cycling in rat ventricular myocytes using methods of electrophysiology, Ca2+ imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca2+ current, flattening of Ca2+ transients voltage dependency and enhanced frequency of spontaneous Ca2+ sparks while reducing the sarcoplasmic reticulum Ca2+ content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca2+, events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (CaMKII) but not at S2808 (PKA). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56α involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation-contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels. PMID:19131648

  17. Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

    PubMed

    Loveday, Chey; Tatton-Brown, Katrina; Clarke, Matthew; Westwood, Isaac; Renwick, Anthony; Ramsay, Emma; Nemeth, Andrea; Campbell, Jennifer; Joss, Shelagh; Gardner, McKinlay; Zachariou, Anna; Elliott, Anna; Ruark, Elise; van Montfort, Rob; Rahman, Nazneen

    2015-09-01

    Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions.

  18. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation.

    PubMed

    Peng, Di; Wang, Zining; Huang, Anfei; Zhao, Yong; Qin, F Xiao-Feng

    2017-01-15

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

  19. A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation

    PubMed Central

    Peng, Di; Wang, Zining; Huang, Anfei

    2017-01-01

    The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A. PMID:27956528

  20. Differential thiol oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle.

    PubMed

    Tan, Pearl Lin; Shavlakadze, Tea; Grounds, Miranda D; Arthur, Peter G

    2015-05-01

    Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.

  1. Calyculins and Related Marine Natural Products as Serine-Threonine Protein Phosphatase PP1 and PP2A Inhibitors and Total Syntheses of Calyculin A, B, and C

    PubMed Central

    Fagerholm, Annika E.; Habrant, Damien; Koskinen, Ari M. P.

    2010-01-01

    Calyculins, highly cytotoxic polyketides, originally isolated from the marine sponge Discodermia calyx by Fusetani and co-workers, belong to the lithistid sponges group. These molecules have become interesting targets for cell biologists and synthetic organic chemists. The serine/threonine protein phosphatases play an essential role in the cellular signalling, metabolism, and cell cycle control. Calyculins express potent protein phosphatase 1 and 2A inhibitory activity, and have therefore become valuable tools for cellular biologists studying intracellular processes and their control by reversible phosphorylation. Calyculins might also play an important role in the development of several diseases such as cancer, neurodegenerative diseases, and type 2-diabetes mellitus. The fascinating structures of calyculins have inspired various groups of synthetic organic chemists to develop total syntheses of the most abundant calyculins A and C. However, with fifteen chiral centres, a cyano-capped tetraene unit, a phosphate-bearing spiroketal, an anti, anti, anti dipropionate segment, an α-chiral oxazole, and a trihydroxylated γ-amino acid, calyculins reach versatility that only few natural products can surpass, and truly challenge modern chemists’ asymmetric synthesis skills. PMID:20161975

  2. Ceramide-Initiated Protein Phosphatase 2A Activation Contributes to Arterial Dysfunction In Vivo

    PubMed Central

    Bharath, Leena P.; Ruan, Ting; Li, Youyou; Ravindran, Anindita; Wan, Xin; Nhan, Jennifer Kim; Walker, Matthew Lewis; Deeter, Lance; Goodrich, Rebekah; Johnson, Elizabeth; Munday, Derek; Mueller, Robert; Kunz, David; Jones, Deborah; Reese, Van; Summers, Scott A.; Babu, Pon Velayutham Anandh; Holland, William L.; Zhang, Quan-Jiang; Abel, E. Dale

    2015-01-01

    Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance. PMID:26253611

  3. miRNA-1246 induces pro-inflammatory responses in mesenchymal stem/stromal cells by regulating PKA and PP2A.

    PubMed

    Bott, Alexander; Erdem, Nese; Lerrer, Shalom; Hotz-Wagenblatt, Agnes; Breunig, Christian; Abnaof, Khalid; Wörner, Angelika; Wilhelm, Heike; Münstermann, Ewald; Ben-Baruch, Adit; Wiemann, Stefan

    2017-01-31

    The tumor microenvironment (TME) has an impact on breast cancer progression by creating a pro-inflammatory milieu within the tumor. However, little is known about the roles of miRNAs in cells of the TME during this process. We identified six putative oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells (MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent of TNFα stimulations and mediated by direct targeting of the tumor-suppressors PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3 phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7, T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancer-related inflammation and breast cancer progression.

  4. Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1.

    PubMed

    Yao, Xiu-Qing; Li, Xia-Chun; Zhang, Xiao-Xue; Yin, Yang-Yang; Liu, Bin; Luo, Dan-Ju; Wang, Qun; Wang, Jian-Zhi; Liu, Gong-Ping

    2012-07-30

    Protein phosphatase-2A (PP2A) activity is significantly suppressed in Alzheimer's disease. We have reported that glycogen synthase kinase-3β (GSK-3β) inhibits PP2A via upregulating the phosphorylation of PP2A catalytic subunit (PP2A(C)). Here we studied the effects of GSK-3β on the inhibitory demethylation of PP2A at leucine-309 (dmL309-PP2A(C)). We found that GSK-3β regulates dmL309-PP2A(C) level by regulating PME-1 and PPMT1. Knockdown of PME-1 or PPMT1 eliminated the effects of GSK-3β on PP2A(C). GSK-3 could negatively regulate PP2A regulatory subunit protein level. We conclude that GSK-3β can inhibit PP2A by increasing the inhibitory L309-demethylation involving upregulation of PME-1 and inhibition of PPMT1.

  5. α1-Antitrypsin Activates Protein Phosphatase 2A to Counter Lung Inflammatory Responses

    PubMed Central

    Geraghty, Patrick; Eden, Edward; Pillai, Manju; Campos, Michael; McElvaney, Noel G.

    2014-01-01

    Rationale: α1-Antitrypsin (A1AT) was identified as a plasma protease inhibitor; however, it is now recognized as a multifunctional protein that modulates immunity, inflammation, proteostasis, apoptosis, and cellular senescence. Like A1AT, protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, regulates similar biologic processes and plays a key role in chronic obstructive pulmonary disease. Objectives: Given their common effects, this study investigated whether A1AT acts via PP2A to alter tumor necrosis factor (TNF) signaling, inflammation, and proteolytic responses in this disease. Methods: PP2A activity was measured in peripheral blood neutrophils from A1AT-deficient (PiZZ) and healthy (PiMM) individuals and in alveolar macrophages from normal (60 mg/kg) and high-dose (120 mg/kg) A1AT-treated PiZZ subjects. PP2A activation was assessed in human neutrophils, airway epithelial cells, and peripheral blood monocytes treated with plasma purified A1AT protein. Similarly, lung PP2A activity was measured in mice administered intranasal A1AT. PP2A was silenced in lung epithelial cells treated with A1AT and matrix metalloproteinase and cytokine production was then measured following TNF-α stimulation. Measurements and Main Results: PP2A was significantly lower in neutrophils isolated from PiZZ compared with PiMM subjects. A1AT protein activated PP2A in human alveolar macrophages, monocytes, neutrophils, airway epithelial cells, and in mouse lungs. This activation required functionally active A1AT protein and protein tyrosine phosphatase 1B expression. A1AT treatment acted via PP2A to prevent p38 and IκBα phosphorylation and matrix metalloproteinase and cytokine induction in TNF-α–stimulated epithelial cells. Conclusions: Together, these data indicate that A1AT modulates PP2A to counter inflammatory and proteolytic responses induced by TNF signaling in the lung. PMID:25341065

  6. Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

    PubMed Central

    Lee, Erwin M.; Harrison, Celeste; Kahl, Richard; Flanagan, Hayley; Panicker, Nikita; Mashkani, Baratali; Don, Anthony S.; Morris, Jonathan; Toop, Hamish; Lock, Richard B.; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Moore, Andrew; Ashman, Leonie K.; Skelding, Kathryn A.; Enjeti, Anoop; Verrills, Nicole M.

    2016-01-01

    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. PMID:27329844

  7. Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development.

    PubMed

    Kawamoto, M; Fujiwara, A; Yasumasu, I

    2000-08-01

    In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP-dependent protein kinase (A kinase), Ca2+/calmodulin-dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid-dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise.

  8. Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay, the PP2A inhibition assay and HPLC with fluorimetric detection.

    PubMed

    Prassopoulou, Eleanna; Katikou, Panagiota; Georgantelis, Dimitrios; Kyritsakis, Apostolos

    2009-02-01

    An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 microg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.

  9. Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor.

    PubMed

    Okamura, Hirohiko; Yoshida, Kaya; Yang, Di; Haneji, Tatsuji

    2013-05-01

    Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation.

  10. Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney.

    PubMed

    Li, M; Guo, H; Damuni, Z

    1995-02-14

    Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.

  11. [Changes of activity and expression of protein phosphatase type 2A during the apoptosis of NB4 and MR2 cells induced by arsenic trioxide].

    PubMed

    Xu, Xi-Hui; Ouyang, Jian; Xie, Pin-Hao; Chen, Jun-Hao

    2008-10-01

    This study was aimed to investigate the change of expression and activity of protein phosphatases type 2A (PP2A) during the apoptosis of NB4 and MR2 cells induced by Arsenic trioxide (ATO). NB4 and MR2 cells were incubated with Okadaic acid (OKA) (0.5 nmol/L), ATO (0.5 - 2.0 micromol/L), and the combination of OKA and ATO at the same doses as in the single-agent treatment respectively. Then the proliferation of NB4 and MR2 cells was determined by MTT assay, the morphologic changes of cells were evaluated by Wright's staining, the apoptosis rates were detected by flow cytometry. At last, the activities of PP2A were evaluated by the serine/threonine phosphatase assay system, and the levels of PP2A subunits were detected by Western blot analysis. The results showed that ATO inhibited proliferation of NB4 and MR2 cells, and the inhibition rates of ATO on the two cells significantly increased after the addition of OKA. OKA could augment the apoptosis of NB4 and MR2 cells induced by ATO. During the apoptosis of NB4 and MR2 cells, the activity of PP2A decreased with increasing concentration of ATO, and OKA augmented the inhibitory effect of ATO on the activity. The level of PP2A structural subunit (PP2A-A) decreased during ATO-induced apoptosis of NB4 and MR2 cells, that expressions of B and C subunits of PP2A were relatively unaltered. It is concluded that the activity of PP2A decreases with increasing concentration of ATO during the apoptosis of NB4 and MR2 cells, and the decrease of the activity of PP2A maybe is related to the repression of expression of PP2A -A subunit; the inhibition of the activity of PP2A can promote the ATO induced apoptosis of NB4 and MRL cells.

  12. The Ubiquitin E3 Ligase NOSIP Modulates Protein Phosphatase 2A Activity in Craniofacial Development

    PubMed Central

    Hoffmeister, Meike; Prelle, Carola; Küchler, Philipp; Kovacevic, Igor; Moser, Markus; Müller-Esterl, Werner; Oess, Stefanie

    2014-01-01

    Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans. PMID:25546391

  13. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

    PubMed Central

    Janssens, V; Goris, J

    2001-01-01

    Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon. PMID:11171037

  14. PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.

    PubMed

    Wandzioch, Ewa; Pusey, Michelle; Werda, Amy; Bail, Sophie; Bhaskar, Aishwarya; Nestor, Mariya; Yang, Jing-Jing; Rice, Lyndi M

    2014-08-15

    Protein phosphatase 2A (PP2A) negatively regulates tumorigenic signaling pathways, in part, by supporting the function of tumor suppressors like p53. The PP2A methylesterase PME-1 limits the activity of PP2A by demethylating its catalytic subunit. Here, we report the finding that PME-1 overexpression correlates with increased cell proliferation and invasive phenotypes in endometrial adenocarcinoma cells, where it helps maintain activated ERK and Akt by inhibiting PP2A. We obtained evidence that PME-1 could bind and regulate protein phosphatase 4 (PP4), a tumor-promoting protein, but not the related protein phosphatase 6 (PP6). When the PP2A, PP4, or PP6 catalytic subunits were overexpressed, inhibiting PME-1 was sufficient to limit cell proliferation. In clinical specimens of endometrial adenocarcinoma, PME-1 levels were increased and we found that PME-1 overexpression was sufficient to drive tumor growth in a xenograft model of the disease. Our findings identify PME-1 as a modifier of malignant development and suggest its candidacy as a diagnostic marker and as a therapeutic target in endometrial cancer.

  15. Structural Mechanism of Demethylation and Inactivation of Protein Phosphatase 2A

    SciTech Connect

    Xing,Y.; Li, Z.; Chen, Y.; Stock, J.; Jeffrey, P.; Shi, Y.

    2008-01-01

    Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that plays a role in many biological processes. Reversible carboxyl methylation of the PP2A catalytic subunit is an essential regulatory mechanism for its function. Demethylation and negative regulation of PP2A is mediated by a PP2A-specific methylesterase PME-1, which is conserved from yeast to humans. However, the underlying mechanism of PME-1 function remains enigmatic. Here we report the crystal structures of PME-1 by itself and in complex with a PP2A heterodimeric core enzyme. The structures reveal that PME-1 directly binds to the active site of PP2A and that this interaction results in the activation of PME-1 by rearranging the catalytic triad into an active conformation. Strikingly, these interactions also lead to inactivation of PP2A by evicting the manganese ions that are required for the phosphatase activity of PP2A. These observations identify a dual role of PME-1 that regulates PP2A activation, methylation, and holoenzyme assembly in cells.

  16. CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection

    PubMed Central

    Cvrljevic, Anna; Khan, Mohd Moin; Treise, Irina; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Au-Yeung, Byron; Sittig, Eleonora; Laajala, Teemu Daniel; Chen, Yiling; Oeder, Sebastian; Calzada-Wack, Julia; Horsch, Marion; Aittokallio, Tero; Busch, Dirk H.; Ollert, Markus W.; Neff, Frauke; Beckers, Johannes; Gailus-Durner, Valerie; Fuchs, Helmut; de Angelis, Martin Hrabě; Chen, Zhi; Lahesmaa, Riitta; Westermarck, Jukka

    2016-01-01

    The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2AHOZ) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2AHOZ mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4+ T-cells and CD8+ effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2AHOZ as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects. PMID:27100879

  17. Protein phosphatase 2A regulates interleukin-2 receptor complex formation and JAK3/STAT5 activation.

    PubMed

    Ross, Jeremy A; Cheng, Hanyin; Nagy, Zsuzsanna S; Frost, Jeffrey A; Kirken, Robert A

    2010-02-05

    Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rbeta, but not IL-2Rgamma. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rbeta. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rbeta, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rbeta by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rgamma in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.

  18. Protein phosphatase 2A is essential to maintain active Wnt signaling and its Aβ tumor suppressor subunit is not expressed in colon cancer cells.

    PubMed

    Carmen Figueroa-Aldariz, M; Castañeda-Patlán, M Cristina; Santoyo-Ramos, Paula; Zentella, Alejandro; Robles-Flores, Martha

    2015-11-01

    Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and β isoforms, cancer cells which have altered Wnt signaling do not express the Aβ isoform mRNA. Remarkably, we found that the Aβ protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aβ expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aβ isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with β-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in β-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.

  19. A role for protein phosphatase 2A in regulating p38 mitogen activated protein kinase activation and tumor necrosis factor-alpha expression during influenza virus infection.

    PubMed

    Law, Anna H Y; Tam, Alex H M; Lee, Davy C W; Lau, Allan S Y

    2013-04-02

    Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

  20. Oxidative Impairment of Hippocampal Long-term Potentiation Involves Activation of Protein Phosphatase 2A and Is Prevented by Ketone Bodies

    PubMed Central

    Maalouf, Marwan; Rho, Jong M.

    2008-01-01

    Previous studies have shown that ketone bodies (KB) exert antioxidant effects in experimental models of neurological disease. In the present study, we explored the effects of the KB acetoacetate (ACA) and β-hydroxybutyrate (BHB) on impairment of hippocampal long-term potentiation (LTP) in rats by hydrogen peroxide (H2O2) using electrophysiological, fluorescence imaging and enzyme assay techniques. We found that: (1) a combination of ACA and BHB (1 mM each) prevented impairment of LTP by H2O2 (200 μM); (2) KB significantly lowered intracellular levels of reactive oxygen species (ROS) — measured with the fluorescent indicator carboxy-H2DCFDA — in CA1 pyramidal neurons exposed to H2O2; (3) the effect of KB on LTP was replicated by the protein phosphatase 2A (PP2A) inhibitor fostriecin; (4) KB prevented impairment of LTP by the PP2A activator C6 ceramide; (5) fostriecin did not prevent the increase in ROS levels in CA1 pyramidal neurons exposed to H2O2, and C6 ceramide did not increase ROS levels; (6) PP2A activity was enhanced by both H2O2and rotenone – a mitochondrial complex I inhibitor that increases endogenous superoxide production; and (7) KB inhibited PP2A activity in protein extracts from brain tissue treated with either H2O2 or ceramide. We propose that oxidative impairment of hippocampal LTP is associated with PP2A activation, and that KB prevent this impairment in part by inducing PP2A inhibition through an antioxidant mechanism. PMID:18646208

  1. Protein serine/threonine Phosphotase-2A is differentially expressed and regulates eye development in vertebrates.

    PubMed

    Liu, W-B; Hu, X-H; Zhang, X-W; Deng, M-X; Nie, L; Hui, S-S; Duan, W; Tao, M; Zhang, C; Liu, J; Hu, W-F; Huang, Z-X; Li, L; Yi, M; Li, T-T; Wang, L; Liu, Y; Liu, S-J; Li, D W-C

    2013-09-01

    Protein serine/threonine phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that protein phosphatase-2A is important for normal eye development in goldfish.

  2. The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

    NASA Technical Reports Server (NTRS)

    Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

    1999-01-01

    Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

  3. FAM122A, a new endogenous inhibitor of protein phosphatase 2A

    PubMed Central

    Fan, Li; Liu, Man-Hua; Guo, Meng; Hu, Chuan-Xi; Yan, Zhao-Wen; Chen, Jing; Chen, Guo-Qiang; Huang, Ying

    2016-01-01

    The regulation of the ubiquitously expressed protein phosphatase 2A (PP2A) is essential for various cellular functions such as cell proliferation, transformation, and fate determination. In this study, we demonstrate that the highly conserved protein in mammals, designated FAM122A, directly interacts with PP2A-Aα and B55α rather than B56α subunits, and inhibits the phosphatase activity of PP2A-Aα/B55α/Cα complex. Further, FAM122A potentiates the degradation of catalytic subunit PP2A-Cα with the increased poly-ubiquitination. In agreement, FAM122A silencing inhibits while its overexpression enhances cell growth and colony-forming ability. Collectively, we identify FAM122A as a new endogenous PP2A inhibitor and its physiological and pathophysiological significances warrant to be further investigated. PMID:27588481

  4. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

    PubMed

    Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

    2002-03-15

    We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

  5. Yeast Protein Phosphatase 2A-Cdc55 Regulates the Transcriptional Response to Hyperosmolarity Stress by Regulating Msn2 and Msn4 Chromatin Recruitment

    PubMed Central

    Reiter, Wolfgang; Klopf, Eva; De Wever, Veerle; Anrather, Dorothea; Petryshyn, Andriy; Roetzer, Andreas; Niederacher, Gerhard; Roitinger, Elisabeth; Dohnal, Ilse; Görner, Wolfram; Mechtler, Karl; Brocard, Cécile

    2013-01-01

    We have identified Cdc55, a regulatory B subunit of protein phosphatase 2A (PP2A), as an essential activating factor for stress gene transcription in Saccharomyces cerevisiae. The presence of PP2A-Cdc55 is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. We show that PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 during hyperosmolarity stress. PP2A-Cdc55 also enhances Msn2-dependent transactivation, required for extended chromatin recruitment of the transcription factor. We analyzed a possible direct regulatory role for PP2A-Cdc55 on the phosphorylation status of Msn2. Detailed mass spectrometric and genetic analysis of Msn2 showed that stress exposure causes immediate transient dephosphorylation of Msn2 which is not dependent on PP2A-Cdc55 activity. Furthermore, the Hog1 mitogen-activated protein kinase pathway activity is not influenced by PP2A-Cdc55. We therefore propose that the PP2A-Cdc55 phosphatase is not involved in cytosolic stress signal perception but is involved in a specific intranuclear mechanism to regulate Msn2 and Msn4 nuclear accumulation and chromatin association under stress conditions. PMID:23275436

  6. Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

    NASA Astrophysics Data System (ADS)

    Deng, Yuanjun; Guo, Yanyan; Liu, Ping; Zeng, Rui; Ning, Yong; Pei, Guangchang; Li, Yueqiang; Chen, Meixue; Guo, Shuiming; Li, Xiaoqing; Han, Min; Xu, Gang

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) contributes to the emergence of fibroblasts and plays a significant role in renal interstitial fibrosis. Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and regulates many signaling pathways. However, the significance of PP2A in EndMT is poorly understood. In present study, the role of PP2A in EndMT was evaluated. We demonstrated that PP2A activated in endothelial cells (EC) during their EndMT phenotype acquisition and in the mouse model of obstructive nephropathy (i.e., UUO). Inhibition of PP2A activity by its specific inhibitor prevented EC undergoing EndMT. Importantly, PP2A activation was dependent on tyrosine nitration at 127 in the catalytic subunit of PP2A (PP2Ac). Our renal-protective strategy was to block tyrosine127 nitration to inhibit PP2A activation by using a mimic peptide derived from PP2Ac conjugating a cell penetrating peptide (CPP: TAT), termed TAT-Y127WT. Pretreatment withTAT-Y127WT was able to prevent TGF-β1-induced EndMT. Administration of the peptide to UUO mice significantly ameliorated renal EndMT level, with preserved density of peritubular capillaries and reduction in extracellular matrix deposition. Taken together, these results suggest that inhibiting PP2Ac nitration using a mimic peptide is a potential preventive strategy for EndMT in renal fibrosis.

  7. Protein phosphatase 2A is a critical regulator of protein kinase C zeta signaling targeted by SV40 small t to promote cell growth and NF-kappaB activation.

    PubMed Central

    Sontag, E; Sontag, J M; Garcia, A

    1997-01-01

    We have reported that inhibition of protein phosphatase 2A (PP2A) by expression of SV40 small t stimulates the mitogenic MAP kinase cascade. Here, we show that SV40 small t can substitute for tumor necrosis factor-alpha (TNF-alpha) or serum and stimulate atypical protein kinase C zeta (PKC zeta) activity, resulting in MEK activation, cell proliferation and NF-kappaB-dependent gene transcriptional activation in CV-1 and NIH 3T3 cells. These effects were abrogated by co-expression of kinase-deficient PKC zeta and inhibition of phosphatidylinositol 3-kinase p85alpha-p110 by wortmannin, LY294002 and a dominant-negative mutant of p85alpha. In contrast, expression of kinase-inactive ERK2 inhibited small t-dependent cell growth but was unable to abolish small t-induced NF-kappaB transactivation. Our results provide the first in vivo evidence for a critical regulatory role of PP2A in bifunctional PKC zeta signaling pathways controlled by phosphatidylinositol 3-kinase. Constitutive activation of PKC zeta and NF-kappaB following inhibition of PP2A supports new mechanisms by which SV40 small t promotes cell growth and transformation. By establishing PP2A as a key player in the response of cells to growth factors and stress signals like TNF-alpha, our findings could explain why PP2A is a primary target utilized during SV40 infection to alter cellular behavior. PMID:9312025

  8. Structure and Mechanism of the Phosphotyrosyl Phosphatase Activator

    SciTech Connect

    Chao,Y.; Xing, Y.; Chen, Y.; Xu, Y.; Lin, Z.; Li, Z.; Jeffrey, P.; Stock, J.; Shi, Y.

    2006-01-01

    Phosphotyrosyl phosphatase activator (PTPA), also known as PP2A phosphatase activator, is a conserved protein from yeast to human. Here we report the 1.9 {angstrom} crystal structure of human PTPA, which reveals a previously unreported fold consisting of three subdomains: core, lid, and linker. Structural analysis uncovers a highly conserved surface patch, which borders the three subdomains, and an associated deep pocket located between the core and the linker subdomains. The conserved surface patch and the deep pocket are responsible for binding to PP2A and ATP, respectively. PTPA and PP2A A-C dimer together constitute a composite ATPase. PTPA binding to PP2A results in a dramatic alteration of substrate specificity, with enhanced phosphotyrosine phosphatase activity and decreased phosphoserine phosphatase activity. This function of PTPA strictly depends on the composite ATPase activity. These observations reveal significant insights into the function and mechanism of PTPA and have important ramifications for understanding PP2A function.

  9. Zinc binds to and directly inhibits protein phosphatase 2A in vitro.

    PubMed

    Xiong, Yan; Luo, Dan-Ju; Wang, Xiu-Lian; Qiu, Mei; Yang, Yang; Yan, Xiong; Wang, Jian-Zhi; Ye, Qi-Fa; Liu, Rong

    2015-06-01

    Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn(2+) has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn(2+) on PP2A and their direct interaction in vitro. The results showed that Zn(2+) mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 μmol/L) of Zn(2+) inhibited PP2A directly. Further Zn(2+)-IDA-agarose affinity binding assays showed that Zn(2+) bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn(2+) inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.

  10. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle

    PubMed Central

    1995-01-01

    Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport. PMID:7896877

  11. Piperine induces autophagy by enhancing protein phosphotase 2A activity in a rotenone-induced Parkinson's disease model

    PubMed Central

    Liu, Jia; Chen, Min; Wang, Xue; Wang, Yi; Duan, Chunli; Gao, Ge; Lu, Lingling; Wu, Xia; Wang, Xiaomin; Yang, Hui

    2016-01-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but there are few treatments currently available. The autophagy pathway plays an important role in the pathogenesis of PD; modulating this pathway is considered to be a promising treatment strategy. Piperine (PIP) is a Chinese medicine with anti-inflammatory and antioxidant effects. The present study investigated the neuroprotective effects of PIP on rotenone-induced neurotoxicity in SK-N-SH cells, primary rat cortical neurons, and in a mouse model. Mice were administered rotenone (10mg/kg) for 6 weeks; PIP (25mg/kg, 50mg/kg) was subsequently administered for 4 weeks. We found that PIP treatment attenuated rotenone-induced motor deficits, and rescued the loss of dopaminergic neurons in the substantia nigra. PIP increased cell viability and restored mitochondrial functioning in SK-N-SH cells and primary neurons. In addition, PIP induced autophagy by inhibiting mammalian target of rapamycin complex 1(mTORC1) via activation of protein phosphotase 2A (PP2A). However, inhibiting PP2A activity with okadaic acid reduced these protective effects, suggesting that PP2A is a target of PIP. These findings demonstrate that PIP exerts neuroprotective effects in PD models via induction of autophagy, and may be an effective agent for PD treatment. PMID:27572322

  12. Piperine induces autophagy by enhancing protein phosphotase 2A activity in a rotenone-induced Parkinson's disease model.

    PubMed

    Liu, Jia; Chen, Min; Wang, Xue; Wang, Yi; Duan, Chunli; Gao, Ge; Lu, Lingling; Wu, Xia; Wang, Xiaomin; Yang, Hui

    2016-09-20

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but there are few treatments currently available. The autophagy pathway plays an important role in the pathogenesis of PD; modulating this pathway is considered to be a promising treatment strategy. Piperine (PIP) is a Chinese medicine with anti-inflammatory and antioxidant effects. The present study investigated the neuroprotective effects of PIP on rotenone-induced neurotoxicity in SK-N-SH cells, primary rat cortical neurons, and in a mouse model. Mice were administered rotenone (10mg/kg) for 6 weeks; PIP (25mg/kg, 50mg/kg) was subsequently administered for 4 weeks. We found that PIP treatment attenuated rotenone-induced motor deficits, and rescued the loss of dopaminergic neurons in the substantia nigra. PIP increased cell viability and restored mitochondrial functioning in SK-N-SH cells and primary neurons. In addition, PIP induced autophagy by inhibiting mammalian target of rapamycin complex 1(mTORC1) via activation of protein phosphotase 2A (PP2A). However, inhibiting PP2A activity with okadaic acid reduced these protective effects, suggesting that PP2A is a target of PIP. These findings demonstrate that PIP exerts neuroprotective effects in PD models via induction of autophagy, and may be an effective agent for PD treatment.

  13. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

    PubMed

    Ogris, E; Du, X; Nelson, K C; Mak, E K; Yu, X X; Lane, W S; Pallas, D C

    1999-05-14

    Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

  14. Tau pathology involves protein phosphatase 2A in Parkinsonism-dementia of Guam

    PubMed Central

    Arif, Mohammad; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Garruto, Ralph M.; Iqbal, Khalid

    2014-01-01

    Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr307 and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr307 in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr307. These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5–PP2A complex, and its phosphorylation at Tyr307 by Src. PMID:24395787

  15. Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A

    NASA Technical Reports Server (NTRS)

    Jackson, K.; Soll, D.; Evans, M. L. (Principal Investigator)

    1999-01-01

    The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

  16. Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix.

    PubMed

    Okamura, Hirohiko; Yoshida, Kaya; Ochiai, Kazuhiko; Haneji, Tatsuji

    2011-09-01

    The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A Cα in osteoblast differentiation. Administration of 1nM OA to the calvarial region in mice increased bone mineral density, as shown by μCT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A Cα was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A Cα in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A Cα. Accordingly, the silencing of PP2A Cα in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, Bsp, and OCN. Our data indicate that PP2A Cα plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes.

  17. Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B′

    PubMed Central

    Berry, Meera; Gehring, Walter

    2000-01-01

    Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, we performed a yeast two-hybrid screen. A Drosophila homologue of the regulatory subunit (B′/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B′) specifically interacted with the SCR homeodomain. The N-terminal arm within the SCR homeodomain was shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses revealed that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain were active or inactive, respectively. Inactivity of the phosphorylated mimic form was attributed to impaired DNA binding. Specific ablation of dPP2A,B′ gene activity by double-stranded RNA-mediated genetic interference resulted in embryos without salivary glands, an SCR null phenotype. Our data demonstrate an essential role for Drosophila PP2A,B′ in positively modulating SCR function. PMID:10856239

  18. Nitric oxide modulates chromatin folding in human endothelial cells via protein phosphatase 2A activation and class II histone deacetylases nuclear shuttling.

    PubMed

    Illi, Barbara; Dello Russo, Claudio; Colussi, Claudia; Rosati, Jessica; Pallaoro, Michele; Spallotta, Francesco; Rotili, Dante; Valente, Sergio; Ragone, Gianluca; Martelli, Fabio; Biglioli, Paolo; Steinkuhler, Christian; Gallinari, Paola; Mai, Antonello; Capogrossi, Maurizio C; Gaetano, Carlo

    2008-01-04

    Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27-275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.

  19. Phosphorylated protein phosphatase 2A determines poor outcome in patients with metastatic colorectal cancer

    PubMed Central

    Cristóbal, I; Manso, R; Rincón, R; Caramés, C; Zazo, S; del Pulgar, T G; Cebrián, A; Madoz-Gúrpide, J; Rojo, F; García-Foncillas, J

    2014-01-01

    Background: Protein phosphatase 2A (PP2A) is a tumour suppressor frequently inactivated in human cancer and its tyrosine-307 phosphorylation has been reported as a molecular inhibitory mechanism. Methods: Expression of phosphorylated PP2A (p-PP2A) was evaluated in 250 metastatic colorectal cancer (CRC) patients. Chi-square, Kaplan–Meier and Cox analyses were used to determine correlations with clinical and molecular parameters and impact on clinical outcomes. Results: High p-PP2A levels were found in 17.2% cases and were associated with ECOG performance status (P=0.001) and presence of synchronous metastasis at diagnosis (P=0.035). This subgroup showed substantially worse overall survival (OS) (median OS, 6.0 vs 26.2 months, P<0.001) and progression-free survival (PFS) (median PFS, 3.8 vs 13.3 months, P<0.001). The prognostic impact of p-PP2A was particularly evident in patients aged <70 years (P<0.001). Multivariate analysis revealed that p-PP2A retained its prognostic impact for OS (hazard ratio 2.7; 95% confidence interval, 1.8–4.1; P<0.001) and PFS (hazard ratio 3.0; 95% confidence interval, 1.8–5.0; P<0.001). Conclusions: Phosphorylated PP2A is an alteration that determines poor outcome in metastatic CRC and represents a novel potential therapeutic target in this disease, thus enabling to define a subgroup of patients who could benefit from future treatments based on PP2A activators. PMID:25003662

  20. Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

    PubMed Central

    Deng, Yuanjun; Guo, Yanyan; Liu, Ping; Zeng, Rui; Ning, Yong; Pei, Guangchang; Li, Yueqiang; Chen, Meixue; Guo, Shuiming; Li, Xiaoqing; Han, Min; Xu, Gang

    2016-01-01

    Endothelial-to-mesenchymal transition (EndMT) contributes to the emergence of fibroblasts and plays a significant role in renal interstitial fibrosis. Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and regulates many signaling pathways. However, the significance of PP2A in EndMT is poorly understood. In present study, the role of PP2A in EndMT was evaluated. We demonstrated that PP2A activated in endothelial cells (EC) during their EndMT phenotype acquisition and in the mouse model of obstructive nephropathy (i.e., UUO). Inhibition of PP2A activity by its specific inhibitor prevented EC undergoing EndMT. Importantly, PP2A activation was dependent on tyrosine nitration at 127 in the catalytic subunit of PP2A (PP2Ac). Our renal-protective strategy was to block tyrosine127 nitration to inhibit PP2A activation by using a mimic peptide derived from PP2Ac conjugating a cell penetrating peptide (CPP: TAT), termed TAT-Y127WT. Pretreatment withTAT-Y127WT was able to prevent TGF-β1-induced EndMT. Administration of the peptide to UUO mice significantly ameliorated renal EndMT level, with preserved density of peritubular capillaries and reduction in extracellular matrix deposition. Taken together, these results suggest that inhibiting PP2Ac nitration using a mimic peptide is a potential preventive strategy for EndMT in renal fibrosis. PMID:26805394

  1. A protein phosphatase 2A catalytic subunit modulates blue light-induced chloroplast avoidance movements through regulating actin cytoskeleton in Arabidopsis.

    PubMed

    Wen, Feng; Wang, Jinqian; Xing, Da

    2012-08-01

    Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.

  2. Negative control of BAK1 by protein phosphatase 2A during plant innate immunity.

    PubMed

    Segonzac, Cécile; Macho, Alberto P; Sanmartín, Maite; Ntoukakis, Vardis; Sánchez-Serrano, José Juan; Zipfel, Cyril

    2014-09-17

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP-triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co-receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B'η/ζ inhibits immune responses triggered by several PAMPs and anti-bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A-based regulation leads to increased steady-state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface-localized immune receptor complexes.

  3. Negative control of BAK1 by protein phosphatase 2A during plant innate immunity

    PubMed Central

    Segonzac, Cécile; Macho, Alberto P; Sanmartín, Maite; Ntoukakis, Vardis; Sánchez-Serrano, José Juan; Zipfel, Cyril

    2014-01-01

    Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP-triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co-receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B’η/ζ inhibits immune responses triggered by several PAMPs and anti-bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A-based regulation leads to increased steady-state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface-localized immune receptor complexes. PMID:25085430

  4. Nilotinib induces autophagy in hepatocellular carcinoma through AMPK activation.

    PubMed

    Yu, Hui-Chuan; Lin, Chen-Si; Tai, Wei-Tien; Liu, Chun-Yu; Shiau, Chung-Wai; Chen, Kuen-Feng

    2013-06-21

    Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.

  5. CIP2A modulates cell-cycle progression in human cancer cells by regulating the stability and activity of Plk1.

    PubMed

    Kim, Jae-Sung; Kim, Eun Ju; Oh, Jeong Su; Park, In-Chul; Hwang, Sang-Gu

    2013-11-15

    Abnormal cell-cycle control can lead to aberrant cell proliferation and cancer. The oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) is an inhibitor of protein phosphatase 2A (PP2A) that stabilizes c-Myc. However, the precise role of CIP2A in cell division is not understood. Herein, we show that CIP2A is required for mitotic progression by regulating the polo-like kinase (Plk1). With mitotic entry, CIP2A translocated from the cytoplasm to the nucleus, where it was enriched at spindle poles. CIP2A depletion delayed mitotic progression, resulting in mitotic abnormalities independent of PP2A activity. Unexpectedly, CIP2A interacted directly with the polo-box domain of Plk1 during mitosis. This interaction was required to maintain Plk1 stability by blocking APC/C-Cdh1-dependent proteolysis, thereby enhancing the kinase activity of Plk1 during mitosis. We observed strong correlation and in vivo interactions between these two proteins in multiple human cancer specimens. Overall, our results established a novel function for CIP2A in facilitating the stability and activity of the pivotal mitotic kinase Plk1 in cell-cycle progression and tumor development.

  6. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    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.

  7. Protein Phosphatase 2A Reactivates FOXO3a through a Dynamic Interplay with 14-3-3 and AKT

    PubMed Central

    Singh, Amrik; Ye, Min; Bucur, Octavian; Zhu, Shudong; Tanya Santos, Maria; Rabinovitz, Isaac; Wei, Wenyi; Gao, Daming; Hahn, William C.

    2010-01-01

    Forkhead box transcription factor FOXO3a, a key regulator of cell survival, is regulated by reversible phosphorylation and subcellular localization. Although the kinases regulating FOXO3a activity have been characterized, the role of protein phosphatases (PP) in the control of FOXO3a subcellular localization and function is unknown. In this study, we detected a robust interaction between FOXO3a and PP2A. We further demonstrate that 14-3-3, while not impeding the interaction between PP2A and FOXO3a, restrains its activity toward AKT phosphorylation sites T32/S253. Disruption of PP2A function revealed that after AKT inhibition, PP2A-mediated dephosphorylation of T32/S253 is required for dissociation of 14-3-3, nuclear translocation, and transcriptional activation of FOXO3a. Our findings reveal that distinct phosphatases dephosphorylate conserved AKT motifs within the FOXO family and that PP2A is entwined in a dynamic interplay with AKT and 14-3-3 to directly regulate FOXO3a subcellular localization and transcriptional activation. PMID:20110348

  8. The Role of PP2A Methylation in Susceptibility and Resistance to TBI and AD-Induced Neurodegeneration

    DTIC Science & Technology

    2014-10-01

    methylation affects sensitivity to behavioral impairments resulting traumatic brain injury caused by shockwave exposure. The primary bases for this...of these transgenes will exert similar effects with respect to shockwave -induced impairments. In the past year of the project, we identified shockwave ...increases. We also identified shockwave -induced behavioral impairments in these animals and similarly assessed the effect of PME over expression on

  9. The Role of PP2A Methylation in Susceptibility and Resistance to TBI and AD-Induced Neurodegeneration

    DTIC Science & Technology

    2015-06-01

    from shockwave exposure, and to compare those results with the effects of the same manipulations on the sensitivity to Alzheimer’s disease (AD) - like...our hypothesis is that they exert similar effects on shockwave -induced impairments. This effort has required a substantial investment in developing...equipment and protocols for exposing mice to a range of shockwave exposure conditions that mimic militarily relevant exposures. We found changes in

  10. The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis.

    PubMed Central

    Lin, F C; Arndt, K T

    1995-01-01

    We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102. At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis. These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis. Images PMID:7796803

  11. Activation of asparaginyl endopeptidase leads to Tau hyperphosphorylation in Alzheimer disease.

    PubMed

    Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Tung, Yunn Chyn; Liu, Fei; Iqbal, Khalid

    2013-06-14

    Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I2(PP2A). In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2(PP2A) is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I2NTF) and the C-terminal fragment (I2CTF). Both I2NTF and I2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I2(PP2A) translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I2(PP2A), except when I2(PP2A) was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I2(PP2A), inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I2(PP2A)-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.

  12. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  13. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  14. Physical association of GPR54 C-terminal with protein phosphatase 2A

    SciTech Connect

    Evans, Barry J.; Wang Zixuan; Mobley, La'Tonya; Khosravi, Davood; Fujii, Nobutaka; Navenot, Jean-Marc; Peiper, Stephen C.

    2008-12-26

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  15. Protein Phosphatase 2A in the Regulatory Network Underlying Biotic Stress Resistance in Plants

    PubMed Central

    Durian, Guido; Rahikainen, Moona; Alegre, Sara; Brosché, Mikael; Kangasjärvi, Saijaliisa

    2016-01-01

    Biotic stress factors pose a major threat to plant health and can significantly deteriorate plant productivity by impairing the physiological functions of the plant. To combat the wide range of pathogens and insect herbivores, plants deploy converging signaling pathways, where counteracting activities of protein kinases and phosphatases form a basic mechanism for determining appropriate defensive measures. Recent studies have identified Protein Phosphatase 2A (PP2A) as a crucial component that controls pathogenesis responses in various plant species. Genetic, proteomic and metabolomic approaches have underscored the versatile nature of PP2A, which contributes to the regulation of receptor signaling, organellar signaling, gene expression, metabolic pathways, and cell death, all of which essentially impact plant immunity. Associated with this, various PP2A subunits mediate post-translational regulation of metabolic enzymes and signaling components. Here we provide an overview of protein kinase/phosphatase functions in plant immunity signaling, and position the multifaceted functions of PP2A in the tightly inter-connected regulatory network that controls the perception, signaling and responding to biotic stress agents in plants. PMID:27375664

  16. Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A[W

    PubMed Central

    Leivar, Pablo; Antolín-Llovera, Meritxell; Ferrero, Sergi; Closa, Marta; Arró, Montserrat; Ferrer, Albert; Boronat, Albert; Campos, Narciso

    2011-01-01

    Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B′′ regulatory subunits of protein phosphatase 2A (PP2A), designated B′′α and B′′β, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B′′α and B′′β are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B′′α and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B′′β. Our data indicate that PP2A exerts multilevel control on HMGR through the five-member B′′ protein family during normal development and in response to a variety of stress conditions. PMID:21478440

  17. Carcinogenic Aspects of Protein Phosphatase 1 and 2A Inhibitors

    NASA Astrophysics Data System (ADS)

    Fujiki, Hirota; Suganuma, Masami

    Okadaic acid is functionally a potent tumor promoter working through inhibition of protein phosphatases 1 and 2A (PP1 and PP2A), resulting in sustained phosphorylation of proteins in cells. The mechanism of tumor promotion with oka-daic acid is thus completely different from that of the classic tumor promoter phorbol ester. Other potent inhibitors of PP1 and PP2A - such as dinophysistoxin-1, calyculins A-H, microcystin-LR and its derivatives, and nodularin - were isolated from marine organisms, and their structural features including the crystal structure of the PP1-inhibitor complex, tumor promoting activities, and biochemical and biological effects, are here reviewed. The compounds induced tumor promoting activity in three different organs, including mouse skin, rat glandular stomach and rat liver, initiated with three different carcinogens. The results indicate that inhibition of PP1 and PP2A is a general mechanism of tumor promotion applicable to various organs. This study supports the concept of endogenous tumor promoters in human cancer development.

  18. Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm

    PubMed Central

    Yi, Kexi; Wang, Xu; Emmett, Mark R.; Marshall, Alan G.; Stewart, Murray

    2009-01-01

    The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward. PMID:19458186

  19. Berberine Attenuates Axonal Transport Impairment and Axonopathy Induced by Calyculin A in N2a Cells

    PubMed Central

    Abid, Morad Dirhem Naji; Yan, Huanhuan; Huang, Hao; Wan, Limin; Feng, Zuohua; Chen, Juan

    2014-01-01

    Berberine is a primary component of the most functional extracts of Coptidis rhizome used in traditional Chinese medicine for centuries. Recent reports indicate that Berberine has the potential to prevent and treat Alzheimer's disease (AD). The previous studies reported that Calyculin A (CA) impaired the axonal transport in neuroblastoma-2a (N2a) cells. Berberine attenuated tau hyperphosphorylation and cytotoxicity induced by CA. Our study aimed at investigating the effects of Berberine on the axonal transport impairment induced by CA in N2a cells. The results showed that Berberine could protect the cell from CA -induced toxicity in metabolism and viability, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, Berberine could reverse CA-induced axonal transport impairment significantly. Berberine also partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307, a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde and the activity of superoxide dismutase, markers of oxidative stress, induced by CA. The present work for the first time demonstrates that Berberine may play a role in protecting against CA-induced axonal transport impairment by modulating the activity of PP-2A and oxidative stress. Our findings also suggest that Berberine may be a potential therapeutic drug for AD. PMID:24713870

  20. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  1. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  2. Structure of a Protein Phosphatase 2A Holoenzyme: Insights into B55-Mediated Tau Dephosphorylation

    SciTech Connect

    Xu, Y.; Chen, Y; Zhang, P; Jeffrey, P; Shi, Y

    2008-01-01

    Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular physiology. Members of the regulatory B/B55/PR55 family are thought to play a key role in the dephosphorylation of Tau, whose hyperphosphorylation contributes to Alzheimer's disease. The underlying mechanisms of the PP2A-Tau connection remain largely enigmatic. Here, we report the complete reconstitution of a Tau dephosphorylation assay and the crystal structure of a heterotrimeric PP2A holoenzyme involving the regulatory subunit B?. We show that B? specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The B? subunit comprises a seven-bladed ? propeller, with an acidic, substrate-binding groove located in the center of the propeller. The ? propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding ? hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau.

  3. Role for regulated phosphatase activity in generating mitotic oscillations in Xenopus cell-free extracts.

    PubMed

    Zhang, Tongli; Tyson, John J; Novák, Béla

    2013-12-17

    Although current textbook explanations of cell-cycle control in eukaryotes emphasize the periodic activation of cyclin-dependent protein kinases (CDKs), recent experimental observations suggest a significant role for the periodic activation and inactivation of a CDK-counteracting protein phosphatase 2A with a B55δ subunit (PP2A:B55δ), during mitotic cycles in frog-egg extracts and early embryos. In this paper, we extend an earlier mathematical model of embryonic cell cycles to include experimentally motivated roles for PP2A:B55δ and its regulation by Greatwall kinase. Our model is consistent with what is already known about the regulation of CDK and PP2A:B55δ in frog eggs, and it suggests a previously undescribed role for the Greatwall-PP2A:B55δ interaction in creating a toggle switch for activation of the anaphase-promoting complex as embryonic cells exit mitosis and return to interphase.

  4. Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina.

    PubMed

    Azad, Md Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2006-11-30

    The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

  5. Cross Talk between Wnt/β-Catenin and CIP2A/Plk1 Signaling in Prostate Cancer: Promising Therapeutic Implications

    PubMed Central

    Rojo, Federico; Madoz-Gúrpide, Juan

    2016-01-01

    Aberrant activation of the Wnt/β-catenin pathway and polo-like kinase 1 (Plk1) overexpression represent two common events in prostate cancer with relevant functional implications. This minireview analyzes their potential therapeutic significance in prostate cancer based on their role as androgen receptor (AR) signaling regulators and the pivotal role of the tumor suppressor protein phosphatase 2A (PP2A) modulating these pathways. PMID:27090640

  6. REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling

    PubMed Central

    Dennis, Michael D.; Coleman, Catherine S.; Berg, Arthur; Jefferson, Leonard S.; Kimball, Scot R.

    2014-01-01

    The protein kinase mTOR (mechanistic target of rapamycin) in complex 1 (mTORC1) promotes cell growth and proliferation in response to anabolic stimuli, including growth factors and nutrients. Growth factors activate mTORC1 by stimulating the kinase Akt, which phosphorylates and inhibits the tuberous sclerosis complex (TSC; which is comprised of TSC1, TSC2, and TBC1D7), thereby stimulating the mTORC1 activator Rheb. Here, we identified the mechanism through which REDD1 (regulated in DNA damage and development 1) represses the mTORC1 signaling pathway. We found that REDD1 promoted the protein phosphatase 2A (PP2A)-dependent dephosphorylation of Akt at Thr308 but not at Ser473. Consistent with previous studies showing that phosphorylation of Akt on Thr308, but not Ser473, is necessary for phosphorylation of TSC2, we observed a REDD1-dependent reduction in the phosphorylation of TSC2 and subsequently in the activity of Rheb. REDD1 and PP2A coimmunoprecipitated with Akt from wild-type but not REDD1-knockout mouse embryonic fibroblasts, suggesting that REDD1 may act as a targeting protein for the catalytic subunit of PP2A. Furthermore, binding to both Akt and PP2A was essential for REDD1 to repress signaling to mTORC1. Overall, the results demonstrate that REDD1 acts not just as a repressor of mTORC1, but also as a constant modulator of the phosphorylation of Akt in response to growth factors and nutrients. PMID:25056877

  7. ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

    PubMed Central

    Karampelias, Michael; Neyt, Pia; De Groeve, Steven; Aesaert, Stijn; Coussens, Griet; Rolčík, Jakub; Bruno, Leonardo; De Winne, Nancy; Van Minnebruggen, Annemie; Van Montagu, Marc; Ponce, María Rosa; Micol, José Luis; Friml, Jiří; De Jaeger, Geert; Van Lijsebettens, Mieke

    2016-01-01

    The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical–basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity. PMID:26888284

  8. ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling.

    PubMed

    Karampelias, Michael; Neyt, Pia; De Groeve, Steven; Aesaert, Stijn; Coussens, Griet; Rolčík, Jakub; Bruno, Leonardo; De Winne, Nancy; Van Minnebruggen, Annemie; Van Montagu, Marc; Ponce, María Rosa; Micol, José Luis; Friml, Jiří; De Jaeger, Geert; Van Lijsebettens, Mieke

    2016-03-08

    The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

  9. Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

    PubMed

    Sun, Lei; Pham, Tiffany T; Cornell, Timothy T; McDonough, Kelli L; McHugh, Walker M; Blatt, Neal B; Dahmer, Mary K; Shanley, Thomas P

    2017-01-01

    Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2A(fl/fl)) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2A(fl/fl) mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/β, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/β hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-β/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/β production compared with control BMDMs. Serum IFN-β levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-β-dependent pathways.

  10. Protein Phosphatase 2A Signaling in Human Prostate Cancer

    DTIC Science & Technology

    2012-06-01

    immunoblot and malachite green based assay, respectively. We observe that LNCaP- shPPP2CA cells have low PP2ACα expression (Figure 1A) and activity...regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 2001;353:417-39. (6) Jennbacken K, Gustavsson H...cancer cells - - - shPPP2CA. Expression and activity of catalytic subunit of PP2A (PP2ACα) was determined by immunoblot and melachite green - based

  11. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2.

    PubMed

    Eum, Sung Yong; Jaraki, Dima; András, Ibolya E; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs.

  12. On the regulation of protein phosphatase 2A and its role in controlling entry into and exit from mitosis.

    PubMed

    Hunt, Tim

    2013-05-01

    The process of mitosis involves a comprehensive reorganization of the cell: chromosomes condense, the nuclear envelope breaks down, the mitotic spindle is assembled, cells round up and release their ties to the substrate and so on and so forth. This reorganization is triggered by the activation of the protein kinase, Cyclin-Dependent Kinase 1 (CDK1). The end of mitosis is marked by the proteolysis of the cyclin subunit of CDK1, which terminates kinase activity. At this point, the phosphate moieties that altered the properties of hundreds of proteins to bring about the cellular reorganization are removed by protein phosphatases. At least one protein phosphatase, PP2A-B55, is completely shut off in mitosis. Depletion of this particular form of PP2A accelerates entry into mitosis, and blocks exit from mitosis. Control of this phosphatase is achieved by an inhibitor protein (α-endosulfine or ARPP-19) that becomes inhibitory when phosphorylated by a protein kinase called Greatwall, which is itself a substrate of CDK1. Failure to inhibit PP2A-B55 causes arrest of the cell cycle in G2 phase. I will discuss the role of this control mechanism in the control of mitosis.

  13. Targeting inhibitor 2 of protein phosphatase 2A as a therapeutic strategy for prostate cancer treatment

    PubMed Central

    Mukhopadhyay, Archana; Tabanor, Kayann; Chaguturu, Rathnam; Aldrich, Jane V

    2013-01-01

    Inhibitor 2 of protein phosphatase 2A (I2PP2A), a biological inhibitor of the cellular serine/threonine protein phosphatase PP2A, is associated with numerous cellular processes that often lead to the formation and progression of cancer. In this study we hypothesized that targeting the inhibition of I2PP2A’s multiple functions in prostate cancer cells might prevent cancer progression. We have investigated the effect of the small chain C6-ceramide, known to be a bioactive tumor suppressor lipid, on I2PP2A function, thereby affecting c-Myc signaling and histone acetylation in cells. Our data indicated that C6-ceramide treatment of prostate cancer cells induces cell death in PC-3, DU145, and LNCaP cells, but not normal prostate epithelial cells. C6-ceramide was able to disrupt the association between PP2A and I2PP2A. C6-ceramide inhibits I2PP2A’s upregulation of c-Myc and downregulation of histone acetylation in prostate cancer cells. Our data indicated that targeting cancer related signaling pathways through I2PP2A using ceramide as an anti-I2PP2A agent could have beneficial effects as a therapeutic approach to prevent prostate cancer. PMID:24025258

  14. Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function

    PubMed Central

    Okamura, Hirohiko; Yoshida, Kaya; Morimoto, Hiroyuki; Teramachi, Jumpei; Ochiai, Kazuhiko; Haneji, Tatsuji; Yamamoto, Akihito

    2017-01-01

    The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes. PMID:28241467

  15. Atypical Protein Phosphatase 2A Gene Families Do Not Expand via Paleopolyploidization1[OPEN

    PubMed Central

    2017-01-01

    Protein phosphatase 2A (PP2A) presents unique opportunities for analyzing molecular mechanisms of functional divergence between gene family members. The canonical PP2A holoenzyme regulates multiple eukaryotic signaling pathways by dephosphorylating target proteins and contains a catalytic (C) subunit, a structural/scaffolding (A) subunit, and a regulatory (B) subunit. Genes encoding PP2A subunits have expanded into multigene families in both flowering plants and mammals, and the extent to which different isoform functions may overlap is not clearly understood. To gain insight into the diversification of PP2A subunits, we used phylogenetic analyses to reconstruct the evolutionary histories of PP2A gene families in Arabidopsis (Arabidopsis thaliana). Genes encoding PP2A subunits in mammals represent ancient lineages that expanded early in vertebrate evolution, while flowering plant PP2A subunit lineages evolved much more recently. Despite this temporal difference, our data indicate that the expansion of PP2A subunit gene families in both flowering plants and animals was driven by whole-genome duplications followed by nonrandom gene loss. Selection analysis suggests that the expansion of one B subunit gene family (B56/PPP2R5) was driven by functional diversification rather than by the maintenance of gene dosage. We also observed reduced expansion rates in three distinct B subunit subclades. One of these subclades plays a highly conserved role in cell division, while the distribution of a second subclade suggests a specialized function in supporting beneficial microbial associations. Thus, while whole-genome duplications have driven the expansion and diversification of most PP2A gene families, members of functionally specialized subclades quickly revert to singleton status after duplication events. PMID:28034953

  16. Phosphorylated nuclear receptor CAR forms a homodimer to repress its constitutive activity for ligand activation.

    PubMed

    Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya; Negishi, Masahiko

    2017-03-06

    Nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and disease development such as diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase PP2A dephosphorylates threonine 38 to activate CAR. Here we have demonstrated that CAR undergoes its homodimer-monomer conversion to regulate this dephosphorylation. By co-expressing two differently-tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes and mouse livers, co-immunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homo-dimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomer, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and in direct activation of CAR.

  17. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    SciTech Connect

    Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

    2011-07-15

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKC{delta}) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKC{delta} negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKC{delta}-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 {mu}M) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKC{delta} specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 {mu}M Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKC{delta} kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKC{delta} inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKC{delta} and PP2A activity.

  18. MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac

    PubMed Central

    Du, Haijuan; Wu, Kuanlin; Didoronkute, Alma; Levy, Marcus V. A.; Todi, Nimish; Shchelokova, Anna; Massiah, Michael A.

    2014-01-01

    MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts. PMID:25207814

  19. Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

    PubMed

    Chao, Ting-Ting; Wang, Cheng-Yi; Chen, Yen-Lin; Lai, Chih-Cheng; Chang, Fang-Yu; Tsai, Yi-Ting; Chao, Chung-Hao H; Shiau, Chung-Wai; Huang, Yuh-Chin T; Yu, Chong-Jen; Chen, Kuen-Feng

    2015-02-10

    Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.

  20. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    SciTech Connect

    Eum, Sung Yong Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  1. Whole genome sequencing identifies a deletion in protein phosphatase 2A that affects its stability and localization in Chlamydomonas reinhardtii.

    PubMed

    Lin, Huawen; Miller, Michelle L; Granas, David M; Dutcher, Susan K

    2013-01-01

    Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.

  2. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    PubMed Central

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  3. Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II.

    PubMed

    Adhikari, Deepak; Diril, M Kasim; Busayavalasa, Kiran; Risal, Sanjiv; Nakagawa, Shoma; Lindkvist, Rebecca; Shen, Yan; Coppola, Vincenzo; Tessarollo, Lino; Kudo, Nobuaki R; Kaldis, Philipp; Liu, Kui

    2014-09-29

    In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.

  4. Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins

    SciTech Connect

    Xing,Y.; Xu, Y.; Chen, Y.; Jeffrey, P.; Chao, Y.; Lin, Z.; Li, Z.; Strack, S.; Stock, J.; Shi, Y.

    2006-01-01

    The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 {angstrom} resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

  5. Roles of phosphotase 2A in nociceptive signal processing.

    PubMed

    Wang, Yun; Lei, Yongzhong; Fang, Li; Mu, Yonggao; Wu, Jing; Zhang, Xuan

    2013-09-08

    Multiple protein kinases affect the responses of dorsal horn neurons through phosphorylation of synaptic receptors and proteins involved in intracellular signal transduction pathways, and the consequences of this modulation may be spinal central sensitization. In contrast, the phosphatases catalyze an opposing reaction of de-phosphorylation, which may also modulate the functions of crucial proteins in signaling nociception. This is an important mechanism in the regulation of intracellular signal transduction pathways in nociceptive neurons. Accumulated evidence has shown that phosphatase 2A (PP2A), a serine/threonine specific phosphatase, is implicated in synaptic plasticity of the central nervous system and central sensitization of nociception. Therefore, targeting protein phosphotase 2A may provide an effective and novel strategy for the treatment of clinical pain. This review will characterize the structure and functional regulation of neuronal PP2A and bring together recent advances on the modulation of PP2A in targeted downstream substrates and relevant multiple nociceptive signaling molecules.

  6. Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    PubMed Central

    Signorelli, Janetti R.; Díaz, Emilce S.; Fara, Karla; Barón, Lina; Morales, Patricio

    2013-01-01

    There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the

  7. Celery extract inhibits mouse CYP2A5 and human CYP2A6 activities via different mechanisms.

    PubMed

    Deng, Xiao; Pu, Qianghong; Wang, Erhao; Yu, Chao

    2016-12-01

    Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7-hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose-dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time- and nicotinamide adenine dinucleotide phosphate (NADPH)-independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH-dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5-methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism-based inhibition for CYP2A6.

  8. Celery extract inhibits mouse CYP2A5 and human CYP2A6 activities via different mechanisms

    PubMed Central

    Deng, Xiao; Pu, Qianghong; Wang, Erhao; Yu, Chao

    2016-01-01

    Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7-hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose-dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time- and nicotinamide adenine dinucleotide phosphate (NADPH)-independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH-dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5-methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism-based inhibition for CYP2A6. PMID:28101244

  9. Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism.

    PubMed

    Dunagan, Mitzi; Chaudhry, Kamaljit; Samak, Geetha; Rao, R K

    2012-12-15

    Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200-600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

  10. The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

    PubMed Central

    Lai, Tai-Yu; Yang, Yu-San; Hong, Wei-Fu; Chiang, Chi-Wu

    2016-01-01

    The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role. PMID:26684356

  11. Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

    PubMed Central

    Bátori, Róbert; Bécsi, Bálint; Nagy, Dénes; Kónya, Zoltán; Hegedűs, Csaba; Bordán, Zsuzsanna; Verin, Alexander; Lontay, Beáta; Erdődi, Ferenc

    2017-01-01

    The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement. PMID:28300193

  12. microRNA-183 plays as oncogenes by increasing cell proliferation, migration and invasion via targeting protein phosphatase 2A in renal cancer cells

    SciTech Connect

    Qiu, Mingning Liu, Lei Chen, Lieqian Tan, Guobin Liang, Ziji Wang, Kangning Liu, Jianjun Chen, Hege

    2014-09-12

    Highlights: • miR-183 was up-regulated in renal cancer tissues. • Inhibition of endogenous miR-183 suppressed renal cancer cell growth and metastasis. • miR-183 increased cell growth and metastasis. • miR-183 regulated renal cancer cell growth and metastasis via directly targeting tumor suppressor protein phosphatase 2A. - Abstract: The aim of this study was to investigate the function of miR-183 in renal cancer cells and the mechanisms miR-183 regulates this process. In this study, level of miR-183 in clinical renal cancer specimens was detected by quantitative real-time PCR. miR-183 was up- and down-regulated in two renal cancer cell lines ACHN and A498, respectively, and cell proliferation, Caspase 3/7 activity, colony formation, in vitro migration and invasion were measured; and then the mechanisms of miR-183 regulating was analyzed. We found that miR-183 was up-regulated in renal cancer tissues; inhibition of endogenous miR-183 suppressed in vitro cell proliferation, colony formation, migration, and invasion and stimulated Caspase 3/7 activity; up-regulated miR-183 increased cell growth and metastasis and suppressed Caspase 3/7 activity. We also found that miR-183 directly targeted tumor suppressor, specifically the 3′UTR of three subunits of protein phosphatase 2A (PP2A-Cα, PP2A-Cβ, and PP2A-B56-γ) transcripts, inhibiting their expression and regulated the downstream regulators p21, p27, MMP2/3/7 and TIMP1/2/3/4. These results revealed the oncogenes role of miR-183 in renal cancer cells via direct targeting protein phosphatase 2A.

  13. Therapeutic relevance of the protein phosphatase 2A in cancer

    PubMed Central

    Bhanumathy, Kalpana Kalyanasundaram; Lee, Joo Sang; Parameswaran, Sreejit; Furber, Levi; Abuhussein, Omar; Paul, James M.; McDonald, Megan; Templeton, Shaina D.; Shukla, Hersh; El Zawily, Amr M.; Boyd, Frederick; Alli, Nezeka; Mousseau, Darrell D.; Geyer, Ron; Bonham, Keith; Anderson, Deborah H.; Yan, Jiong; Yu-Lee, Li-Yuan; Weaver, Beth A.; Uppalapati, Maruti; Ruppin, Eytan; Sablina, Anna; Freywald, Andrew; Vizeacoumar, Franco J.

    2016-01-01

    Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits. PMID:27557495

  14. Dephosphorylation of JC virus agnoprotein by protein phosphatase 2A: Inhibition by small t antigen

    PubMed Central

    Sariyer, Ilker K.; Khalili, Kamel; Safak, Mahmut

    2009-01-01

    Previous studies have demonstrated that the JC virus (JCV) late regulatory protein agnoprotein is phosphorylated by the serine/threonine-specific protein kinase-C (PKC) and mutants of this protein at the PKC phosphorylation sites exhibit defects in the viral replication cycle. We have now investigated whether agnoprotein phosphorylation is regulated by PP2A, a serine/threonine-specific protein phosphatase and whether JCV small t antigen (Sm t-Ag) is involved in this regulation. Protein–protein interaction studies demonstrated that PP2A associates with agnoprotein and dephosphorylates it at PKC-specific sites. Sm t-Ag was also found to interact with PP2A and this interaction inhibited the dephosphorylation of agnoprotein by PP2A. The interaction domains of Sm t-Ag and agnoprotein with PP2A were mapped, as were the interaction domains of Sm t-Ag with agnoprotein. The middle portion of Sm t-Ag (aa 82–124) was found to be critical for the interaction with both agnoprotein and PP2A and the N-terminal region of agnoprotein for interaction with Sm t-Ag. To further understand the role of Sm t-Ag in JCV regulation, a stop codon was introduced at Ser90 immediately after splice donor site of the JCV early gene and the functional consequences of this mutation were investigated. The ability of this mutant virus to replicate was substantially reduced compared to WT. Next, the functional significance of PP2A in JCV replication was examined by siRNA targeting. Downregulation of PP2A caused a significant reduction in the level of JCV replication. Moreover, the impact of Sm t-Ag on agnoprotein phosphorylation was investigated by creating a double mutant of JCV, where Sm t-Ag stop codon mutant was combined with an agnoprotein triple phosphorylation mutant (Ser7, Ser11 and Thr21 to Ala). Results showed that double mutant behaves much like the triple phosphorylation mutant of agnoprotein during viral replication cycle, which suggests that agnoprotein might be an important target of

  15. TORC2 Signaling Is Antagonized by Protein Phosphatase 2A and the Far Complex in Saccharomyces cerevisiae

    PubMed Central

    Pracheil, Tammy; Thornton, Janet; Liu, Zhengchang

    2012-01-01

    The target of rapamycin (TOR) kinase, a central regulator of eukaryotic cell growth, exists in two essential, yet distinct, TOR kinase complexes in the budding yeast Saccharomyces cerevisiae: rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell viability. However, it is unclear whether the essential function of Lst8 is linked to TORC1, TORC2, or both. To that end, we carried out a genetic screen to isolate lst8 deletion suppressor mutants. Here we report that mutations in SAC7 and FAR11 suppress lethality of lst8Δ and TORC2-deficient (tor2-21) mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2. More importantly, characterization of lst8Δ bypass mutants reveals a role for protein phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that Far11, a member of the Far3-7-8-9-10-11 complex involved in pheromone-induced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and deletions of components of the Far3-7-8-9-10-11 complex and PP2A rescue growth defects in lst8Δ and tor2-21 mutants. In addition, loss of the regulatory B′ subunit of PP2A Rts1 or Far11 restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11 orthologs FAM40A/B exist in a complex with PP2A known as STRIPAK, suggesting a conserved functional association of PP2A and Far11. Antagonism of TORC2 signaling by PP2A-Far11 represents a novel regulatory mechanism for controlling spatial cell growth of yeast. PMID:22298706

  16. Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

    PubMed

    Majd, Shohreh; Power, John H T; Koblar, Simon A; Grantham, Hugh J M

    2016-08-01

    Abnormal tau phosphorylation (p-tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p-tau is controlled by Glycogen Synthase Kinase (GSK)-3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p-tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser(396) and Ser(262) after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK-3β sensitive but AMPK insensitive residues, Ser(202) /Thr(205) (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK-3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p-AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p-tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK-3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation.

  17. Purinergic receptor-mediated rapid depletion of nuclear phosphorylated Akt depends on pleckstrin homology domain leucine-rich repeat phosphatase, calcineurin, protein phosphatase 2A, and PTEN phosphatases.

    PubMed

    Mistafa, Oras; Ghalali, Aram; Kadekar, Sandeep; Högberg, Johan; Stenius, Ulla

    2010-09-03

    Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21(cip1) complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.

  18. Modulation of plant HMG-CoA reductase by protein phosphatase 2A

    PubMed Central

    Antolín-Llovera, Meritxell; Leivar, Pablo; Arró, Montserrat; Ferrer, Albert; Boronat, Albert

    2011-01-01

    The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B″ regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR. PMID:21701259

  19. DMH1 increases glucose metabolism through activating Akt in L6 rat skeletal muscle cells.

    PubMed

    Xie, Xin; Xu, Xiao-Ming; Li, Na; Zhang, Yong-Hui; Zhao, Yu; Ma, Chun-Yan; Dong, De-Li

    2014-01-01

    DMH1(4-[6-(4-Isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a compound C analogue with the structural modifications at the 3- and 6-positions in pyrazolo[1,5-a]pyrimidine backbone. Compound C was reported to inhibit both AMPK and Akt. Our preliminary work found that DMH1 activated Akt. Since Akt was involved in glucose metabolism, we aimed to identify the effects of DMH1 on glucose metabolism in L6 rat muscle cells and the potential mechanism. Results showed that DMH1 increased lactic acid release and glucose consumption in L6 rat muscle cells in a dose-dependent manner. DMH1 activated Akt in L6 cells. Akt inhibitor inhibited DMH1-induced Akt activation and DMH1-induced increases of glucose uptake and consumption. DMH1 had no cytotoxicity in L6 cells, but inhibited mitochondrial function and reduced ATP production. DMH1 showed no effect on AMPK, but in the presence of Akt inhibitor, DMH1 significantly activated AMPK. Compound C inhibited DMH1-induced Akt activation in L6 cells. Compound C inhibited DMH1-induced increase of glucose uptake, consumption and lactic acid release in L6 cells. DMH1 inhibited PP2A activity, and PP2A activator forskolin reversed DMH1-induced Akt activation. We concluded that DMH1 increased glucose metabolism through activating Akt and DMH1 activated Akt through inhibiting PP2A activity in L6 rat muscle cells. In view of the analogue structure of DMH1 and compound C and the contrasting effects of DMH1 and compound C on Akt, the present study provides a novel leading chemical structure targeting Akt with potential use for regulating glucose metabolism.

  20. Resveratrol Attenuates Formaldehyde Induced Hyperphosphorylation of Tau Protein and Cytotoxicity in N2a Cells

    PubMed Central

    He, Xiaping; Li, Zhenhui; Rizak, Joshua D.; Wu, Shihao; Wang, Zhengbo; He, Rongqiao; Su, Min; Qin, Dongdong; Wang, Jingkun; Hu, Xintian

    2017-01-01

    Recent studies have demonstrated that formaldehyde (FA)—induced neurotoxicity is important in the pathogenesis of Alzheimer's disease (AD). Elevated levels of FA have been associated with memory impairments and the main hallmarks of AD pathology, including β-amyloid plaques, tau protein hyperphosphorylation, and neuronal loss. Resveratrol (Res), as a polyphenol anti-oxidant, has been considered to have therapeutic potential for the treatment of AD. However, it has not been elucidated whether Res can exert its neuroprotective effects against FA-induced neuronal damages related to AD pathology. To answer this question, the effects of Res were investigated on Neuro-2a (N2a) cells prior to and after FA exposure. The experiments found that pre-treatment with Res significantly decreased FA-induced cytotoxicity, reduced cell apoptosis rates, and inhibited the hyperphosphorylation of tau protein at Thr181 in a dose-dependent manner. Further tests revealed that this effect was associated with the suppression of glycogen synthase kinase (GSK-3β) and calmodulin-dependent protein kinase II (CaMKII) activities, both of which are important kinases for tau protein hyperphosphorylation. In addition, Res was found to increase the activity of phosphoseryl/phosphothreonyl protein phosphatase-2A (PP2A). In summary, these findings provide evidence that Res protects N2a cells from FA-induced damages and suggests that inhibition of GSK-3β and CaMKII and the activation of PP2A by Res protect against the hyperphosphorylation and/or mediates the dephosphorylation of tau protein, respectively. These possible mechanisms underlying the neuroprotective effects of Res against FA-induced damages provide another perspective on AD treatment via inhibition of tau protein hyperhosphorylation. PMID:28197064

  1. Suppression of Scant Identifies Endos as a Substrate of Greatwall Kinase and a Negative Regulator of Protein Phosphatase 2A in Mitosis

    PubMed Central

    Rangone, Hélène; Wegel, Eva; Gatt, Melanie K.; Yeung, Eirene; Flowers, Alexander; Debski, Janusz; Dadlez, Michal; Janssens, Veerle; Carpenter, Adelaide T. C.; Glover, David M.

    2011-01-01

    Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop. PMID:21852956

  2. Altered protein phosphatase 2A methylation and Tau phosphorylation in the young and aged brain of methylenetetrahydrofolate reductase (MTHFR) deficient mice

    PubMed Central

    Sontag, Jean-Marie; Wasek, Brandi; Taleski, Goce; Smith, Josephine; Arning, Erland; Sontag, Estelle; Bottiglieri, Teodoro

    2014-01-01

    Common functional polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, a key enzyme in folate and homocysteine metabolism, influence risk for a variety of complex disorders, including developmental, vascular, and neurological diseases. MTHFR deficiency is associated with elevation of homocysteine levels and alterations in the methylation cycle. Here, using young and aged Mthfr knockout mouse models, we show that mild MTHFR deficiency can lead to brain-region specific impairment of the methylation of Ser/Thr protein phosphatase 2A (PP2A). Relative to wild-type controls, decreased expression levels of PP2A and leucine carboxyl methyltransferase (LCMT1) were primarily observed in the hippocampus and cerebellum, and to a lesser extent in the cortex of young null Mthfr−/− and aged heterozygous Mthfr+/− mice. A marked down regulation of LCMT1 correlated with the loss of PP2A/Bα holoenzymes. Dietary folate deficiency significantly decreased LCMT1, methylated PP2A and PP2A/Bα levels in all brain regions examined from aged Mthfr+/+ mice, and further exacerbated the regional effects of MTHFR deficiency in aged Mthfr+/− mice. In turn, the down regulation of PP2A/Bα was associated with enhanced phosphorylation of Tau, a neuropathological hallmark of Alzheimer’s disease (AD). Our findings identify hypomethylation of PP2A enzymes, which are major CNS phosphatases, as a novel mechanism by which MTHFR deficiency and Mthfr gene-diet interactions could lead to disruption of neuronal homeostasis, and increase the risk for a variety of neuropsychiatric disorders, including age-related diseases like sporadic AD. PMID:25202269

  3. The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator-role of Ceramide in MU anti-tumor activity

    PubMed Central

    Qin, Jingdong; Kilkus, John; Dawson, Glyn

    2015-01-01

    Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomelinase2 (NSMase2), generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow. PMID:26548718

  4. The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator-role of Ceramide in MU anti-tumor activity.

    PubMed

    Qin, Jingdong; Kilkus, John; Dawson, Glyn

    2016-02-01

    Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.

  5. Cip2a promotes cell cycle progression in triple-negative breast cancer cells by regulating the expression and nuclear export of p27Kip1.

    PubMed

    Liu, H; Qiu, H; Song, Y; Liu, Y; Wang, H; Lu, M; Deng, M; Gu, Y; Yin, J; Luo, K; Zhang, Z; Jia, X; Zheng, G; He, Z

    2016-10-03

    Triple-negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets; as a consequence, TNBC exhibits poor clinical outcome. In this study, we showed that cancerous inhibitor of protein phosphatase 2A (Cip2a) represents a promising target in TNBC because Cip2a was highly expressed in TNBC cells and tumor tissues, and its expression showed an inverse correlation with overall survival in patients with TNBC. We found that inhibition of Cip2a in TNBC cells induced cell cycle arrest at the G2/M phase, inhibited cell proliferation and delayed tumor growth in the xenograft model. Moreover, Cip2a markedly decreased the expression and nuclear localization of p27Kip1 and this is critical for the ability of Cip2a to promote TNBC progression. Mechanistically, our studies showed that Cip2a promoted p27Kip1 phosphoration at Ser10 via inhibiting Akt-associated PP2A activity, which seems to relocalize p27Kip1 to the cytoplasm in TNBC cells. On the other hand, Cip2a also recruited c-myc to mediate the transcriptional inhibition of p27Kip1. Notably, we observed negative correlation between Cip2a and p27Kip1 expression in TNBC specimens. In addition, our data showed that Cip2a depletion could sensitize TNBC to PARP inhibition. Collectively, these data suggested that Cip2a effectively promotes TNBC cell cycle progression and tumor growth via regulation of PP2A/c-myc/p27Kip1 signaling, which could serve as a potential therapeutic target for TNBC patients.Oncogene advance online publication, 3 October 2016; doi:10.1038/onc.2016.355.

  6. Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

    PubMed

    Takahashi, Toshimasa; Shibasaki, Tadao; Takahashi, Harumi; Sugawara, Kenji; Ono, Aika; Inoue, Naoko; Furuya, Toshio; Seino, Susumu

    2013-10-22

    Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

  7. Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein.

    PubMed Central

    Takai, A; Sasaki, K; Nagai, H; Mieskes, G; Isobe, M; Isono, K; Yasumoto, T

    1995-01-01

    Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper, we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding, ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-3H]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-3H]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (Ki) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the Ki value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tautomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported Ki values. PMID:7702557

  8. Secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis.

    PubMed

    Park, Min Chul; Kang, Taehee; Jin, Da; Han, Jung Min; Kim, Sang Bum; Park, Yun Jung; Cho, Kiwon; Park, Young Woo; Guo, Min; He, Weiwei; Yang, Xiang-Lei; Schimmel, Paul; Kim, Sunghoon

    2012-03-13

    Although adaptive systems of immunity against tumor initiation and destruction are well investigated, less understood is the role, if any, of endogenous factors that have conventional functions. Here we show that glycyl-tRNA synthetase (GRS), an essential component of the translation apparatus, circulates in serum and can be secreted from macrophages in response to Fas ligand that is released from tumor cells. Through cadherin (CDH)6 (K-cadherin), GRS bound to different ERK-activated tumor cells, and released phosphatase 2A (PP2A) from CDH6. The activated PP2A then suppressed ERK signaling through dephosphorylation of ERK and induced apoptosis. These activities were inhibited by blocking GRS with a soluble fragment of CDH6. With in vivo administration of GRS, growth of tumors with a high level of CDH6 and ERK activation were strongly suppressed. Our results implicate a conventional cytoplasmic enzyme in translation as an intrinsic component of the defense against ERK-activated tumor formation.

  9. Role of the immunoreceptor tyrosine-based activation motif of latent membrane protein 2A (LMP2A) in Epstein-Barr virus LMP2A-induced cell transformation.

    PubMed

    Fukuda, Makoto; Kawaguchi, Yasushi

    2014-05-01

    Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is widely expressed in EBV-associated malignancies. We demonstrate that LMP2A has a transformation ability. This study shows that LMP2A-induced transformation in several human nonhematopoietic cell lines was blocked in those cells expressing an immunoreceptor tyrosine-based activation motif (ITAM) LMP2A mutant. The Syk inhibitor or Syk-specific small interfering RNA (siRNA) inhibited LMP2A-induced transformation. These results indicate that the interaction of the LMP2A ITAM with Syk is a key step for LMP2A-mediated transformation.

  10. Hypotonicity activates a voltage-dependent membrane conductance in N2a neuroblastoma cells.

    PubMed

    Taruno, Akiyuki; Marunaka, Yoshinori

    2017-03-04

    To maintain cellular and bodily homeostasis, cells respond to extracellular stimuli including osmotic stress by activating various ion channels, which have been implicated in many physiological and pathophysiological conditions. However, cellular osmosensory mechanisms remain elusive. Here, we report a novel voltage-dependent current in N2a cells activated by exposure to hypotonic stress. After a hypotonic challenge, N2a cells sequentially develop two distinct currents. The volume-regulated anion channel (VRAC) current emerges first and, after a delay, activation of a previously uncharacterized strongly outwardly rectifying current follows. The latter, delayed current (Id) is insensitive to NPPB, a nonspecific blocker of Cl(-) channels, and intracellular Mg(2+), which inhibits VRAC and swelling-activated TRPM3 and TRPM7 channels. Replacement of extracellular Na(+) with NMDG(+) reduces inward tail currents, suggesting that Id is mediated by cations. Finally, Id shows voltage-dependent activation with slow activation kinetics and half-maximal activation at +76 mV. These pharmacological and biophysical characteristics of Id are distinct from those of known osmotic cell swelling-activated ion channels. In conclusion, our data identify and characterize a novel osmotically-activated, voltage-dependent ion channel in N2a cells.

  11. Activation of NR2A receptors induces ischemic tolerance through CREB signaling.

    PubMed

    Terasaki, Yasukazu; Sasaki, Tsutomu; Yagita, Yoshiki; Okazaki, Shuhei; Sugiyama, Yukio; Oyama, Naoki; Omura-Matsuoka, Emi; Sakoda, Saburo; Kitagawa, Kazuo

    2010-08-01

    Previous exposure to a nonlethal ischemic insult protects the brain against subsequent harmful ischemia. N-methyl-D-aspartate (NMDA) receptors are a highly studied target of neuroprotection after ischemia. Recently, NMDA receptor subtypes were implicated in neuronal survival and death. We focused on the contribution of NR2A and cyclic-AMP response element (CRE)-binding protein (CREB) signaling to ischemic tolerance using primary cortical neurons. Ischemia in vitro was modeled by oxygen-glucose deprivation (OGD). Ischemic tolerance was induced by applying 45-mins OGD 24 h before 180-mins OGD. Sublethal OGD also induced cross-tolerance against lethal glutamate and hydrogen peroxide. After sublethal OGD, expression of phosphorylated CREB and CRE transcriptional activity were significantly increased. When CRE activity was inhibited by CREB-S133A, a mutant CREB, ischemic tolerance was abolished. Inhibiting NR2A using NVP-AAM077 attenuated preconditioning-induced neuroprotection and correlated with decreased CRE activity levels. Activating NR2A using bicuculline and 4-aminopiridine induced resistance to lethal ischemia accompanied by elevated CRE activity levels, and this effect was abolished by NVP-AAM077. Elevated brain-derived neurotrophic factor (BDNF) transcriptional activities were observed after sublethal OGD and administration of bicuculline and 4-aminopiridine. NR2A-containing NMDA receptors and CREB signaling have important functions in the induction of ischemic tolerance. This may provide potential novel therapeutic strategies to treat ischemic stroke.

  12. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  13. Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure

    PubMed Central

    Kho, Changwon; Lee, Ahyoung; Jeong, Dongtak; Oh, Jae Gyun; Gorski, Przemek A.; Fish, Kenneth; Sanchez, Roberto; DeVita, Robert J.; Christensen, Geir; Dahl, Russell; Hajjar, Roger J.

    2015-01-01

    Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure. PMID:26068603

  14. Epstein-Barr virus latent membrane protein 2A contributes to anoikis resistance through ERK activation.

    PubMed

    Iwakiri, Dai; Minamitani, Takeharu; Samanta, Mrinal

    2013-07-01

    Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.

  15. TOSPAC calculations in support of the COVE 2A benchmarking activity; Yucca Mountain Site Characterization Project

    SciTech Connect

    Gauthier, J.H.; Zieman, N.B.; Miller, W.B.

    1991-10-01

    The purpose of the the Code Verification (COVE) 2A benchmarking activity is to assess the numerical accuracy of several computer programs for the Yucca Mountain Site Characterization Project of the Department of Energy. This paper presents a brief description of the computer program TOSPAC and a discussion of the calculational effort and results generated by TOSPAC for the COVE 2A problem set. The calculations were performed twice. The initial calculations provided preliminary results for comparison with the results from other COVE 2A participants. TOSPAC was modified in response to the comparison and the final calculations included a correction and several enhancements to improve efficiency. 8 refs.

  16. CIP2A Promotes Proliferation of Spermatogonial Progenitor Cells and Spermatogenesis in Mice

    PubMed Central

    Mäkelä, Juho-Antti; Hobbs, Robin M.; Mannermaa, Leni; Kallajoki, Markku; Chan, Edward K.; Pandolfi, Pier Paolo; Toppari, Jorma; Westermarck, Jukka

    2012-01-01

    Protein phosphatase 2A (PP2A) is a critical regulator of protein serine/threonine phosphorylation. However, the physiological and developmental roles of different PP2A complexes are very poorly understood. Here, we show that a newly characterized PP2A inhibitory protein CIP2A is co-expressed with ki-67 and with self-renewal protein PLZF in the spermatogonial progenitor cell (SPC) population in the testis. CIP2A and PLZF expression was shown also to correlate Ki-67 expression in human testicular spermatogonia. Functionally, CIP2A mutant mouse testes exhibited smaller number of PLZF-positive SPCs and reduced sperm counts. Moreover, seminiferous tubuli cells isolated from CIP2A mutant mice showed reduced expression of Plzf and other renewal genes Oct-4 and Nanog at mRNA level. However, PLZF-deficient testes did not show altered CIP2A expression. Importantly, spermatogonia-specific restoration of CIP2A expression rescued PLZF expression and sperm production defects observed in CIP2A mutant mice. Taken together, these results reveal first physiological function for an emerging human oncoprotein CIP2A, and provide insights into maintenance of PLZF-positive progenitors. Moreover, demonstration that CIP2A expression can be systematically inhibited without severe consequences to normal mouse development and viability may have clinical relevance regarding targeting of oncogenic CIP2A for future cancer therapies. PMID:22461891

  17. Protein Phosphatase 2A Signaling in Human Prostate Cancer

    DTIC Science & Technology

    2014-08-01

    phosphatidylinositol 3’-kinase and Akt/protein kinase B. Cancer Res 1999;59:1449-53. (14) Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad...growth and sig- nalling. Biochem J 2001;353:417–39. 15. Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad via PP2A-dependent suppression of

  18. Molecular level activation insights from a NR2A/NR2B agonist.

    PubMed

    Ieong Tou, Weng; Chang, Su-Sen; Wu, Dongchuan; Lai, Ted Weita; Wang, Yu Tian; Hsu, Chung Y; Chen, Calvin Yu-Chian

    2014-01-01

    N-methyl D-aspartate receptors (NMDARs), a subclass of glutamate receptors have broad actions in neural transmission for major brain functions. Overactivation of NMDARs leading to "excitotoxicity" is the underlying mechanism of neuronal death in a number of neurological diseases, especially stroke. Much research effort has been directed toward developing pharmacological agents to modulate NMDAR actions for treating neurological diseases, in particular stroke. Here, we report that Alliin, a sulfoxide in fresh garlic, exhibits affinity toward NR2A as well as NR2B receptors based on virtual screening. Biological activities of Alliin on these two receptors were confirmed in electrophysiological studies. Ligand-binding site closure, a structural change precluding ion channel opening, was observed with Alliin during 100 ns molecular dynamics simulation. Alliin interactions with NR2A and NR2B suggest that residues E/A413, H485, T690, and Y730 may play important roles in the conformation shift. Activation of NR2A and NR2B by Alliin can be differentiated from that caused by glutamate, the endogenous neurotransmitter. These characteristic molecular features in NR2A and NR2B activation provide insight into structural requirements for future development of novel drugs with selective interaction with NR2A and NR2B for treating neurological diseases, particularly stroke.

  19. Is the O2(a1Δg) Venus nightglow emission controlled by solar activity?

    NASA Astrophysics Data System (ADS)

    Soret, Lauriane; Gérard, Jean-Claude

    2015-12-01

    Several past studies showed that the O2(a1Δg) Venus nightglow emission at 1.27 μm is highly variable on a timescale of hours. We examine whether the intensity of this emission shows a more global trend linked to solar activity.

  20. CRL4-DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-08-18

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility.

  1. Oxidative stress in cardiomyocytes contributes to decreased SERCA2a activity in rats with metabolic syndrome.

    PubMed

    Balderas-Villalobos, Jaime; Molina-Muñoz, Tzindilu; Mailloux-Salinas, Patrick; Bravo, Guadalupe; Carvajal, Karla; Gómez-Viquez, Norma L

    2013-11-01

    Ca(+) mishandling due to impaired activity of cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) has been associated with the development of left ventricular diastolic dysfunction in insulin-resistant cardiomyopathy. However, the molecular causes underlying SERCA2a alterations induced by insulin resistance and related metabolic disorders, such as metabolic syndrome (MetS), are not completely understood. In this study, we used a sucrose-fed rat model of MetS to test the hypothesis that decreased SERCA2a activity is mediated by elevated oxidative stress produced in the MetS heart. Production of ROS and cytosolic Ca(2+) concentration were recorded in left ventricular myocytes using confocal imaging. The level of SERCA2a oxidation was determined in left ventricular homogenates by biotinylated iodoacetamide labeling. Compared with control rats, sucrose-fed rats exhibited several characteristics of MetS, including central obesity, insulin resistance, hyperinsulinemia, and hypertriglyceridemia. Moreover, relative to myocytes from control rats, myocytes from MetS rats exhibited elevated basal production of ROS accompanied by slowed cytosolic Ca(2+) removal, reflected by prolonged Ca(2+) transients. The slowed cytosolic Ca(2+) removal was associated with a significant decrease in SERCA2a-mediated Ca(2+) reuptake and increased SERCA2a oxidation. Importantly, myocytes from MetS rats treated with the antioxidant N-acetylcysteine showed normal ROS levels and SERCA2a-mediated Ca(2+) reuptake as well as accelerated cytosolic Ca(2+) removal. These data suggest that elevated oxidative stress may induce oxidative modifications on SERCA2a leading to abnormal function of this protein in the MetS heart.

  2. The Human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation.

    PubMed

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-09-01

    Bile acids (BA) are essential modulators of lipid, glucose, and cholesterol homeostasis, but they exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically relevant BA detoxification process. The present study characterized the BA-conjugating activity of the little-studied human UGTs of subfamily 2A: UGT2A1, 2A2, and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of six major bile acids: chenodeoxycholic acid (CDCA), cholic acid (CA), lithocholic acid (LCA), deoxycholic acid (DCA), hyocholic acid (HCA) and hyodeoxycholic acid (HDCA). UGT2A3 exhibited detectable but very low activity with all the tested BA substrates. UGT2A1 was highly efficient in forming LCA-3 and LCA-24G, CDCA-24, DCA-24, HCA-24, and HDCA-24G, whereas UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation and also was able to generate HDCA-6G, HDCA-24G, LCA-24G, and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 µM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 µM range. We demonstrate the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiologic importance of these reactions to BA disposition remains, however, to be clarified in vivo.

  3. The human UDP-glucuronosyltransferase UGT2A1 and UGT2A2 enzymes are highly active in bile acid glucuronidation

    PubMed Central

    Perreault, Martin; Gauthier-Landry, Louis; Trottier, Jocelyn; Verreault, Mélanie; Caron, Patrick; Finel, Moshe; Barbier, Olivier

    2013-01-01

    Bile acids (BA) are essential modulators of lipid, glucose and cholesterol homeostasis, but exert cytotoxic effects in the cholestatic liver. Glucuronidation, catalyzed by the UDP-glucuronosyltransferase (UGT) enzymes is a pharmacologically-relevant BA detoxification process. The present study aimed at characterizing the BA-conjugating activity of the little-studied human UGTs of subfamily 2A, UGT2A1, 2A2 and 2A3. Recombinant UGT2As, expressed in baculovirus-infected insect cells, were assayed for the glucuronidation of 6 major bile acids, chenodeoxycholic (CDCA), cholic (CA), lithocholic (LCA), deoxycholic (DCA), hyocholic (HCA) and hyodeoxycholic (HDCA) acids. UGT2A3 exhibited detectable, but very low, activity with all the tested BAs substrates. UGT2A1 was highly efficient in forming LCA-3 and -24G, CDCA-24, DCA-24, HCA-24 and HDCA-24G, while UGT2A2 was the most active enzyme for CA-24G and CDCA-24G formation, and was also able to generate HDCA-6G, HDCA-24G, LCA-24G and HCA-24G. The Km values of UGT2A1 varied between 102.2 ± 14.3 μM and 2.4 ± 1.2 mM. With the exception of CA-24G, a low affinity substrate for UGT2A2, all the Km values for UGT2A2 were in the 100 to 400 μM range. In conclusion, the present study demonstrates the high reactivity of the human UGT2A1 and UGT2A2 for bile acid glucuronidation. The physiological importance of these reactions to BA disposition remains, however, to be clarified in vivo. PMID:23756265

  4. Antioxidative and antiproliferative activities of novel pyrido[1,2-a]benzimidazoles.

    PubMed

    Tireli, Martina; Starčević, Kristina; Martinović, Tamara; Pavelić, Sandra Kraljević; Karminski-Zamola, Grace; Hranjec, Marijana

    2017-02-01

    A series of pyrido[1,2-a]benzimidazoles has been designed, and novel examples are synthesized and evaluated for their potential antiproliferative activity against four human tumour cell lines-cervical (HeLa), colorectal (SW620), breast (MCF-7) and hepatocellular carcinoma (HepG2). In addition, their antioxidative potency has been evaluated by in vitro spectrophotometric assays. Preliminary structure-activity relationships among the synthesized compounds are discussed. Evaluation of their antioxidative capacity has shown that two compounds (25 and 26) possess promising reducing characteristics and free radical scavenging activity. Selective antiproliferative effect in the single-digit micromolar range was observed for compound 25 on MCF-7 [Formula: see text] and HeLa [Formula: see text] cell lines, comparable to the standards 5-fluorouracil and cisplatin. The combination of the radical scavenging activity and antiproliferative activity of compound 25 positions this compound as a potential lead candidate for further optimization.

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

    PubMed Central

    Sontag, Jean-Marie; Nunbhakdi-Craig, Viyada; Mitterhuber, Martina; Ogris, Egon; Sontag, Estelle

    2010-01-01

    Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase LCMT1, and demethylation by the methylesterase PME-1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. Here, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of PME-1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes. PMID:21044074

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

    PubMed

    Sontag, Jean-Marie; Nunbhakdi-Craig, Viyada; Mitterhuber, Martina; Ogris, Egon; Sontag, Estelle

    2010-12-01

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

  7. Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network.

    PubMed

    de Crozé, Noémie; Maczkowiak, Frédérique; Monsoro-Burq, Anne H

    2011-01-04

    The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates.

  8. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A.

    PubMed

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.

  9. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  10. Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation.

    PubMed

    Gao, Yanzhe; Yao, Jianhong; Poudel, Sumeet; Romer, Eric; Abu-Niaaj, Lubna; Leffak, Michael

    2014-12-26

    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.

  11. Modulation of Receptor Phosphorylation Contributes to Activation of Peroxisome Proliferator Activated Receptor α by Dehydroepiandrosterone and Other Peroxisome Proliferators

    PubMed Central

    Tamasi, Viola; Miller, Kristy K. Michael; Ripp, Sharon L.; Vila, Ermin; Geoghagen, Thomas E.; Prough, Russell A.

    2008-01-01

    Dehydroepiandrosterone (DHEA), a C19 human adrenal steroid, activates peroxisome proliferator-activated receptor α (PPARα) in vivo but does not ligand-activate PPARα in transient transfection experiments. We demonstrate that DHEA regulates PPARα action by altering both the levels and phosphorylation status of the receptor. Human hepatoma cells (HepG2) were transiently transfected with the expression plasmid encoding PPARα and a plasmid containing two copies of fatty acyl coenzyme oxidase (FACO) peroxisome-proliferator activated receptor responsive element consensus oligonucleotide in a luciferase reporter gene. Nafenopin treatment increased reporter gene activity in this system, whereas DHEA treatment did not. Okadaic acid significantly decreased nafenopin-induced reporter activity in a concentration-dependent manner. Okadaic acid treatment of primary rat hepatocytes decreased both DHEA- and nafenopin-induced FACO activity in primary rat hepatocytes. DHEA induced both PPARα mRNA and protein levels, as well as PP2A message in primary rat hepatocytes. Western blot analysis showed that the serines at positions 12 and 21 were rapidly dephosphorylated upon treatment with DHEA and nafenopin. Results using specific protein phosphatase inhibitors suggested that protein phosphatase 2A (PP2A) is responsible for DHEA action, and protein phosphatase 1 might be involved in nafenopin induction. Mutation of serines at position 6, 12, and 21 to an uncharged alanine residue significantly increased transcriptional activity, whereas mutation to negative charged aspartate residues (mimicking receptor phosphorylation) decreased transcriptional activity. DHEA action involves induction of PPARα mRNA and protein levels as well as increased PPARα transcriptional activity through decreasing receptor phosphorylation at serines in the AF1 region. PMID:18079279

  12. More potent inhibition of human CYP2A6 than mouse CYP2A5 enzyme activities by derivatives of phenylethylamine and benzaldehyde.

    PubMed

    Rahnasto, M; Raunio, H; Poso, A; Juvonen, R O

    2003-05-01

    1. A rapid 96-well plate assay method was developed and validated to measure liver microsomal coumarin 7-hydroxylation in vitro. 2. The method was used to test inhibition of human and mouse CYP2A enzymes by three phenylethylamine derivatives 2-(p-tolyl)-ethylamine, amphetamine, 2-phenylethylamine and benzaldehyde, and two of its derivatives, 4-methylbenzaldehyde and 4-methoxybenzaldehyde. 3. The benzaldehyde derivatives were more potent inhibitors of CYP2A5 than the phenylethylamines. The K(ic) value of 4-methylbenzaldehyde was 3.4 micro M and for 4-methoxybenzaldehyde it was 0.86 micro M for CYP2A5. 4. Amphetamine is a weak inhibitor of CYP2A6, whereas benzaldehyde is a suicide inhibitor with K(inact) = 0.16 min(-1) and K(I) = 18 micro M. The K(ic) values of 2-phenylethylamine, 2-(p-tolyl)-ethylamine, 4-methylbenzaldehyde and 4-methoxybenzaldehyde were 1.13, 0.23, 0.36 and 0.73 micro M for CYP2A6, respectively. 5. Novel potent inhibitors were found for CYP2A6 and, except for 4-methoxybenzaldehyde, all the compounds inhibited CYP2A5 and CYP2A6 enzymes differentially. These data add to the refinement of CYP2A enzyme active sites and provide chemical leads for developing novel chemical inhibitors of the CYP2A6 enzyme.

  13. Triggering neurotrophic factor actions through adenosine A2A receptor activation: implications for neuroprotection

    PubMed Central

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    G protein coupled receptors and tropomyosin-related kinase (Trk) receptors have distinct structure and transducing mechanisms; therefore, cross-talk among them was unexpected. Evidence has, however, accumulated showing that tonic adenosine A2A receptor activity is a required step to allow synaptic actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. An enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor in aged animals, which might prove particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as glial derived neurotrophic factor at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurones will be reviewed and discussed in the light of therapeutic strategies for neurodegenerative diseases. PMID:19508402

  14. Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

    PubMed Central

    Bhattacharyya, Samarjit; Raote, Ishier; Bhattacharya, Aditi; Miledi, Ricardo; Panicker, Mitradas M.

    2006-01-01

    Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or “primed” by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations ≈10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders. PMID:17005723

  15. CoRoT-2a Magnetic Activity: Hints for Possible Star-Planet Interaction

    NASA Astrophysics Data System (ADS)

    Pagano, Isabella; Lanza, Antonino F.; Leto, Giuseppe; Messina, Sergio; Barge, Pierre; Baglin, Annie

    2009-09-01

    CoRoT-2a is a young (≈0.5 Gyr) G7V star accompanied by a transiting hot-Jupiter, discovered by the CoRoT satellite (Alonso et al. Astron Astrophys 482:L21, 2008; Bouchy et al. Astron Astrophys 482:L25, 2008). An analysis of its photospheric activity, based on spot modelling techniques previously developed by our group for the analysis of the Sun as a star, shows that the active regions on CoRoT-2a arised within two active longitudes separated by about 180° and rotating with periods of 4.5221 and 4.5543 days, respectively, at epoch of CoRoT observations (112 continous days centered at ≈2007.6). We show that the total spotted area oscillates with a period of about 28.9 days, a value close to 10 times the synodic period of the planet with respect to the active longitude pattern rotating in 4.5221 days. Moreover, the variance of the stellar flux is modulated in phase with the planet orbital period. This suggests a possible star-planet magnetic interaction, a phenomenon already seen in other extrasolar planetary systems hosting hot-Jupiters.

  16. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    NASA Astrophysics Data System (ADS)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  17. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  18. Inefficient Chronic Activation of Parietal Cells in Ae2a,b−/− Mice

    PubMed Central

    Recalde, Sergio; Muruzábal, Francisco; Looije, Norbert; Kunne, Cindy; Burrell, María A.; Sáez, Elena; Martínez-Ansó, Eduardo; Salas, January T.; Mardones, Pablo; Prieto, Jesús; Medina, Juan F.; Elferink, Ronald P.J. Oude

    2006-01-01

    In parietal cells, basolateral Ae2 Cl−/HCO3− exchanger (Slc4a2) appears to compensate for luminal H+ pumping while providing Cl− for apical secretion. In mouse and rat, mRNA variants Ae2a, Ae2b1, Ae2b2, and Ae2c2 are all found in most tissues (although the latter at very low levels), whereas Ae2c1 is restricted to the stomach. We studied the acid secretory function of gastric mucosa in mice with targeted disruption of Ae2a, Ae2b1, and Ae2b2 (but not Ae2c) isoforms. In the oxyntic mucosa of Ae2a,b−/− mice, total Ae2 protein was nearly undetectable, indicating low gastric expression of the Ae2c isoforms. In Ae2a,b−/− mice basal acid secretion was normal, whereas carbachol/histamine-stimulated acid secretion was impaired by 70%. These animals showed increased serum gastrin levels and hyperplasia of G cells. Immunohistochemistry and electron microscopy revealed baseline activation of parietal cells with fusion of intracellular H+/K+-ATPase-containing vesicles with the apical membrane and degenerative changes (but not substantial apoptosis) in a subpopulation of these cells. Increased expression of proliferating cell nuclear antigen in the oxyntic glands suggested enhanced Ae2a,b−/− parietal cell turnover. These data reveal a critical role of Ae2a-Ae2b1-Ae2b2 isoforms in stimulated gastric acid secretion whereas residual Ae2c isoforms could account to a limited extent for basal acid secretion. PMID:16816370

  19. Flavonol-induced changes in PIN2 polarity and auxin transport in the Arabidopsis thaliana rol1-2 mutant require phosphatase activity

    PubMed Central

    Kuhn, Benjamin M.; Nodzyński, Tomasz; Errafi, Sanae; Bucher, Rahel; Gupta, Shibu; Aryal, Bibek; Dobrev, Petre; Bigler, Laurent; Geisler, Markus; Zažímalová, Eva; Friml, Jiří; Ringli, Christoph

    2017-01-01

    The phytohormone auxin is a major determinant and regulatory component important for plant development. Auxin transport between cells is mediated by a complex system of transporters such as AUX1/LAX, PIN, and ABCB proteins, and their localization and activity is thought to be influenced by phosphatases and kinases. Flavonols have been shown to alter auxin transport activity and changes in flavonol accumulation in the Arabidopsis thaliana rol1-2 mutant cause defects in auxin transport and seedling development. A new mutation in ROOTS CURL IN NPA 1 (RCN1), encoding a regulatory subunit of the phosphatase PP2A, was found to suppress the growth defects of rol1-2 without changing the flavonol content. rol1-2 rcn1-3 double mutants show wild type-like auxin transport activity while levels of free auxin are not affected by rcn1-3. In the rol1-2 mutant, PIN2 shows a flavonol-induced basal-to-apical shift in polar localization which is reversed in the rol1-2 rcn1-3 to basal localization. In vivo analysis of PINOID action, a kinase known to influence PIN protein localization in a PP2A-antagonistic manner, revealed a negative impact of flavonols on PINOID activity. Together, these data suggest that flavonols affect auxin transport by modifying the antagonistic kinase/phosphatase equilibrium. PMID:28165500

  20. Potential anticancer activity of carvone in N2a neuroblastoma cell line.

    PubMed

    Aydın, Elanur; Türkez, Hasan; Keleş, Mevlüt Sait

    2015-08-01

    Carvone (CVN) is a monocyclic monoterpene found in the essential oils of Mentha spicata var. crispa (Lamiaceae) and Carum carvi L. (Apiaceae) plants and has been reported to have antioxidant, antimicrobial, anticonvulsant, and antitumor activities. The beneficial health properties of CVN have encouraged us to look into its anticancer activity. To the best of our knowledge, reports are not available on the anticancer activity of CVN in cultured primary rat neuron and N2a neuroblastoma (NB) cells. Therefore, the present study is an attempt toward exploring the potential anticancer activity of CVN, if any, in cultured primary rat neuron and N2a NB cells. Our results indicated that CVN (only at 25 mg/L) treatment led to an increase in the total antioxidant capacity levels in cultured primary rat neuron cells compared with control cells. Also, CVN (at concentrations higher than 100 mg/L) treatment led to an increase in the total oxidative stress levels in both cell types. The mean values of the total scores of cells showing DNA damage (for comet assay) were not found to be significantly different from the control values in both cells (p > 0.05). On the other hand, after 24 h treatment with CVN, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay showed that CVN application significantly reduced the cell viability rates in both cell types at concentrations higher than 100 mg/L. Summarizing, our data suggest that CVN represents little potential for promising anticancer agent to improve brain tumors therapy.

  1. Reduced sulfotransferase SULT2A1 activity in patients with Alzheimer's disease.

    PubMed

    Vaňková, M; Hill, M; Velíková, M; Včelák, J; Vacínová, G; Lukášová, P; Vejražková, D; Dvořáková, K; Rusina, R; Holmerová, I; Jarolímová, E; Vaňková, H; Bendlová, B

    2015-01-01

    Steroids are important components in the pathophysiology of Alzheimer's disease (AD). Although their role has been studied, the corresponding metabolomic data is limited. In the present study we evaluate the role of steroid sulfotransferase SULT2A1 in the pathophysiology of AD on the basis of circulating steroids (measured by GC-MS), in which the sulfation catalyzed by SULT2A1 dominates over glucuronidation (pregnenolone/sulfate, DHEA/sulfate, androstenediol/sulfate and 5alpha-reduced pregnane and androstane catabolites). To estimate a general trend of SUL2A1 activity in AD patients we compared the ratios of steroid conjugates to their unconjugated counterparts (C/U) in controls (11 men and 22 women) and AD patients (18 men and 16 women) for individual circulating steroids after adjustment for age and BMI using ANCOVA model including the factors AD status and gender. Decreased C/U ratio for the C19 steroids demonstrate an association between attenuated sulfation of C19 steroids in adrenal zona reticularis and the pathophysiology of AD.

  2. 5-HT2A receptor activation is necessary for CO2-induced arousal

    PubMed Central

    Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

    2015-01-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  3. Involvement of serotonin 2A receptor activation in modulating medial prefrontal cortex and amygdala neuronal activation during novelty-exposure.

    PubMed

    Hervig, Mona El-Sayed; Jensen, Nadja Cecilie Hvid; Rasmussen, Nadja Bredo; Rydbirk, Rasmus; Olesen, Mikkel Vestergaard; Hay-Schmidt, Anders; Pakkenberg, Bente; Aznar, Susana

    2017-03-02

    The medial prefrontal cortex (PFC) plays a major role in executive function by exerting a top-down control onto subcortical areas. Novelty-induced frontal cortex activation is 5-HT2A receptor (5-HT2AR) dependent. Here, we further investigated how blockade of 5-HT2ARs in mice exposed to a novel open-field arena affects medial PFC activation and basolateral amygdala (BLA) reactivity. We used c-Fos immunoreactivity (IR) as a marker of neuronal activation and stereological quantification for obtaining the total number of c-Fos-IR neurons as a measure of regional activation. We further examined the impact of 5-HT2AR blockade on the striatal-projecting BLA neurons. Systemic administration of ketanserin (0.5mg/kg) prior to novel open-field exposure resulted in reduced total numbers of c-Fos-IR cells in dorsomedial PFC areas and the BLA. Moreover, there was a positive correlation between the relative time spent in the centre of the open-field and BLA c-Fos-IR in the ketanserin-treated animals. Unilateral medial PFC lesions blocked this effect, ascertaining an involvement of this frontal cortex area. On the other hand, medial PFC lesioning exacerbated the more anxiogenic-like behaviour of the ketanserin-treated animals, upholding its involvement in modulating averseness. Ketanserin did not affect the number of activated striatal-projecting BLA neurons (measured by number of Cholera Toxin b (CTb) retrograde labelled neurons also being c-Fos-IR) following CTb injection in the ventral striatum. These results support a role of 5-HT2AR activation in modulating mPFC and BLA activation during exposure to a novel environment, which may be interrelated. Conversely, 5-HT2AR blockade does not seem to affect the amygdala-striatal projection.

  4. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  5. Cloning and characterization of human IC53-2, a novel CDK5 activator binding protein.

    PubMed

    Xie, Yi Hu; He, Xiang Huo; Tang, Yun Tian; Li, Jin Jun; Pan, Zhi Mei; Qin, Wen Xin; Wan, Da Fang; Gu, Jian Ren

    2003-04-01

    We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No.AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431 and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellular carcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate that IC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.

  6. Maintained activity of glycogen synthase kinase-3{beta} despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    SciTech Connect

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-04-30

    Glycogen synthase kinase-3{beta} (GSK3{beta}) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3{beta}. However, the inactive form of GSK3{beta} which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3{beta} substrates, such as {beta}-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3{beta} at serine-9 and other substrates including tau, {beta}-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3{beta} inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3{beta} may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3{beta} inhibitors could be a valuable drug candidate in AD.

  7. USP2a positively regulates TCR-induced NF-κB activation by bridging MALT1-TRAF6.

    PubMed

    Li, Yi; He, Xiao; Wang, Shuai; Shu, Hong-Bing; Liu, Yu

    2013-01-01

    The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.

  8. Oncogenic nexus of cancerous inhibitor of protein phosphatase 2A (CIP2A): An oncoprotein with many hands

    PubMed Central

    De, Pradip; Carlson, Jennifer; Leyland-Jones, Brian; Dey, Nandini

    2014-01-01

    Oncoprotein CIP2A a Cancerous Inhibitor of PP2A forms an “oncogenic nexus” by virtue of its control on PP2A and MYC stabilization in cancer cells. The expression and prognostic function of CIP2A in different solid tumors including colorectal carcinoma, head & neck cancers, gastric cancers, lung carcinoma, cholangiocarcinoma, esophageal cancers, pancreatic carcinoma, brain cancers, breast carcinoma, bladder cancers, ovarian carcinoma, renal cell carcinomas, tongue cancers, cervical carcinoma, prostate cancers, and oral carcinoma as well as a number of hematological malignancies are just beginning to emerge. Herein, we reviewed the recent progress in our understanding of (1) how an “oncogenic nexus” of CIP2A participates in the tumorigenic transformation of cells and (2) how we can prospect/view the clinical relevance of CIP2A in the context of cancer therapy. The review will try to understand the role of CIP2A (a) as a biomarker in cancers and evaluate the prognostic value of CIP2A in different cancers (b) as a therapeutic target in cancers and (c) in drug response and developing chemo-resistance in cancers. PMID:25015035

  9. Rhesus lymphocryptovirus latent membrane protein 2A activates {beta}-catenin signaling and inhibits differentiation in epithelial cells

    SciTech Connect

    Siler, Catherine A.; Raab-Traub, Nancy

    2008-08-01

    Rhesus lymphocryptovirus (LCV) is a {gamma}-herpesvirus closely related to Epstein-Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and {beta}-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3{beta} inactivation and accumulation of {beta}-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of {beta}-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on {beta}-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis.

  10. Axonal pruning is actively regulated by the microtubule-destabilizing protein kinesin superfamily protein 2A.

    PubMed

    Maor-Nof, Maya; Homma, Noriko; Raanan, Calanit; Nof, Aviv; Hirokawa, Nobutaka; Yaron, Avraham

    2013-04-25

    Extensive axonal pruning and neuronal cell death are critical events for the development of the nervous system. Like neuronal cell death, axonal elimination occurs in discrete steps; however, the regulators of these processes remain mostly elusive. Here, we identify the kinesin superfamily protein 2A (KIF2A) as a key executor of microtubule disassembly and axonal breakdown during axonal pruning. Knockdown of Kif2a, but not other microtubule depolymerization or severing proteins, protects axonal microtubules from disassembly upon trophic deprivation. We further confirmed and extended this result to demonstrate that the entire degeneration process is delayed in neurons from the Kif2a knockout mice. Finally, we show that the Kif2a-null mice exhibit normal sensory axon patterning early during development, but abnormal target hyperinnervation later on, as they compete for limited skin-derived trophic support. Overall, these findings reveal a central regulatory mechanism of axonal pruning during development.

  11. Protein Phosphatase 2A Signaling in Human Prostate Cancer

    DTIC Science & Technology

    2011-06-01

    been shown to be involved in androgen-independent growth of human prostate cancer cells (Carson et al., 1999; Grethe and Porn -Ares, 2006; Murillo et... Porn -Ares MI. (2006). p38 MAPK regulates phosphorylation of Bad via PP2A- dependent suppression of the MEK1/2-ERK1/2 survival pathway in TNF-alpha...threonine phosphatases implicated in cell growth and sig- nalling. Biochem J 2001;353:417–39. 15. Grethe S, Porn -Ares MI. p38 MAPK regulates

  12. Alzheimer disease and amyotrophic lateral sclerosis: An etiopathogenic connection

    PubMed Central

    Wang, Xiaochuan; Blanchard, Julie; Grundke-Iqbal, Inge; Wegiel, Jerzy; Deng, Han-Xiang; Siddique, Teepu; Iqbal, Khalid

    2013-01-01

    The etiopathogenesis of neither the sporadic form of Alzheimer disease (AD) nor of amyotrophic lateral sclerosis (ALS) are well understood. The activity of protein phosphatase-2A (PP2A), which regulates the phosphorylation of tau and neurofilaments, is negatively regulated by the myeloid leukemia-associated protein SET, also known as inhibitor-2 of PP2A, I2PP2A. In AD brain PP2A activity is compromised, probably because I2PP2A is overexpressed and is selectively cleaved at asparagine 175 into an N-terminal fragment, I2NTF, and a C-terminal fragment, I2CTF, and both fragments inhibit PP2A. Here we analyzed the spinal cords from ALS and control cases for I2PP2A cleavage and PP2A activity. As observed in AD brain, we found a selective increase in the cleavage of I2PP2A into I2NTF and I2CTF and inhibition of the activity and not the expression of PP2A in the spinal cords of ALS cases. To test the hypothesis that both AD and ALS could be triggered by I2CTF, a cleavage product of I2PP2A, we transduced by intracerebroventricular injections newborn rats with adeno-associated virus serotype 1 (AAV1) containing human I2CTF. AAV1- I2CTF produced reference memory impairment and tau pathology, and intraneuronal accumulation of Aβ by 5–8 months, and motor deficit and hyperphosphorylation and proliferation of neurofilaments, tau and TDP-43 pathologies, degeneration and loss of motor neurons and axons in the spinal cord by 10–14 months in rats. These findings suggest a previously undiscovered etiopathogenic relationship between sporadic forms of AD and ALS that is linked to I2PP2A and the potential of I2PP2A-based therapeutics for these diseases. PMID:24136402

  13. Alzheimer disease and amyotrophic lateral sclerosis: an etiopathogenic connection.

    PubMed

    Wang, Xiaochuan; Blanchard, Julie; Grundke-Iqbal, Inge; Wegiel, Jerzy; Deng, Han-Xiang; Siddique, Teepu; Iqbal, Khalid

    2014-02-01

    The etiopathogenesis of neither the sporadic form of Alzheimer disease (AD) nor of amyotrophic lateral sclerosis (ALS) is well understood. The activity of protein phosphatase-2A (PP2A), which regulates the phosphorylation of tau and neurofilaments, is negatively regulated by the myeloid leukemia-associated protein SET, also known as inhibitor-2 of PP2A, I2(PP2A). In AD brain, PP2A activity is compromised, probably because I2(PP2A) is overexpressed and is selectively cleaved at asparagine 175 into an N-terminal fragment, I2NTF, and a C-terminal fragment, I2CTF, and both fragments inhibit PP2A. Here, we analyzed the spinal cords from ALS and control cases for I2(PP2A) cleavage and PP2A activity. As observed in AD brain, we found a selective increase in the cleavage of I2(PP2A) into I2NTF and I2CTF and inhibition of the activity and not the expression of PP2A in the spinal cords of ALS cases. To test the hypothesis that both AD and ALS could be triggered by I2CTF, a cleavage product of I2(PP2A), we transduced by intracerebroventricular injections newborn rats with adeno-associated virus serotype 1 (AAV1) containing human I2CTF. AAV1-I2CTF produced reference memory impairment and tau pathology, and intraneuronal accumulation of Aβ by 5-8 months, and motor deficit and hyperphosphorylation and proliferation of neurofilaments, tau and TDP-43 pathologies, degeneration and loss of motor neurons and axons in the spinal cord by 10-14 months in rats. These findings suggest a previously undiscovered etiopathogenic relationship between sporadic forms of AD and ALS that is linked to I2(PP2A) and the potential of I2(PP2A)-based therapeutics for these diseases.

  14. Obscurin Targets Ankyrin-B and Protein Phosphatase 2A to the Cardiac M-line*

    PubMed Central

    Cunha, Shane R.; Mohler, Peter J.

    2008-01-01

    Ankyrin-B targets ion channels and transporters in excitable cells. Dysfunction in ankyrin-B-based pathways results in defects in cardiac physiology. Despite a wealth of knowledge regarding the role of ankyrin-B for cardiac function, little is known regarding the mechanisms underlying ankyrin-B regulation. Moreover, the pathways underlying ankyrin-B targeting in heart are unclear. We report that alternative splicing regulates ankyrin-B localization and function in cardiomyocytes. Specifically, we identify a novel exon (exon 43′) in the ankyrin-B regulatory domain that mediates interaction with the Rho-GEF obscurin. Ankyrin-B transcripts harboring exon 43′ represent the primary cardiac isoform in human and mouse. We demonstrate that ankyrin-B and obscurin are co-localized at the M-line of myocytes and co-immunoprecipitate from heart. We define the structural requirements for ankyrin-B/obscurin interaction to two motifs in the ankyrin-B regulatory domain and demonstrate that both are critical for obscurin/ankyrin-B interaction. In addition, we demonstrate that interaction with obscurin is required for ankyrin-B M-line targeting. Specifically, both obscurin-binding motifs are required for the M-line targeting of a GFP-ankyrin-B regulatory domain. Moreover, this construct acts as a dominant-negative by competing with endogenous ankyrin-B for obscurin-binding at the M-line, thus providing a powerful new tool to evaluate the function of obscurin/ankyrin-B interactions. With this new tool, we demonstrate that the obscurin/ankyrin-B interaction is critical for recruitment of PP2A to the cardiac M-line. Together, these data provide the first evidence for the molecular basis of ankyrin-B and PP2A targeting and function at the cardiac M-line. Finally, we report that ankyrin-B R1788W is localized adjacent to the ankyrin-B obscurin-binding motif and increases binding activity for obscurin. In summary, our new findings demonstrate that ANK2 is subject to alternative splicing

  15. Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

    PubMed Central

    Loza-Muller, Lloyd; Rodríguez-Corona, Ulises; Sobol, Margarita; Rodríguez-Zapata, Luis C.; Hozak, Pavel; Castano, Enrique

    2015-01-01

    Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter. PMID:26594224

  16. Suppression of piriform cortex activity in rat by corticotropin-releasing factor 1 and serotonin 2A/C receptors.

    PubMed

    Narla, Chakravarthi; Dunn, Henry A; Ferguson, Stephen S G; Poulter, Michael O

    2015-01-01

    The piriform cortex (PC) is richly innervated by corticotropin-releasing factor (CRF) and serotonin (5-HT) containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the Layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC) either mimicked or blocked CRF modulation, respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of Layer II pyramidal neurons. CRF had highly variable effects on interneurons within Layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and 5-HT, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviors mediated through the olfactory cortex.

  17. Electrophysiological characterization of V2a interneurons and their locomotor-related activity in the neonatal mouse spinal cord.

    PubMed

    Zhong, Guisheng; Droho, Steven; Crone, Steven A; Dietz, Shelby; Kwan, Alex C; Webb, Watt W; Sharma, Kamal; Harris-Warrick, Ronald M

    2010-01-06

    The V2a class of Chx10-expressing interneurons has been implicated in frequency-dependent control of left-right phase during locomotion in the mouse. We have used the Chx10::CFP mouse line to further investigate the properties and locomotion-related activity of V2a interneurons in the isolated neonatal spinal cord. V2a interneurons can be divided into three classes, based on their tonic, phasic, or delayed-onset responses to step depolarization. Electrical coupling is found only between neurons of same class and helps to synchronize neuronal activity within the class. Serotonin (5-HT) excites isolated tonic V2a interneurons by depolarizing the neurons and increasing their membrane input resistance, with no significant effects on action potential properties, a mechanism distinct from 5-HT excitation of commissural interneurons. During NMDA-/5-HT-induced locomotor-like activity, patch-clamp recordings and two-photon calcium imaging experiments show that approximately half of V2a interneurons fire rhythmically with ventral root-recorded motor activity; the rhythmic V2a interneurons fired during one half of the cycle, in phase with either the ipsilateral or the contralateral L2 ventral root bursts. The percentage of rhythmically firing V2a interneurons increases during higher-frequency fictive locomotion, and they become significantly more rhythmic in their firing during the locomotor cycle; this may help to explain the frequency-dependent shift in left-right coupling in Chx10::DTA mice, which lack these neurons. Our results together with data from the accompanying paper (Dougherty and Kiehn, 2009) reinforce earlier proposals that the V2a interneurons are components of the hindlimb central pattern generator, helping to organize left-right locomotor coordination in the neonatal mouse spinal cord.

  18. Structure–Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins

    PubMed Central

    Twiner, Michael J.; Doucette, Gregory J.; Pang, Yucheng; Fang, Chao; Forsyth, Craig J.; Miles, Christopher O.

    2016-01-01

    Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure–activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs’ activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors. PMID:27827901

  19. Co-activation of NR2A and NR2B subunits induces resistance to fear extinction.

    PubMed

    Leaderbrand, Katherine; Corcoran, Kevin A; Radulovic, Jelena

    2014-09-01

    Unpredictable stress is known to profoundly enhance susceptibility to fear and anxiety while reducing the ability to extinguish fear when threat is no longer present. Accordingly, partial aversive reinforcement, via random exposure to footshocks, induces fear that is resistant to extinction. Here we sought to determine the hippocampal mechanisms underlying susceptibility versus resistance to context fear extinction as a result of continuous (CR) and partial (PR) reinforcement, respectively. We focused on N-methyl-D-aspartate receptor (NMDAR) subunits 2A and B (NR2A and NR2B) as well as their downstream signaling effector, extracellular signal-regulated kinase (ERK), based on their critical role in the acquisition and extinction of fear. Pharmacological inactivation of NR2A, but not NR2B, blocked extinction after CR, whereas inactivation of NR2A, NR2B, or both subunits facilitated extinction after PR. The latter finding suggests that co-activation of NR2A and NR2B contributes to persistent fear following PR. In contrast to CR, PR increased membrane levels of ERK and NR2 subunits after the conditioning and extinction sessions, respectively. In parallel, nuclear activation of ERK was significantly reduced after the extinction session. Thus, co-activation and increased surface expression of NR2A and NR2B, possibly mediated by ERK, may cause persistent fear. These findings suggest that patients with post-traumatic stress disorder (PTSD) may benefit from antagonism of specific NR2 subunits.

  20. Activation of α2A-adrenergic signal transduction in chondrocytes promotes degenerative remodelling of temporomandibular joint

    PubMed Central

    Jiao, Kai; Zeng, Guang; Niu, Li-Na; Yang, Hong-xu; Ren, Gao-tong; Xu, Xin-yue; Li, Fei-fei; Tay, Franklin R.; Wang, Mei-qing

    2016-01-01

    This study tested whether activation of adrenoreceptors in chondrocytes has roles in degenerative remodelling of temporomandibular joint (TMJ) and to determine associated mechanisms. Unilateral anterior crossbite (UAC) was established to induce TMJ degeneration in rats. Saline vehicle, α2- and β-adrenoreceptor antagonists or agonists were injected locally into the TMJ area of UAC rats. Cartilage degeneration, subchondral bone microarchitecture and the expression of adrenoreceptors, aggrecans, matrix metalloproteinases (MMPs) and RANKL by chondrocytes were evaluated. Chondrocytes were stimulated by norepinephrine to investigate signal transduction of adrenoreceptors. Increased α2A-adrenoreceptor expression was observed in condylar cartilage of UAC rats, together with cartilage degeneration and subchondral bone loss. Norepinephrine depresses aggrecans expression but stimulates MMP-3, MMP-13 and RANKL production by chondrocytes through ERK1/2 and PKA pathway; these effects were abolished by an α2A-adrenoreceptor antagonist. Furthermore, inhibition of α2A-adrenoreceptor attenuated degenerative remodelling in the condylar cartilage and subchondral bone, as revealed by increased cartilage thickness, proteoglycans and aggrecan expression, and decreased MMP-3, MMP-13 and RANKL expressions in cartilage, increased BMD, BV/TV, and decreased Tb.Sp in subchondral bone. Conversely, activation of α2A-adrenoreceptor intensified aforementioned degenerative changes in UAC rats. It is concluded that activation of α2A-adrenergic signal in chondrocytes promotes TMJ degenerative remodelling by chondrocyte-mediated pro-catabolic activities. PMID:27452863

  1. MicroRNA-1 and -133 Increase Arrhythmogenesis in Heart Failure by Dissociating Phosphatase Activity from RyR2 Complex

    PubMed Central

    Belevych, Andriy E.; Sansom, Sarah E.; Terentyeva, Radmila; Ho, Hsiang-Ting; Nishijima, Yoshinori; Martin, Mickey M.; Jindal, Hitesh K.; Rochira, Jennifer A.; Kunitomo, Yukiko; Abdellatif, Maha; Carnes, Cynthia A.; Elton, Terry S.; Györke, Sandor; Terentyev, Dmitry

    2011-01-01

    In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing

  2. Imidazo[1,2-a]Pyridine-3-Carboxamides Are Active Antimicrobial Agents against Mycobacterium avium Infection In Vivo

    PubMed Central

    Moraski, Garrett C.; Cheng, Yong; Cho, Sanghyun; Cramer, Jeffrey W.; Godfrey, Alexander; Masquelin, Thierry; Franzblau, Scott G.; Miller, Marvin J.

    2016-01-01

    A panel of six imidazo[1,2-a]pyridine-3-carboxamides (IAPs) were shown to have low-micromolar activity against Mycobacterium avium strains. Compound ND-10885 (compound 2) showed significant activity in the lung, spleen, and liver in a mouse M. avium infection model. A combined regimen consisting of ND-10885 (compound 2) and rifampin was additive in its anti-M. avium activity in the lung. Our data indicate that IAPs represent a new class of antibiotics that are active against M. avium and could potentially serve as an effective addition to a combined treatment regimen. PMID:27216051

  3. Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation.

    PubMed

    Gómez-Villafuertes, Rosa; Pintor, Jesús; Miras-Portugal, María Teresa; Gualix, Javier

    2014-11-01

    Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn(2+) ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells

  4. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  5. Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker.

    PubMed

    Park, Hyun I; Lee, Seakwoo; Ullah, Asad; Cao, Qiang; Sang, Qing-Xiang Amy

    2010-01-15

    Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 ( approximately 90muM). Their IC(50) values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon's plot; however, the inhibition mechanism of endometase was noncompetitive with a K(i) value of 240muM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.

  6. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms

    PubMed Central

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.

    2002-01-01

    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  7. A2A Adenosine Receptor Antagonism Enhances Synaptic and Motor Effects of Cocaine via CB1 Cannabinoid Receptor Activation

    PubMed Central

    Tozzi, Alessandro; de Iure, Antonio; Marsili, Valentina; Romano, Rosaria; Tantucci, Michela; Di Filippo, Massimiliano; Costa, Cinzia; Napolitano, Francesco; Mercuri, Nicola Biagio; Borsini, Franco; Giampà, Carmen; Fusco, Francesca Romana; Picconi, Barbara; Usiello, Alessandro; Calabresi, Paolo

    2012-01-01

    Background Cocaine increases the level of endogenous dopamine (DA) in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs) have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. Principal Findings Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. Conclusions The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine. PMID:22715379

  8. Deletion of striatal adenosine A(2A) receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning.

    PubMed

    Singer, Philipp; Wei, Catherine J; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K

    2013-04-01

    Following early clinical leads, the adenosine A(2A)R receptor (A(2A)R) has continued to attract attention as a potential novel target for treating schizophrenia, especially against the negative and cognitive symptoms of the disease because of A(2A)R's unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through (i) the antagonistic interaction with the dopamine D(2) receptor, and (ii) the regulation of glutamate release and N-methyl-d-aspartate receptor function, striatal A(2A)R is ideally positioned to fine-tune the dopamine-glutamate balance, the disturbance of which is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A(2A)Rs in the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A(2A)R knockout (st-A(2A)R-KO) on latent inhibition (LI) and prepulse inhibition (PPI) - behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A(2A)R-KO mice, although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning - namely, conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A(2A)Rs - a finding that may undermine the hypothesized importance of A(2A)R in the genesis and/or treatment of schizophrenia.

  9. Interactions between glycogen synthase kinase 3beta, protein kinase B, and protein phosphatase 2A in tau phosphorylation in mouse N2a neuroblastoma cells.

    PubMed

    Zhou, Xin-Wen; Winblad, Bengt; Guan, Zhizhong; Pei, Jin-Jing

    2009-01-01

    In this study, we investigated how tau phosphorylation is regulated by protein kinase glycogen synthase kinase 3beta (GSK3 beta), protein kinase B (PKB), and protein phosphatase 2A (PP2A) in mouse N2a neuroblastoma cells. Results showed that GSK3 beta overexpression significantly increased PKB phosphorylation at the S473 site but not the T308 site. Neither GSK3 beta nor PKB overexpression could reduce the PP2AC phosphorylation at the Y307 site. In contrast, either PKB or GSK3 beta knockdown could increase PP2A phosphorylation at the Y307 site. PP2AC knockdown increased GSK3 beta phosphorylation at the S9 site but not at the Y216 site, and PKB phosphorylation at the T308 site but not at the S473 site. Tau phosphorylation at the S396 site was increased by GSK3 beta or PKB overexpression. Tau phosphorylation at the S214 site was only induced by PKB overexpression in the study. While GSK3 beta knockdown decreased tau phosphorylation at the S396 site, PKB knockdown increased tau phosphorylation at both the S396 and S214 sites. PP2AC knockdown decreased tau phosphorylation at the S396 and S214 sites. These findings suggest that tau phosphorylation at the S396 and S214 sites is differentially regulated by GSK3 beta, PKB, and PP2A in N2a cells. The final phosphorylation state of tau is possibly caused by the synergic action of the three enzymes.

  10. Activation of A1, A2A, or A3 adenosine receptors attenuates lung ischemia-reperfusion injury

    PubMed Central

    Gazoni, Leo M.; Walters, Dustin M.; Unger, Eric B.; Linden, Joel; Kron, Irving L.; Laubach, Victor E.

    2010-01-01

    Objective Adenosine and the activation of specific adenosine receptors are implicated in the attenuation of inflammation and organ ischemia-reperfusion (IR) injury. We hypothesized that activation of A1, A2A, or A3 adenosine receptors would provide protection against lung IR injury. Methods Using an isolated, ventilated, blood-perfused rabbit lung model, lungs underwent 18 hours cold ischemia followed by 2 hours reperfusion. Lungs were administered either vehicle, adenosine, or selective A1, A2A, or A3 receptor agonists (CCPA, ATL-313, or IB-MECA, respectively) alone or with their respective antagonists (DPCPX, ZM241385, or MRS1191) during reperfusion. Results Compared to the vehicle-treated control group, treatment with A1, A2A, or A3 agonists significantly improved function (increased lung compliance and oxygenation and decreased pulmonary artery pressure), decreased neutrophil infiltration by myeloperoxidase activity, decreased edema, and reduced TNF-α production. Adenosine treatment was also protective but not to the level of the agonists. When each agonist was paired with its respective antagonist, all protective effects were blocked. The A2A agonist reduced pulmonary artery pressure and myeloperoxidase activity and increased oxygenation to a greater degree than the A1 or A3 agonists. Conclusions Selective activation of A1, A2A, or A3 adenosine receptors provides significant protection against lung IR injury. The decreased elaboration of the potent proinflammatory cytokine, TNF-α, and decreased neutrophil sequestration likely contribute to the overall improvement in pulmonary function. These results provide evidence for the therapeutic potential of specific adenosine receptor agonists in lung transplant recipients. PMID:20398911

  11. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  12. Soluble ST2: A new and promising activity marker in ulcerative colitis

    PubMed Central

    Díaz-Jiménez, David; Núñez, Lucía E; Beltrán, Caroll J; Candia, Enzo; Suazo, Cristóbal; Álvarez-Lobos, Manuel; González, María-Julieta; Hermoso, Marcela A; Quera, Rodrigo

    2011-01-01

    AIM: To correlate circulating soluble ST2 (sST2) levels with the severity of ulcerative colitis (UC) and serum levels of pro-inflammatory cytokines, and to demonstrate the predictive power of sST2 levels for differentiation between active and inactive UC. METHODS: We recruited 153 patients: 82 with UC, 26 with Crohn’s disease (CD) and 43 disease controls [non-inflammatory bowel disease (IBD)]. Subjects were excluded if they had diagnosis of asthma, autoimmune diseases or hypertension. The serum levels of sST2 and pro-inflammatory cytokines [pg/mL; median (25th-75th)] as well as clinical features, endoscopic and histological features, were subjected to analyses. The sST2 performance for discrimination between active and inactive UC, non-IBD and healthy controls (HC) was determined with regard to sensitivity and specificity, and Spearman’s rank correlation coefficient (r). To validate the method, the area under the curve (AUC) of receiver-operator characteristic (ROC) was determined (AUC, 95% CI) and the total ST2 content of the colonic mucosa in UC patients was correlated with circulating levels of sST2. RESULTS: The serum sST2 value was significantly higher in patients with active [235.80 (90.65-367.90) pg/mL] rather than inactive UC [33.19 (20.04-65.32) pg/mL], based on clinical, endoscopic and histopathological characteristics, as well as compared with non-IBD and HC (P < 0.001). The median level of sST2 in CD patients was 54.17 (35.02-122.0) pg/mL, significantly higher than that of the HC group only (P < 0.01). The cutoff was set at 74.87 pg/mL to compare active with inactive UC in a multicenter cohort of patients. Values of sensitivity, specificity, and ability to correctly classify UC, according to activity, were 83.33%, 83.33% and 83.33%, respectively. The AUC of the ROC curve to assess the ability of this molecule to discriminate between active vs inactive UC was 0.92 (0.86-0.97, P < 0.0001). The serum levels of sST2 in patients with UC significantly

  13. NG2, a member of chondroitin sulfate proteoglycans family mediates the inflammatory response of activated microglia.

    PubMed

    Gao, Q; Lu, J; Huo, Y; Baby, N; Ling, E A; Dheen, S T

    2010-01-20

    Activation of microglial cells, the resident immune cells of the CNS causes neurotoxicity through the release of a wide array of inflammatory mediators including proinflammatory cytokines, chemokines and reactive oxygen species. In this study, we have investigated the expression of NG2 (also known as CSPG4), one of the members of transmembrane chondroitin sulfate proteoglycans family, in microglial cells and its role on inflammatory reaction of microglia by analyzing the expression of the proinflammation cytokines (interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)), chemokines (stromal cell-derived factor-1alpha and monocyte chemotactic protein-1) and inducible nitric oxide synthase (iNOS). NG2 expression was not detectable in microglial cells expressing OX-42 in the brains of 1-day old postnatal rat pups and adult rats; it was, however, induced in activated microglial cells in pups and adult rats injected with lipopolysaccharide (LPS). In vitro analysis further confirmed that LPS induced the expression of NG2 in primary microglial cells and this was inhibited by dexamethasone. It has been well demonstrated that LPS induces the expression of iNOS and proinflammatory cytokines in microglia. However in this study, LPS did not induce the mRNA expression of iNOS and cytokines including IL-1beta, and TNF-alpha in microglial cells transfected with CSPG4 siRNA. On the contrary, mRNA expression of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1alpha (SDF-1alpha) was significantly increased in LPS-activated microglial cells after CSPG4 siRNA transfection in comparison with the control. The above results indicate that NG2 mediates the induction of iNOS and inflammatory cytokine expression, but not the chemokine expression in activated microglia.

  14. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  15. Antibacterial activities of Groebke-Blackburn-Bienaymé derived imidazo[1,2-a]pyridin-3-amines

    PubMed Central

    Shukla, Nikunj M.; Salunke, Deepak B.; Yoo, Euna; Mutz, Cole A.; Balakrishna, Rajalakshmi; David, Sunil A.

    2012-01-01

    We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke-Blackburn-Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure-activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency. PMID:22925449

  16. Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus.

    PubMed

    Paulson, Matthew S; Yang, Huiling; Shih, I-hung; Feng, Joy Y; Mabery, Eric M; Robinson, Margaret F; Zhong, Weidong; Delaney, William E

    2009-08-01

    The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2'-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61-109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC(50)) regardless of genotype or subtype. Importantly, we observed a significant correlation (p<0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.

  17. CacyBP/SIP phosphatase activity in neuroblastoma NB2a and colon cancer HCT116 cells.

    PubMed

    Kilanczyk, Ewa; Wasik, Urszula; Filipek, Anna

    2012-08-01

    Recently, we have reported that CacyBP/SIP could be a novel phosphatase for ERK1/2 kinase. In this work, we analyzed the CacyBP/SIP phosphatase activity toward ERK1/2 in 2 cell lines of different origin. We showed that overexpression of CacyBP/SIP in NB2a cells resulted in a lower level of phosphorylated ERK1/2 (P-ERK1/2) in the nuclear fraction while such overexpression in HCT116 cells had no effect on the level of P-ERK1/2. Moreover, we found that overexpression of CacyBP/SIP resulted in higher phosphatase activity in the nuclear fraction obtained from NB2a cells when compared with HCT116 cells. Using 2-D electrophoresis we showed that the pattern of spots representing CacyBP/SIP differed in these 2 cell lines and was probably due to a different phosphorylation state of this protein. We also established that after overexpression of CacyBP/SIP in NB2a cells, the amount of nuclear β-catenin was low, while it remained high in HCT116 cells. Since NB2a cells have differentiation potential and HCT116 cells do not, our data suggest that different activity of CacyBP/SIP in these 2 cell lines might affect the ERK1/2 pathway in the differentiation or proliferation processes.

  18. Biochemical profile of YM992, a novel selective serotonin reuptake inhibitor with 5-HT2A receptor antagonistic activity.

    PubMed

    Hatanaka, K; Nomura, T; Hidaka, K; Takeuchi, H; Yatsugi, S; Fujii, M; Yamaguchi, T

    1996-01-01

    YM992, (S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride, exhibited the biochemical profile of a selective serotonin (5-HT) reuptake inhibitor (SSRI) with 5-HT2A receptor antagonistic activity. YM922 showed the same high affinity as fluoxetine against the 5-HT reuptake site (Ki = 21 nM) and a similar affinity to that of crazodone against the 5-HT2A receptor (Ki = 86 nM). In other receptor binding studies, an affinity for the adrenergic alpha 1 receptor (Ki = 200 nM) and 5-HT2C receptor (Ki = 680 nM) was observed. In a monoamine uptake study, YM992 showed a selective 5-HT uptake inhibition (IC50 = 0.15 microM), but only very weakly inhibited both noradrenaline (NA) and dopamine (DA) uptake (IC50 = 3.1 microM (NA), > 10 microM (DA)). YM992 was also found to potently inhibit the aggregation of human platelets (IC50 = 1.9 microM), revealing antagonistic activity for the 5-HT2A receptor in vitro. Enhanced serotonergic neurotransmission, in particular that mediated by the 5-HT1A receptor, has recently been reported to be important in the long-term treatment of depressive disorders with antidepressants. In addition, some 5-HT1A receptor-mediated responses are known to be potentiated by co-administration of 5-HT2A receptor antagonists. Thus, YM992, having both selective 5-HT reuptake inhibition and 5-HT2A antagonistic activity, might show potent therapeutic activity as a novel antidepressant in comparison with conventional SSRIs.

  19. Groundwater flow code verification ``benchmarking`` activity (COVE-2A): Analysis of participants` work

    SciTech Connect

    Dykhuizen, R.C.; Barnard, R.W.

    1992-02-01

    The Nuclear Waste Repository Technology Department at Sandia National Laboratories (SNL) is investigating the suitability of Yucca Mountain as a potential site for underground burial of nuclear wastes. One element of the investigations is to assess the potential long-term effects of groundwater flow on the integrity of a potential repository. A number of computer codes are being used to model groundwater flow through geologic media in which the potential repository would be located. These codes compute numerical solutions for problems that are usually analytically intractable. Consequently, independent confirmation of the correctness of the solution is often not possible. Code verification is a process that permits the determination of the numerical accuracy of codes by comparing the results of several numerical solutions for the same problem. The international nuclear waste research community uses benchmarking for intercomparisons that partially satisfy the Nuclear Regulatory Commission (NRC) definition of code verification. This report presents the results from the COVE-2A (Code Verification) project, which is a subset of the COVE project.

  20. The MATROSHKA experiment: results and comparison from extravehicular activity (MTR-1) and intravehicular activity (MTR-2A/2B) exposure.

    PubMed

    Berger, Thomas; Bilski, Paweł; Hajek, Michael; Puchalska, Monika; Reitz, Günther

    2013-12-01

    Astronauts working and living in space are exposed to considerably higher doses and different qualities of ionizing radiation than people on Earth. The multilateral MATROSHKA (MTR) experiment, coordinated by the German Aerospace Center, represents the most comprehensive effort to date in radiation protection dosimetry in space using an anthropomorphic upper-torso phantom used for radiotherapy treatment planning. The anthropomorphic upper-torso phantom maps the radiation distribution as a simulated human body installed outside (MTR-1) and inside different compartments (MTR-2A: Pirs; MTR-2B: Zvezda) of the Russian Segment of the International Space Station. Thermoluminescence dosimeters arranged in a 2.54 cm orthogonal grid, at the site of vital organs and on the surface of the phantom allow for visualization of the absorbed dose distribution with superior spatial resolution. These results should help improve the estimation of radiation risks for long-term human space exploration and support benchmarking of radiation transport codes.

  1. Transcriptional activation of the SH2D2A gene is dependent on a cyclic adenosine 5'-monophosphate-responsive element in the proximal SH2D2A promoter.

    PubMed

    Dai, Ke-Zheng; Johansen, Finn-Eirik; Kolltveit, Kristin Melkevik; Aasheim, Hans-Christian; Dembic, Zlatko; Vartdal, Frode; Spurkland, Anne

    2004-05-15

    The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.

  2. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells.

    PubMed

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X; Huang, Guowei

    2015-10-20

    Alzheimer's disease (AD) is a common neurodegenerative disease resulting in progressive dementia, and is a principal cause of dementia among older adults. Folate acts through one-carbon metabolism to support the methylation of multiple substrates. We hypothesized that folic acid supplementation modulates DNA methyltransferase (DNMT) activity and may alter amyloid β-peptide (Aβ) production in AD. Mouse Neuro-2a cells expressing human APP695 were incubated with folic acid (2.8-40 μmol/L), and with or without zebularine (the DNMT inhibitor). DNMT activity, cell viability, Aβ and DNMTs expression were then examined. The results showed that folic acid stimulated DNMT gene and protein expression, and DNMT activity. Furthermore, folic acid decreased Aβ protein production, whereas inhibition of DNMT activity by zebularine increased Aβ production. The results indicate that folic acid induces methylation potential-dependent DNMT enzymes, thereby attenuating Aβ production.

  3. Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism.

    PubMed

    Lee, Heejeong; Hwang, Jae-Sam; Lee, Jaeho; Kim, Jae Il; Lee, Dong Gun

    2015-02-01

    Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.

  4. The downregulation of thioredoxin accelerated Neuro2a cell apoptosis induced by advanced glycation end product via activating several pathways.

    PubMed

    Ren, Xiang; Ma, Haiying; Qiu, Yuanyuan; Liu, Bo; Qi, Hui; Li, Zeyu; Kong, Hui; Kong, Li

    2015-08-01

    Thioredoxin (Trx), a 12 kDa protein, has different functions in different cellular environments, playing important anti-oxidative and anti-apoptotic roles and regulating the expression of transcription factors. Advanced glycation end products (AGEs) are a heterogeneous group of irreversible adducts from glucose-protein condensation reactions and are considered crucial to the development of diabetic nephropathy, retinopathy, neurodegeneration and atherosclerosis. The aim of this study was to use a Trx inhibitor to investigate the effects and mechanism of Trx down-regulation on AGE-induced Neuro2a cell apoptosis. Neuro2a cells were cultured in vitro and treated with different conditions. The apoptosis and proliferation of Neuro2a cells were detected using flow cytometry, DNA-Ladder and CCK8 assays. Rho 123 was used to detect the mitochondrial membrane potential. ROS generation and caspase3 activity were detected using a DCFH-DA probe and micro-plate reader. Western blotting and real-time PCR were used to detect the expression of proteins and genes. We found that the down-regulation of thioredoxin could accelerate AGE-induced apoptosis in Neuro2a cells. A possible underlying mechanism is that the down-regulation of thioredoxin stimulated the up-regulation of ASK1, p-JNK, PTEN, and Txnip, as well as the down-regulation of p-AKT, ultimately increasing ROS levels and caspase3 activity.

  5. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  6. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

  7. 2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

    PubMed

    Slee, Deborah H; Moorjani, Manisha; Zhang, Xiaohu; Lin, Emily; Lanier, Marion C; Chen, Yongsheng; Rueter, Jaimie K; Lechner, Sandra M; Markison, Stacy; Malany, Siobhan; Joswig, Tanya; Santos, Mark; Gross, Raymond S; Williams, John P; Castro-Palomino, Julio C; Crespo, María I; Prat, Maria; Gual, Silvia; Díaz, José-Luis; Jalali, Kayvon; Sai, Yang; Zuo, Zhiyang; Yang, Chun; Wen, Jenny; O'Brien, Zhihong; Petroski, Robert; Saunders, John

    2008-03-27

    Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.

  8. Quantitative structure-activity relationship of phenoxyphenyl-methanamine compounds with 5HT2A, SERT, and hERG activities.

    PubMed

    Mente, Scot; Gallaschun, Randall; Schmidt, Anne; Lebel, Lorrie; Vanase-Frawley, Michelle; Fliri, Anton

    2008-12-01

    QSAR models have been used to evaluate activities for compounds in the phenoxyphenyl-methanamine (PPMA) class of compounds. These models utilize Hammett-type donating-withdrawing substituent values as well as simple parameters to describe substituent size and elucidate the SAR of the 'A' and 'B' rings. Using this methodology, intuitive QSAR relationships were found for the three biological activities with R(2) values of 0.73, 0.45, and 0.58 for 5HT(2A), SerT, and hERG activities.

  9. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    PubMed Central

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

  10. Design and synthesis of new imidazo[1,2-a]pyridine and imidazo[1,2-a]pyrazine derivatives with antiproliferative activity against melanoma cells.

    PubMed

    Garamvölgyi, Rita; Dobos, Judit; Sipos, Anna; Boros, Sándor; Illyés, Eszter; Baska, Ferenc; Kékesi, László; Szabadkai, István; Szántai-Kis, Csaba; Kéri, György; Őrfi, László

    2016-01-27

    Melanoma is an aggressive form of skin cancer and it is generally associated with poor prognosis in patients with late-stage disease. Due to the increasing occurrence of melanoma, there is a need for the development of novel therapies. A new series of diarylamide and diarylurea derivatives containing imidazo[1,2-a]pyridine or imidazo[1,2-a]pyrazine scaffold was designed and synthesized to investigate their in vitro efficacy against the A375P human melanoma cell line. We found several compounds expressing submicromolar IC50 values against the A375P cells, from which 15d, 17e, 18c, 18h, 18i demonstrated the highest potencies with IC50 below 0.06 μM.

  11. Usher syndrome type 2 caused by activation of an USH2A pseudoexon: implications for diagnosis and therapy.

    PubMed

    Vaché, Christel; Besnard, Thomas; le Berre, Pauline; García-García, Gema; Baux, David; Larrieu, Lise; Abadie, Caroline; Blanchet, Catherine; Bolz, Hanno Jörn; Millan, Jose; Hamel, Christian; Malcolm, Sue; Claustres, Mireille; Roux, Anne-Françoise

    2012-01-01

    USH2A sequencing in three affected members of a large family, referred for the recessive USH2 syndrome, identified a single pathogenic alteration in one of them and a different mutation in the two affected nieces. As the patients carried a common USH2A haplotype, they likely shared a mutation not found by standard sequencing techniques. Analysis of RNA from nasal cells in one affected individual identified an additional pseudoexon (PE) resulting from a deep intronic mutation. This was confirmed by minigene assay. This is the first example in Usher syndrome (USH) with a mutation causing activation of a PE. The finding of this alteration in eight other individuals of mixed European origin emphasizes the importance of including RNA analysis in a comprehensive diagnostic service. Finally, this mutation, which would not have been found by whole-exome sequencing, could offer, for the first time in USH, the possibility of therapeutic correction by antisense oligonucleotides (AONs).

  12. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

    PubMed

    Luessen, Deborah J; Hinshaw, Tyler P; Sun, Haiguo; Howlett, Allyn C; Marrs, Glen; McCool, Brian A; Chen, Rong

    2016-11-01

    Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

  13. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  14. Inhibition of alpha oscillations through serotonin-2A receptor activation underlies the visual effects of ayahuasca in humans.

    PubMed

    Valle, Marta; Maqueda, Ana Elda; Rabella, Mireia; Rodríguez-Pujadas, Aina; Antonijoan, Rosa Maria; Romero, Sergio; Alonso, Joan Francesc; Mañanas, Miquel Àngel; Barker, Steven; Friedlander, Pablo; Feilding, Amanda; Riba, Jordi

    2016-07-01

    Ayahuasca is an Amazonian psychotropic plant tea typically obtained from two plants, Banisteriopsis caapi and Psychotria viridis. It contains the psychedelic 5-HT2A and sigma-1 agonist N,N-dimethyltryptamine (DMT) plus β-carboline alkaloids with monoamine-oxidase (MAO)-inhibiting properties. Although the psychoactive effects of ayahuasca have commonly been attributed solely to agonism at the 5-HT2A receptor, the molecular target of classical psychedelics, this has not been tested experimentally. Here we wished to study the contribution of the 5-HT2A receptor to the neurophysiological and psychological effects of ayahuasca in humans. We measured drug-induced changes in spontaneous brain oscillations and subjective effects in a double-blind randomized placebo-controlled study involving the oral administration of ayahuasca (0.75mg DMT/kg body weight) and the 5-HT2A antagonist ketanserin (40mg). Twelve healthy, experienced psychedelic users (5 females) participated in four experimental sessions in which they received the following drug combinations: placebo+placebo, placebo+ayahuasca, ketanserin+placebo and ketanserin+ayahuasca. Ayahuasca induced EEG power decreases in the delta, theta and alpha frequency bands. Current density in alpha-band oscillations in parietal and occipital cortex was inversely correlated with the intensity of visual imagery induced by ayahuasca. Pretreatment with ketanserin inhibited neurophysiological modifications, reduced the correlation between alpha and visual effects, and attenuated the intensity of the subjective experience. These findings suggest that despite the chemical complexity of ayahuasca, 5-HT2A activation plays a key role in the neurophysiological and visual effects of ayahuasca in humans.

  15. A2a and a2b adenosine receptors affect HIF-1α signaling in activated primary microglial cells.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Stefanelli, Angela; Bencivenni, Serena; Castillo, Carlos Alberto; Varani, Katia; Gessi, Stefania

    2015-05-15

    Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A2A through p44/42 and A2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A2A and A2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A2B subtype. GLIA 2015.

  16. In Vitro Anti-Neuroblastoma Activity of Thymoquinone Against Neuro-2a Cells via Cell-cycle Arrest.

    PubMed

    Paramasivam, Arumugam; Raghunandhakumar, Subramanian; Priyadharsini, Jayaseelan Vijayashree; Jayaraman, Gopalswamy

    2015-01-01

    We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose- dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

  17. Structure–activity relationships of bumetanide derivatives: correlation between diuretic activity in dogs and inhibition of the human NKCC2A transporter

    PubMed Central

    Lykke, Kasper; Töllner, Kathrin; Römermann, Kerstin; Feit, Peter W; Erker, Thomas; MacAulay, Nanna; Löscher, Wolfgang

    2015-01-01

    Background and Purpose The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na+, K+ and Cl− ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure–activity studies on effects of bumetanide derivatives on NKCC2 are not available. Experimental Approach In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. Key Results Bumetanide blocked hNKCC2A transport with an IC50 of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r2 = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. Conclusions and Implications The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics. PMID:26101812

  18. Magnetic activity in the photosphere of CoRoT-Exo-2a. Active longitudes and short-term spot cycle in a young Sun-like star

    NASA Astrophysics Data System (ADS)

    Lanza, A. F.; Pagano, I.; Leto, G.; Messina, S.; Aigrain, S.; Alonso, R.; Auvergne, M.; Baglin, A.; Barge, P.; Bonomo, A. S.; Boumier, P.; Collier Cameron, A.; Comparato, M.; Cutispoto, G.; de Medeiros, J. R.; Foing, B.; Kaiser, A.; Moutou, C.; Parihar, P. S.; Silva-Valio, A.; Weiss, W. W.

    2009-01-01

    Context: The space experiment CoRoT has recently detected transits by a hot Jupiter across the disc of an active G7V star (CoRoT-Exo-2a) that can be considered as a good proxy for the Sun at an age of approximately 0.5 Gyr. Aims: We present a spot modelling of the optical variability of the star during 142 days of uninterrupted observations performed by CoRoT with unprecedented photometric precision. Methods: We apply spot modelling approaches previously tested in the case of the Sun by modelling total solar irradiance variations, a good proxy for the optical flux variations of the Sun as a star. The best results in terms of mapping of the surface brightness inhomogeneities are obtained by means of maximum entropy regularized models. To model the light curve of CoRoT-Exo-2a, we take into account the photometric effects of both cool spots and solar-like faculae, adopting solar analogy. Results: Two active longitudes initially on opposite hemispheres are found on the photosphere of CoRoT-Exo-2a with a rotation period of 4.522 ± 0.024 days. Their separation changes by ≈80° during the time span of the observations. From this variation, a relative amplitude of the surface differential rotation lower than ~1 percent is estimated. Individual spots form within the active longitudes and show an angular velocity ~1 percent lower than that of the longitude pattern. The total spotted area shows a cyclic oscillation with a period of 28.9 ± 4.3 days, which is close to 10 times the synodic period of the planet as seen by the rotating active longitudes. We discuss the effects of solar-like faculae on our models, finding indications of a facular contribution to the optical flux variations of CoRoT-Exo-2a being significantly smaller than in the present Sun. Conclusions: The implications of such results for the internal rotation of CoRoT-Exo-2a are discussed, based on solar analogy. A possible magnetic star-planet interaction is suggested by the cyclic variation of the spotted

  19. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGES

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; ...

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, ormore » DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  20. K restriction inhibits protein phosphatase 2B (PP2B) and suppression of PP2B decreases ROMK channel activity in the CCD

    PubMed Central

    Zhang, Yan; Lin, Dao-Hong; Wang, Zhi-Jian; Jin, Yan; Yang, Baofeng; Wang, Wen-Hui

    2009-01-01

    We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase (PP)2B but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K depletion did not affect the expression of PP1 and PP2A. Treatment of M-1 cells, mouse cortical collecting duct (CCD) cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of K restriction on PP2B expression and significantly decreased expression of PP2B catalytic subunits. However, GO treatment increased expression of regulatory subunit of PP2B and had no effect on expression of PP1, PP2A, and protein tyrosine phosphatase 1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. Thus superoxide anions or related products may mediate the inhibitory effect of K restriction on the expression of PP2B catalytic subunit. We also used patch-clamp technique to study the effect of inhibiting PP2B on renal outer medullary K (ROMK) channels in the CCD. Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels, and the effect of PP2B inhibitors was abolished by blocking p38 mitogen-activated protein kinase (MAPK) and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or small interfering RNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD. PMID:18184875

  1. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    SciTech Connect

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  2. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  3. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  4. Synthesis and antiviral activity of novel erythrofuranosyl imidazo[1,2-a]pyridine C-nucleosides constructed via palladium coupling of iodoimidazo[1,2-a]pyridines and dihydrofuran.

    PubMed

    Gudmundsson, Kristjan S; Williams, John D; Drach, John C; Townsend, Leroy B

    2003-04-10

    2,5,6-Trichloro-1-(beta-d-ribofuranosyl)benzimidazole (TCRB) and certain analogues have shown significant activity against human cytomegalovirus. The metabolic instability of the glycosidic linkage in TCRB prompted us to synthesize the structurally similar imidazo[1,2-a]pyridine erythrofuranosyl C-nucleosides. As an approach to the synthesis of polychlorinated imidazo[1,2-a]pyridine C-3-erythrofuranosides, a palladium-based methodology for coupling 2,3-dihydrofuran with chlorinated 3-iodoimidazo[1,2-a]pyridines was developed and optimized to give 80-90% yields of 2,6-dichloro- and 2,6,7-trichloro-3-(2,3-dideoxy-2,3-didehydro-d/l-erythrofuranosyl)imidazo[1,2-a]pyridine. Dihydroxylation of these didehydro derivatives with osmium tetroxide or with AD-mix alpha gave a mixture of erythrofuranosyl C-nucleosides that were separated by standard and then chiral chromatography. When screened for anti-HCMV and HSV-1 activity, the alpha-d anomer of 2,6,7-trichloro-3-(erythrofuranosyl)imidazo[1,2-a]pyridine proved to be the most active member of the series, while the beta-anomers all proved to be inactive.

  5. Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

    PubMed

    Tanner, Julie-Anne; Prasad, Bhagwat; Claw, Katrina G; Stapleton, Patricia; Chaudhry, Amarjit; Schuetz, Erin G; Thummel, Kenneth E; Tyndale, Rachel F

    2017-01-01

    Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.

  6. The immunoreceptor tyrosine-based activation motif of Epstein-Barr virus LMP2A is essential for blocking BCR-mediated signal transduction.

    PubMed

    Fruehling, S; Longnecker, R

    1997-09-01

    The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B lymphocytes in vitro. The cytoplasmic amino-terminal domain of LMP2A contains an immunoreceptor tyrosine activation motif (ITAM). ITAMs consist of paired tyrosine and leucine residues and play a central role in signal transduction of the BCR and the T-cell receptor (TCR). To investigate the importance of the LMP2A ITAM, two EBV recombinants were constructed, each containing a tyrosine-to-phenylalanine point mutation at amino acid 74 or 85 within the ITAM of LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were no longer blocked in the LMP2A ITAM mutant LCLs following BCR cross-linking. In addition, the Syk protein tyrosine kinase (PTK) was unable to bind LMP2A in unstimulated LCLs infected with either of the LMP2A ITAM mutants. Analysis of Syk phosphorylation before and after BCR cross-linking in the LMP2A mutant ITAM LCLs compared with wild-type EBV LCLs indicates a specific role of the LMP2A ITAM on the LMP2A-mediated negative effect on the Syk PTK. These data indicate the importance of the LMP2A ITAM motif in the LMP2A-mediated block on BCR signal transduction and position the role of the Syk PTK as being central to the function of LMP2A.

  7. HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

    PubMed Central

    Yu, Nancy; Kakunda, Michael; Pham, Victoria; Lill, Jennie R.; Du, Pan; Wongchenko, Matthew; Yan, Yibing; Firestein, Ron

    2015-01-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study. PMID:25645927

  8. SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A.

    PubMed

    Hung, Man-Hsin; Wang, Cheng-Yi; Chen, Yen-Lin; Chu, Pei-Yi; Hsiao, Yung-Jen; Tai, Wei-Tien; Chao, Ting-Ting; Yu, Hui-Chuan; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-05

    SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227-277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination.

  9. SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A

    PubMed Central

    Hung, Man-Hsin; Wang, Cheng-Yi; Chen, Yen-Lin; Chu, Pei-Yi; Hsiao, Yung-Jen; Tai, Wei-Tien; Chao, Ting-Ting; Yu, Hui-Chuan; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-01

    SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227–277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination. PMID:26575017

  10. Epigenetic Upregulation of Metallothionein 2A by Diallyl Trisulfide Enhances Chemosensitivity of Human Gastric Cancer Cells to Docetaxel Through Attenuating NF-κB Activation

    PubMed Central

    Pan, Yuanming; Lin, Shuye; Xing, Rui; Zhu, Min; Lin, Bonan; Cui, Jiantao; Li, Wenmei; Gao, Jing; Shen, Lin; Zhao, Yuanyuan; Guo, Mingzhou; Wang, Ji Ming

    2016-01-01

    Abstract Aims: Metallothionein 2A (MT2A) and nuclear factor-kappaB (NF-κB) are both involved in carcinogenesis and cancer chemosensitivity. We previously showed decreased expression of MT2A and IκB-α in human gastric cancer (GC) associated with poor prognosis of GC patients. The present study investigated the effect of diallyl trisulfide (DATS), a garlic-derived compound, and docetaxel (DOC) on regulation of MT2A in relation to NF-κB in GC cells. Results: DATS attenuated NF-κB signaling in GC cells, resulting in G2/M cell cycle arrest and apoptosis, culminating in the inhibition of cell proliferation and tumorigenesis in nude mice. The anti-GC effect of DATS was attributable to its capacity to epigenetically upregulate MT2A, which in turn enhanced transcription of IκB-α to suppress NF-κB activation in GC cells. The combination of DATS with DOC exhibited a synergistic anti-GC activity accompanied by MT2A upregulation and NF-κB inactivation. Histopathologic analysis of GC specimens from patients showed a significant increase in MT2A expression following DOC treatment. GC patients with high MT2A expression in tumor specimens showed significantly improved response to chemotherapy and prolonged survival compared with those with low MT2A expression in tumors. Innovation and Conclusion: We conclude that DATS exerts its anti-GC activity and enhances chemosensitivity of GC to DOC by epigenetic upregulation of MT2A to attenuate NF-κB signaling. Our findings delineate a mechanistic basis of MT2A/NF-κB signaling for DATS- and DOC-mediated anti-GC effects, suggesting that MT2A may be a chemosensitivity indicator in GC patients receiving DOC-based treatment and a promising target for more effective treatment of GC by combination of DATS and DOC. Antioxid. Redox Signal. 24, 839–854. PMID:26801633

  11. Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia

    PubMed Central

    Laidlaw, Kamilla M.E.; Berhan, Samuel; Liu, Suhu; Silvestri, Giovannino; Holyoake, Tessa L.; Frank, David A.; Aggarwal, Bharat; Bonner, Michael Y.; Perrotti, Danilo

    2016-01-01

    The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation. PMID:27438151

  12. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

  13. Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.

    PubMed

    Park, Sungshim L; Tiirikainen, Maarit I; Patel, Yesha M; Wilkens, Lynne R; Stram, Daniel O; Le Marchand, Loic; Murphy, Sharon E

    2016-03-01

    Genetic variation in cytochrome P450 2A6 (CYP2A6) gene is the primary contributor to the intraindividual and interindividual differences in nicotine metabolism and has been found to influence smoking intensity. However, no study has evaluated the relationship between CYP2A6 genetic variants and the CYP2A6 activity ratio (total 3-hydroxycotinine/cotinine) and their influence on smoking intensity [total nicotine equivalents (TNE)], across five racial/ethnic groups found to have disparate rates of lung cancer. This study genotyped 10 known functional CYP2A6 genetic or copy number variants in 2115 current smokers from the multiethnic cohort study [African Americans (AA) = 350, Native Hawaiians (NH) = 288, Whites = 413, Latinos (LA) = 437 and Japanese Americans (JA) = 627] to conduct such an investigation. Here, we found that LA had the highest CYP2A6 activity followed by Whites, AA, NH and JA, who had the lowest levels. Adjusting for age, sex, race/ethnicity and body mass index, we found that CYP2A6 diplotypes were predictive of TNE levels, particularly in AA and JA (P trend < 0.0001). However, only in JA did the association remain after accounting for cigarettes per day. Also, it is only in this population that the lower activity ratio supports lower TNE levels, carcinogen exposure and thereby lower risk of lung cancer. Despite the association between nicotine metabolism (CYP2A6 activity phenotype and diplotypes) and smoking intensity (TNE), CYP2A6 levels did not correlate with the higher TNE levels found in AA nor the lower TNE levels found in LA, suggesting that other factors may influence smoking dose in these populations. Therefore, further study in these populations is recommended.

  14. Facile synthesis, structural evaluation, antimicrobial activity and synergistic effects of novel imidazo[1,2-a]pyridine based organoselenium compounds.

    PubMed

    Kumar, Sanjeev; Sharma, Nidhi; Maurya, Indresh K; Bhasin, Aman K K; Wangoo, Nishima; Brandão, Paula; Félix, Vítor; Bhasin, K K; Sharma, Rohit K

    2016-11-10

    A simple and efficient method has been described to synthesize the hitherto unknown imidazo[1,2-a]pyridine selenides (5a-l) by reaction of 2-chloroimidazo [1,2-a]pyridines with aryl/heteroaryl selenols, generated in situ by reduction of various diselenides with hypophosphorous acid. The crystal structures of 3-nitro-2-(phenylselanyl)-imidazo[1,2-a]pyridine (5a), 2-(mesitylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5d) and 3-nitro-2-(pyridin-2-ylselanyl)-imidazo[1,2-a]pyridine (5e) were confirmed by X-ray crystallography and the DFT calculations were performed to determine various structural parameters which were correlated with the X-ray crystal structures. The synthesized compounds were subjected to antimicrobial evaluation and it was found that compounds 5a and 5j were active against gram negative bacterium Escherichia coli whereas compound 5e was active against different fungal strains. Time kill assay was performed to understand the microbial activity of synthesized organoselenium compounds and the toxicity of these compounds was evaluated against human cell lines. Synergistic effects of active compounds 5a and 5e were tested with existing antibiotic drugs which exhibited that the antibiotic combination with synthesized organoselenium compounds efficiently enhanced the antimicrobial activity.

  15. Long non-coding RNA Malat1 promotes neurite outgrowth through activation of ERK/MAPK signalling pathway in N2a cells.

    PubMed

    Chen, Lei; Feng, Peimin; Zhu, Xi; He, Shixu; Duan, Jialan; Zhou, Dong

    2016-11-01

    Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are playing critical roles in neurogenesis, yet the underlying molecular mechanisms remain largely elusive. Neurite outgrowth is an early step in neuronal differentiation and regeneration. Using in vitro differentiation of neuroblastoma-derived Neuro-2a (N2a) cell as a model, we performed expression profiling to identify lncRNAs putatively relevant for neurite outgrowth. We identified that Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was one of the most significantly up-regulated lncRNAs during N2a cell differentiation. Malat1 knockdown resulted in defects in neurite outgrowth as well as enhanced cell death. To pinpoint signalling pathways perturbed by Malat1 depletion, we then performed a reporter-based screening to examine the activities of 50 signalling pathways in Malat1 knockdown cells. We found that Malat1 knockdown resulted in conspicuous inhibition of Mitogen-Activated Protein Kinase (MAPK) signaling pathway as well as abnormal activation of Peroxisome proliferator-activated receptor (PPAR) and P53 signalling pathway. Inhibition of ERK/MAPK pathway with PD98059 potently blocked N2a cell neurite outgrowth, whereas phorbol 12-myristate 13-acetate-induced ERK activation rescued defects in neurite outgrowth and cell death induced by Malat1 depletion. Together, our results established a critical role of Malat1 in the early step of neuronal differentiation through activating ERK/MAPK signalling pathway.

  16. PM2: a partitioning-mining-measuring method for identifying progressive changes in older adults' sleeping activity.

    PubMed

    Lin, Qiang; Zhang, Daqing; Connelly, Kay; Zhou, Xingshe; Ni, Hongbo

    2014-01-01

    As people age, their health typically declines, resulting in difficulty in performing daily activities. Sleep-related problems are common issues with older adults, including shifts in circadian rhythms. A detection method is proposed to identify progressive changes in sleeping activity using a three-step process: partitioning, mining, and measuring. Specifically, the original spatiotemporal representation of each sleeping activity instance was first transformed into a sequence of equal-sized segments, or symbols, via a partitioning process. A data-mining-based algorithm was proposed to find symbols that are not present in all instances of a sleeping activity. Finally, a measuring process was responsible for evaluating the changes in these symbols. Experimental evaluation conducted on a group of datasets of older adults showed that the proposed method is able to identify progressive changes in sleeping activity.

  17. GSK-3β activation mediates Nogo-66-induced inhibition of neurite outgrowth in N2a cells.

    PubMed

    Shen, Jian-ying; Yi, Xu-xia; Xiong, Nan-xiang; Wang, Hai-jun; Duan, Xiao-wei; Zhao, Hong-yang

    2011-11-14

    The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P<0.05). Moreover, we performed RNA interference experiments to knock down GSK-3β expression levels in N2a cells via transient transfection of shRNA plasmids. The inhibition of neurite outgrowth by Nogo-66 was subdued in shRNA cells, compared to the non-RNAi cells (P<0.05). Taken together, these data suggest that GSK-3β is involved in the inhibition by Nogo-66 of neurite outgrowth in N2a cells.

  18. In vitro anti-Giardia lamblia activity of 2-aryl-3-hydroxymethyl imidazo[1,2-a]pyridines and -pyrimidines, individually and in combination with albendazole.

    PubMed

    Velázquez-Olvera, Stephanía; Salgado-Zamora, Héctor; Jiménez-Cardoso, Enedina; Campos-Aldrete, Maria-Elena; Pérez-González, Cuauhtémoc; Ben Hadda, Taibi

    2016-03-01

    Giardiasis is a major diarrheal disease found throughout the world, the causative agent being the flagellate protozoan Giardia intestinalis. Infection is more common in children than in adults. The appearance of drug resistance has complicated the treatment of several parasitic diseases, including giardiasis. Thus, the aim of this investigation was to make an in vitro evaluation of the antigiardia response of synthetic derivatives 2-aryl-3-hydroxymethylimidazo[1,2-a]pyridines 1 and -pyrimidines 2 against trophozoites of Giardia lamblia WB, in comparison with the reference drug, albendazole. Additionally, the synergistic action of albendazole in combination with each of the most active 2-aryl-3-hydroxymethyl imidazo[1,2-a]pyridines and pyrimidines was also assessed. Based on the IC50 values obtained, the best anti-Giardia activity was provided by the 3-hydroxymethyl-4-fluorophenylimidazo[1,2-a]pyrimidine derivative 2c and the corresponding imidazo[1,2-a]pyrimidine with the p-tolyl substituent 2d, followed by 2a and 2b. These four compounds showed effectiveness at a concentration similar to that of albendazole. Regarding synergism, the IC50 of the combination of albendazole with 2a, 2b or 2c gave the best anti-Giardia action, showing greater efficacy than albendazole alone. Hence, G. lamblia WB showed high susceptibility to some 2-aryl-3-hydroxymethyl imidazo[1,2-a] pyrimidines, which acted synergistically when used in combination with albendazole.

  19. Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory

    PubMed Central

    Zhang, Xiao-min; Yan, Xun-yi; Zhang, Bin; Yang, Qian; Ye, Mao; Cao, Wei; Qiang, Wen-bin; Zhu, Li-jun; Du, Yong-lan; Xu, Xing-xing; Wang, Jia-sheng; Xu, Fei; Lu, Wei; Qiu, Shuang; Yang, Wei; Luo, Jian-hong

    2015-01-01

    The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca2+-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation. PMID:26088419

  20. Demethoxycurcumin inhibits energy metabolic and oncogenic signaling pathways through AMPK activation in triple-negative breast cancer cells.

    PubMed

    Shieh, Jiunn-Min; Chen, Yung-Chan; Lin, Ying-Chao; Lin, Jia-Ni; Chen, Wei-Chih; Chen, Yang-Yuan; Ho, Chi-Tang; Way, Tzong-Der

    2013-07-03

    Demethoxycurcumin (DMC), curcumin (Cur), and bisdemethoxycurcumin (BDMC) are major forms of curcuminoids found in the rhizomes of turmeric. This study examined the effects of three curcuminoid analogues on breast cancer cells. The results revealed that DMC demonstrated the most potent cytotoxic effects on breast cancer MDA-MB-231 cells. Compared with estrogen receptor (ER)-positive or HER2-overexpressing breast cancer cells, DMC demonstrated the most efficient cytotoxic effects on triple-negative breast cancer (TNBC) cells. However, nonmalignant MCF-10A cells were unaffected by DMC treatment. The study showed that DMC activated AMPK in TNBC cells. Once activated, AMPK inhibited eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) signaling and mRNA translation via mammalian target of rapamycin (mTOR) and decreased the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). DMC also targeted multiple AMPK downstream pathways. Among these, the dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mTOR inhibition. Moreover, DMC suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation. In addition, DMC also sustained epidermal growth factor receptor (EGFR) activation by suppressing the phosphatases, PP2a and SHP-2. These results suggest that DMC is a potent AMPK activator that acts through a broad spectrum of anti-TNBC activities.

  1. Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients.

    PubMed

    Lucas, C M; Harris, R J; Holcroft, A K; Scott, L J; Carmell, N; McDonald, E; Polydoros, F; Clark, R E

    2015-07-01

    High cancerous inhibitor of PP2A (CIP2A) protein levels at diagnosis of chronic myeloid leukaemia (CML) are predictive of disease progression in imatinib-treated patients. It is not known whether this is true in patients treated with second generation tyrosine kinase inhibitors (2G TKI) from diagnosis, and whether 2G TKIs modulate the CIP2A pathway. Here, we show that patients with high diagnostic CIP2A levels who receive a 2G TKI do not progress, unlike those treated with imatinib (P=<0.0001). 2G TKIs induce more potent suppression of CIP2A and c-Myc than imatinib. The transcription factor E2F1 is elevated in high CIP2A patients and following 1 month of in vivo treatment 2G TKIs suppress E2F1 and reduce CIP2A; these effects are not seen with imatinib. Silencing of CIP2A, c-Myc or E2F1 in K562 cells or CML CD34+ cells reactivates PP2A leading to BCR-ABL suppression. CIP2A increases proliferation and this is only reduced by 2G TKIs. Patients with high CIP2A levels should be offered 2G TKI treatment in preference to imatinib. 2G TKIs disrupt the CIP2A/c-Myc/E2F1 positive feedback loop, leading to lower disease progression risk. The data supports the view that CIP2A inhibits PP2Ac, stabilising E2F1, creating a CIP2A/c-Myc/E2F1 positive feedback loop, which imatinib cannot overcome.

  2. Activation of serotonin 2A receptors underlies the psilocybin-induced effects on α oscillations, N170 visual-evoked potentials, and visual hallucinations.

    PubMed

    Kometer, Michael; Schmidt, André; Jäncke, Lutz; Vollenweider, Franz X

    2013-06-19

    Visual illusions and hallucinations are hallmarks of serotonergic hallucinogen-induced altered states of consciousness. Although the serotonergic hallucinogen psilocybin activates multiple serotonin (5-HT) receptors, recent evidence suggests that activation of 5-HT2A receptors may lead to the formation of visual hallucinations by increasing cortical excitability and altering visual-evoked cortical responses. To address this hypothesis, we assessed the effects of psilocybin (215 μg/kg vs placebo) on both α oscillations that regulate cortical excitability and early visual-evoked P1 and N170 potentials in healthy human subjects. To further disentangle the specific contributions of 5-HT2A receptors, subjects were additionally pretreated with the preferential 5-HT2A receptor antagonist ketanserin (50 mg vs placebo). We found that psilocybin strongly decreased prestimulus parieto-occipital α power values, thus precluding a subsequent stimulus-induced α power decrease. Furthermore, psilocybin strongly decreased N170 potentials associated with the appearance of visual perceptual alterations, including visual hallucinations. All of these effects were blocked by pretreatment with the 5-HT2A antagonist ketanserin, indicating that activation of 5-HT2A receptors by psilocybin profoundly modulates the neurophysiological and phenomenological indices of visual processing. Specifically, activation of 5-HT2A receptors may induce a processing mode in which stimulus-driven cortical excitation is overwhelmed by spontaneous neuronal excitation through the modulation of α oscillations. Furthermore, the observed reduction of N170 visual-evoked potentials may be a key mechanism underlying 5-HT2A receptor-mediated visual hallucinations. This change in N170 potentials may be important not only for psilocybin-induced states but also for understanding acute hallucinatory states seen in psychiatric disorders, such as schizophrenia and Parkinson's disease.

  3. Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines.

    PubMed

    Harris, W; Muñoz, D; Bonner, P L R; Hargreaves, A J

    2009-12-01

    The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca(2+)-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin. The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40-60% of control levels at sub-lethal concentrations (0.1 microM) that were approximately 100-fold lower than their IC(50) values in cytotoxicity assays. Following 24h exposure of N2a cells to 0.3 and 3 microM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 microM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.

  4. Antimicrobial activity of pleurocidin is retained in Plc-2, a C-terminal 12-amino acid fragment.

    PubMed

    Souza, Andre L A; Díaz-Dellavalle, Paola; Cabrera, Andrea; Larrañaga, Patricia; Dalla-Rizza, Marco; De-Simone, Salvatore G

    2013-07-01

    An analysis of a series of five peptides composed of various portions of the pleurocidin (Plc) sequence identified a l2-amino acid fragment from the C-terminus of Plc, designated Plc-2, as the smallest fragment that retained a antimicrobial activity comparable to that of the parent compound. MIC tests in vitro with low-ionic-strength medium showed that Plc-2 has potent activity against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus but not against Enterococcus faecalis. The antifungal activity of the synthetic peptides against phytopathogenic fungi, such as Fusarium oxysporum, Colletotrichum sp., Aspergillus niger and Alternaria sp., also identified Plc-2 as a biologically active peptide. Microscopy studies of fluorescently stained fungi treated with Plc-2 demonstrated that cytoplasmic and nuclear membranes were compromised in all strains of phytopathogenic fungi tested. Together, these results identify Plc-2 as a potential antimicrobial agent with similar properties to its parent compound, pleurocidin. In addition, it demonstrated that the KHVGKAALTHYL residues are critical for the antimicrobial activity described for pleurocidin.

  5. A homology-based model of the human 5-HT2A receptor derived from an in silico activated G-protein coupled receptor

    NASA Astrophysics Data System (ADS)

    Chambers, James J.; Nichols, David E.

    2002-07-01

    A homology-based model of the 5-HT2A receptor was produced utilizing an activated form of the bovine rhodopsin (Rh) crystal structure [1,2]. In silico activation of the Rh structure was accomplished by isomerization of the 11- cis-retinal (1) chromophore, followed by constrained molecular dynamics to relax the resultant high energy structure. The activated form of Rh was then used as a structural template for development of a human 5-HT2A receptor model. Both the 5-HT2A receptor and Rh are members of the G-protein coupled receptor (GPCR) super-family. The resulting homology model of the receptor was then used for docking studies of compounds representing a cross-section of structural classes that activate the 5-HT2A receptor, including ergolines, tryptamines, and amphetamines. The ligand/receptor complexes that ensued were refined and the final binding orientations were observed to be compatible with much of the data acquired through both diversified ligand design and site directed mutagenesis.

  6. Phosphorylation and dephosphorylation of tyrosine 141 regulate stability and degradation of INrf2: a novel mechanism in Nrf2 activation.

    PubMed

    Jain, Abhinav K; Mahajan, Shilpi; Jaiswal, Anil K

    2008-06-20

    INrf2-Nrf2 proteins are sensors of chemical/radiation stress. Nrf2, in response to stresses, is released from INrf2. Nrf2 is translocated into the nucleus where it binds to the antioxidant response element and coordinately activates the expression of a battery of genes that protect cells against oxidative and electrophilic stress. An autoregulatory loop between INrf2 and Nrf2 regulates their cellular abundance. Nrf2 activates INrf2 gene expression, and INrf2 serves as an adapter for degradation of Nrf2. In this report, we demonstrate that mutation of tyrosine 141 in bric-a-bric, tramtrack, broad complex domain to alanine rendered INrf2 unstable and nonfunctional. INrf2Y141A mutant degraded rapidly as compared with wild type INrf2, although it could dimerize and bind Nrf2. De novo synthesized INrf2 protein was phosphorylated at tyrosine 141. Tyrosine 141-phosphorylated INrf2 was highly stable. Treatment with hydrogen peroxide, which is an oxidizing agent, led to dephosphorylation of INrf2Y141, resulting in rapid degradation of INrf2. This resulted in stabilization of Nrf2 and activation of ARE-mediated gene expression. These results demonstrate that stress-induced dephosphorylation of tyrosine 141 is a novel mechanism in Nrf2 activation and cellular protection.

  7. Mechanisms of olfactory toxicity of the herbicide 2,6-dichlorobenzonitrile: Essential roles of CYP2A5 and target-tissue metabolic activation

    SciTech Connect

    Xie Fang; Zhou Xin; Behr, Melissa; Fang Cheng; Horii, Yuichi; Gu Jun; Kannan, Kurunthachalam; Ding Xinxin

    2010-11-15

    The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.

  8. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    SciTech Connect

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; Skaja Robinson, Anne

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.

  9. Abscisic Acid Promotion of Arbuscular Mycorrhizal Colonization Requires a Component of the PROTEIN PHOSPHATASE 2A Complex1[W][OPEN

    PubMed Central

    Charpentier, Myriam; Sun, Jongho; Wen, Jiangqi; Mysore, Kirankumar S.; Oldroyd, Giles E.D.

    2014-01-01

    Legumes can establish intracellular interactions with symbiotic microbes to enhance their fitness, including the interaction with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root epidermal cells to gain access to the root cortex, and this requires the recognition by the host plant of fungus-made mycorrhizal factors. Genetic dissection has revealed the symbiosis signaling pathway that allows the recognition of AM fungi, but the downstream processes that are required to promote fungal infection are poorly understood. Abscisic acid (ABA) has been shown to promote arbuscule formation in tomato (Solanum lycopersicum). Here, we show that ABA modulates the establishment of the AM symbiosis in Medicago truncatula by promoting fungal colonization at low concentrations and impairing it at high concentrations. We show that the positive regulation of AM colonization via ABA requires a PROTEIN PHOSPHATASE 2A (PP2A) holoenzyme subunit, PP2AB′1. Mutations in PP2AB′1 cause reduced levels of AM colonization that cannot be rescued with permissive ABA application. The action of PP2AB′1 in response to ABA is unlinked to the generation of calcium oscillations, as the pp2aB′1 mutant displays a normal calcium response. This contrasts with the application of high concentrations of ABA that impairs mycorrhizal factor-induced calcium oscillations, suggesting different modes of action of ABA on the AM symbiosis. Our work reveals that ABA functions at multiple levels to regulate the AM symbiosis and that a PP2A phosphatase is required for the ABA promotion of AM colonization. PMID:25293963

  10. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

    Li, Wenliang; Xiong, Qian; Yang, Mingkun; Zheng, Peng; Li, Chongyang; Pei, Jianfeng; Ge, Feng

    2012-01-01

    The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells. PMID:22279540

  11. Biological activity of EDQM CRS for Interferon alfa-2a and Interferon alfa-2b - assessment in two in vitro bioassays.

    PubMed

    Silva, M M C G; Gaines-Das, R E; Jones, C; Robinson, C J

    2007-12-01

    The European Directorate for the Quality of Medicines (EDQM) supplies Chemical Reference Substances (CRS) for Interferon (IFN) alfa-2a (CRS I0320300) and for IFN alfa-2b (CRS I0320301) for specified physicochemical tests. However, no information is provided as to their biological activity. In contrast, the World Health Organization (WHO) provides the 2nd International Standards (IS) for IFN alfa-2a (code 95/650) and for IFN alfa-2b (code 95/566), with activity defined in International Units (IU) for calibration of biological activity of preparations of IFN. We have compared the EDQM CRSs with the WHO ISs in two bioassay systems, one measuring the anti-proliferative activity in the Daudi cell line and the other measuring a reporter gene activation in an A549 cell line. In each of these assay systems, the CRSs gave dose - response relations, which were similar to those for the WHO ISs. Estimates of relative activity for each CRS, in terms of the respective IS, showed specific biological activity for the CRSs of the same order as the nominal specific activity for the ISs. However, the estimates of relative activity were not consistent between the two assays systems, emphasizing the need for calibration within each system, if the CRS were to be used as a working standard for bioassays. For structure-activity studies, both physicochemical and biological activity characterisation are required for the same biopharmaceutical preparation. CRS I0320300 and CRS I0320301 may prove useful as working standards for some bioassay systems.

  12. Synthesis, Antibacterial and Antitubercular Activities of Some 5H-Thiazolo[3,2-a]pyrimidin-5-ones and Sulfonic Acid Derivatives.

    PubMed

    Cai, Dong; Zhang, Zhi-Hua; Chen, Yu; Yan, Xin-Jia; Zou, Liang-Jing; Wang, Ya-Xin; Liu, Xue-Qi

    2015-09-10

    A series of 5H-thiazolo[3,2-a]pyrimidin-5-ones were synthesized by the cyclization reactions of S-alkylated derivatives in concentrated H₂SO₄. Upon treatment of S-alkylated derivatives at different temperatures, intramolecular cyclization to 7-(substituted phenylamino)-5H-thiazolo[3,2-a]pyrimidin-5-ones or sulfonation of cyclized products to sulfonic acid derivatives occurred. The structures of the target compounds were confirmed by IR, ¹H-NMR, (13)C-NMR and HRMS studies. The compounds were evaluated for their preliminary in vitro antibacterial activity against some Gram-positive and Gram-negative bacteria and screened for antitubercular activity against Mycobacterium tuberculosis by the broth dilution assay method. Some compounds showed good antibacterial and antitubercular activities.

  13. Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model

    PubMed Central

    Rangasamy, Sampathkumar; Olfers, Shannon; Gerald, Brittany; Hilbert, Alex; Svejda, Sean; Narayanan, Vinodh

    2016-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model ( Mecp2 A140V “knock-in” mutant) expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN) cultures from mutant ( Mecp2 A140V/y) and wild type ( Mecp2 +/y) male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele) and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele). In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V) allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1) treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2   mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i) reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii) MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype (“phenotypic assay”). PMID:27781091

  14. Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model.

    PubMed

    Rangasamy, Sampathkumar; Olfers, Shannon; Gerald, Brittany; Hilbert, Alex; Svejda, Sean; Narayanan, Vinodh

    2016-01-01

    Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model ( Mecp2 A140V "knock-in" mutant) expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN) cultures from mutant ( Mecp2(A140V/y)) and wild type ( Mecp2(+/y)) male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele) and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele). In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V) allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1) treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2  mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i) reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii) MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype ("phenotypic assay").

  15. Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling.

    PubMed

    Séverin, Sonia; Pollitt, Alice Y; Navarro-Nuñez, Leyre; Nash, Craig A; Mourão-Sá, Diego; Eble, Johannes A; Senis, Yotis A; Watson, Steve P

    2011-02-11

    The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

  16. A mutation in the RET proto-oncogene in Hirschsprung's disease affects the tyrosine kinase activity associated with multiple endocrine neoplasia type 2A and 2B.

    PubMed Central

    Cosma, M P; Panariello, L; Quadro, L; Dathan, N A; Fattoruso, O; Colantuoni, V

    1996-01-01

    We demonstrate that a Hirschsprung (HSCR) mutation in the tyrosine kinase domain of the RET proto-oncogene abolishes in cis the tyrosine-phosphorylation associated with the activating mutation in multiple endocrine neoplasia type 2A (MEN2A) in transiently transfected Cos cells. Yet the double mutant RET2AHS retains the ability to form stable dimers, thus dissociating the dimerization from the phosphorylation potential. Co-transfection experiments with single and double mutants carrying plasmids RET2A and RET2AHS in different ratios drastically reduced the phosphorylation levels of the RET2A protein, suggesting a dominant-negative effect of the HSCR mutation. Also, the phosphorylation associated with the multiple endocrine neoplasia type 2B (MEN2B) allele was affected in experiments with single and double mutants carrying plasmids co-transfected under the same conditions. Finally, analysis of the enzymic activity of MEN2A and MEN2B tumours confirmed the relative levels of tyrosine phosphorylation observed in Cos cells, indicating that this condition, in vivo, may account for the RET transforming potential. PMID:8670046

  17. Synthesis of copper and zinc 2-(pyridin-2-yl)imidazo[1,2-a]pyridine complexes and their potential anticancer activity.

    PubMed

    Dam, Jean; Ismail, Zeenat; Kurebwa, Taurai; Gangat, Nadia; Harmse, Leonie; Marques, Helder M; Lemmerer, Andreas; Bode, Moira L; de Koning, Charles B

    2017-01-27

    A small library of novel copper and zinc imidazo[1,2-a]pyridine complexes have been synthesized. Their structures were confirmed by X-ray diffraction crystallography and a selection of these compounds was tested against five cancer cell lines originating from breast cancer (MCF-7 and MDA-MB-231), leukemia (K562 and HL-60) and colorectal cancer (HT-29). The imidazo[1,2-a]pyridines and their zinc complexes showed poor anticancer activity, while the copper complexes were active against the cancer cell lines with IC50 values comparable to and lower than camptothecin. For example, copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 had an IC50 value lower than 1 μM against the HT-29 cells. Fluorescence microscopy with acridine orange, Hoechst 33342 and ethidium bromide, used in a preliminary investigation to evaluate morphological changes showed that copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 caused both apoptosis, necrosis and paraptosis in the MCF-7 and HL-60 cells. A select group of copper N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amines (26, 27, 29 and 31) induced apoptosis, paraptosis and deformed nuclei in MCF-7 cells.

  18. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium

    PubMed Central

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-01-01

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton’s tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca2+ levels ([Ca2+]i), whereas PP2 prolonged the time required for [Ca2+]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca2+. PMID:26659126

  19. Anti-inflammatory effects of Perilla frutescens in activated human neutrophils through two independent pathways: Src family kinases and Calcium.

    PubMed

    Chen, Chun-Yu; Leu, Yann-Lii; Fang, Yu; Lin, Chwan-Fwu; Kuo, Liang-Mou; Sung, Wei-Che; Tsai, Yung-Fong; Chung, Pei-Jen; Lee, Ming-Chung; Kuo, Yu-Ting; Yang, Hsuan-Wu; Hwang, Tsong-Long

    2015-12-14

    The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton's tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca(2+) levels ([Ca(2+)]i), whereas PP2 prolonged the time required for [Ca(2+)]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca(2+).

  20. Multiple forms of phosphatase from human brain: isolation and partial characterization of affi-gel blue nonbinding phosphatase activities.

    PubMed

    Cheng, L Y; Wang, J Z; Gong, C X; Pei, J J; Zaidi, T; Grundke-Iqbal, I; Iqbal, K

    2001-04-01

    Phosphatases extracted from a human brain were resolved into two main groups, namely affi-gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue binding phosphatases were further separated into four different phosphatase activities, designated P1-P4, and described previously. In the present study we describe the affi-gel blue-nonbinding phosphatases which were separated into seven different phosphatase activities, designated P5-P11 by poly-(L-lysine)-agarose and aminohexyl Sepharose 4B chromatographies. These seven phosphatase activities were active toward nonprotein phosphoester. P7-P11 and to some extent P5 could also dephosphorylate a phosphoprotein. They displayed different enzyme kinetics. On the basis of activity peak, the apparent molecular mass as estimated by Sephadex G-200 column chromatography for P5 was 49 kDa; P6, 32 kDa; P7, 150 kDa; P8, 250 kDa; P9, 165 kDa; P10, 90 kDa and P11, 165 kDa. Immunoblot analysis indicated that P8-P11 may belong to PP2B family, whereas P7 may associate with PP2A. The phosphatases P7-P11 were found to be effective in the dephosphorylation of Alzheimer's disease abnormally hyperphosphorylated tau. The resulting dephosphorylated tau regained its activity in promoting the microtubule assembly, suggesting that P7-P11 might regulate the phosphorylation of tau protein in the brain.

  1. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    PubMed

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  2. Cytotoxic activity and probable apoptotic effect of Sph2, a sphigomyelinase hemolysin from Leptospira interrogans strain Lai.

    PubMed

    Zhang, Yi-Xuan; Geng, Yan; Yang, Jun-Wei; Guo, Xiao-Kui; Zhao, Guo-Ping

    2008-02-29

    Our previous work confirmed that Sph2/LA1029 was a sphigomyelinase-like hemolyisn of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. Characteristics of both hemolytic and cytotoxic activities of Sph2 were reported in this paper. Sph2 was a heat-labile neutral hemolysin and had similar hemolytic behavior as the typical sphingomyelinase C of Staphylococcus aureus upon sheep erythrocytes. The cytotoxic activity of Sph2 was shown in mammalian cells such as BALB/C mouse lymphocytes and macrophages, as well as human L-02 liver cells. Transmission electron microscopic observation showed that the Sph2 treated BALB/C mouse lymphocytes were swollen and ruptured with membrane breakage. They also demonstrated condensed chromatin as a high-density area. Cytoskeleton changes were observed via fluorescence confocal microscope in Sph2 treated BALB/C mouse lymphocytes and macrophages, where both cytokine IL-1beta and IL-6 were induced. In addition, typical apoptotic morphological features were observed in Sph2 treated L-02 cells via transmission electron microscope and the percentage of apoptotic cells did increase after the Sph2 treatment detected by flow cytometry. Therefore, Sph2 was likely an apoptosis-inducing factor of human L-02 liver cells.

  3. Cdk2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors.

    PubMed Central

    Hara, E; Hall, M; Peters, G

    1997-01-01

    The helix-loop-helix (HLH) protein Id2 is thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic-helix-loop-helix (bHLH) transcription factors. Id2 acts by forming heterodimers that are unable to bind to specific (E-box) DNA sequences. Here we show that this activity can be overcome by phosphorylation of a serine residue within a consensus target site for cyclin-dependent kinases (Cdks). In vitro, Id2 can be phosphorylated by either cyclin E-Cdk2 or cyclin A-Cdk2 but not by cyclin D-dependent kinases. Analogous phosphorylation occurs in serum-stimulated human diploid fibroblasts at a time in late G1 consistent with the appearance of active cyclin E-Cdk2. The phosphorylation of Id2 in these cells correlates with the restoration of a distinct E-box-dependent DNA-binding complex, suggesting that the levels of this complex are modulated by both the abundance and phosphorylation status of Id2. These data provide a link between cyclin-dependent kinases and bHLH transcription factors that may be critical for the regulation of cell proliferation and differentiation. PMID:9029153

  4. Anti-viral drug treatment along with immune activator IL-2: a control-based mathematical approach for HIV infection

    NASA Astrophysics Data System (ADS)

    Nath Chatterjee, Amar; Roy, Priti Kumar

    2012-02-01

    Recent development in antiretroviral treatment against HIV can help AIDS patients to fight against HIV. But the question that whether the disease is to be partially or totally eradicated from HIV infected individuals still remains unsolved. Usually, the most effective treatment for the disease is HAART which can only control the disease progression. But as the immune system becomes weak, the patients can not fight against other diseases. Immune cells are activated and proliferated by IL-2 after the identification of antigen. IL-2 production is impaired in HIV positive patients and intermitted administration of immune activator IL-2 together with HAART which is a more effective treatment to fight against the disease. Thus, its expediency is essential and is yet to be explored. In this article we anticipated a mathematical model of the effect of IL-2 together with RTIs therapy in HIV positive patients. Our analytical as well as numerical study shows that the optimal schedule of treatment for best result is to be obtained by systematic drug therapy. But at the last stage of treatment, the infection level raises again due to minimisation of drug dosage. Thus we study the perfect adherence of the drugs and found out if RTIs are taken with sufficient interval then for fixed interval of IL-2 therapy, certain amount of drug dosages may be able to sustain the immune system at pre-infection stage and the infected CD4+T cells are going towards extinction.

  5. Hyperthermia-induced seizures alter adenosine A1 and A2A receptors and 5'-nucleotidase activity in rat cerebral cortex.

    PubMed

    León-Navarro, David Agustín; Albasanz, José L; Martín, Mairena

    2015-08-01

    Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia-induced seizures on adenosine A1 and A2A receptors and 5'-nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real-time PCR, and 5'-nucleotidase activity assays. Hyperthermic seizures were induced in 13-day-old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A1 receptor density was observed using [(3) H]DPCPX as radioligand, and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density was detected, using [(3) H]ZM241385 as radioligand, 48 h after hyperthermia-evoked convulsions. These short-term changes in A1 and A2A receptors were also accompanied by a loss of 5'-nucleotidase activity. No significant variations either in A1 or A2A receptor density or 5'-nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A1 and A2A receptors and loss of 5'-nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures. Febrile seizure is one of the most common convulsive disorders in children. The consequences of hyperthermia-induced seizures (animal model of febrile seizures) on adenosine A1 and A2A receptors and 5'-nucleotidase activity have been studied at different periods in cerebral cortical area. A significant increase in A1 receptor density and mRNA coding A1 was observed 48 h after hyperthermia-induced seizures. In contrast, a significant decrease in A2A receptor density and 5'-nucleotidase activity was detected 48 h after convulsions evoked by hyperthermia

  6. Design of narrow-gap TiO2: a passivated codoping approach for enhanced photoelectrochemical activity.

    PubMed

    Gai, Yanqin; Li, Jingbo; Li, Shu-Shen; Xia, Jian-Bai; Wei, Su-Huai

    2009-01-23

    To improve the photoelectrochemical activity of TiO2 for hydrogen production through water splitting, the band edges of TiO2 should be tailored to match with visible light absorption and the hydrogen or oxygen production levels. By analyzing the band structure of TiO2 and the chemical potentials of the dopants, we propose that the band edges of TiO2 can be modified by passivated codopants such as (Mo+C) to shift the valence band edge up significantly, while leaving the conduction band edge almost unchanged, thus satisfying the stringent requirements. The design principle for the band-edge modification should be applicable to other wide-band-gap semiconductors.

  7. Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2.

    PubMed

    Boucabeille, C; Mengin-Lecreulx, D; Henckes, G; Simonet, J M; van Heijenoort, J

    1997-08-15

    Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

  8. Discovery, characterization and in vivo activity of pyocin SD2, a protein antibiotic from Pseudomonas aeruginosa

    PubMed Central

    McCaughey, Laura C.; Josts, Inokentijs; Grinter, Rhys; White, Paul; Byron, Olwyn; Tucker, Nicholas P.; Matthews, Jacqueline M.; Kleanthous, Colin; Whitchurch, Cynthia B.; Walker, Daniel

    2016-01-01

    Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to antibiotic development are urgently required. Pyocins, produced by P. aeruginosa for intraspecies competition, are highly potent protein antibiotics known to actively translocate across the outer membrane of P. aeruginosa. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill P. aeruginosa is a promising approach to antibiotic development. In this work we show the therapeutic potential of a newly identified tRNase pyocin, pyocin SD2, by demonstrating its activity in vivo in a murine model of P. aeruginosa lung infection. In addition, we propose a mechanism of cell targeting and translocation for pyocin SD2 across the P. aeruginosa outer membrane. Pyocin SD2 is concentrated at the cell surface, via binding to the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide (LPS), from where it can efficiently locate its outer membrane receptor FpvAI. This strategy of utilizing both the CPA and a protein receptor for cell targeting is common among pyocins as we show that pyocins S2, S5 and SD3 also bind to the CPA. Additional data indicate a key role for an unstructured N-terminal region of pyocin SD2 in the subsequent translocation of the pyocin into the cell. These results greatly improve our understanding of how pyocins target and translocate across the outer membrane of P. aeruginosa. This knowledge could be useful for the development of novel anti-pseudomonal therapeutics and will also support the development of pyocin SD2 as a therapeutic in its own right. PMID:27252387

  9. Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

    PubMed

    Prasopthum, Aruna; Pouyfung, Phisit; Sarapusit, Songklod; Srisook, Ekaruth; Rongnoparut, Pornpimol

    2015-04-01

    The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

  10. The effects of copper doping on photocatalytic activity at (101) planes of anatase TiO2: A theoretical study

    NASA Astrophysics Data System (ADS)

    Assadi, M. Hussein N.; Hanaor, Dorian A. H.

    2016-11-01

    Copper dopants are varyingly reported to enhance photocatalytic activity at titanium dioxide surfaces through uncertain mechanisms. In order to interpret how copper doping might alter the performance of titanium dioxide photocatalysts in aqueous media we applied density functional theory methods to simulate surface units of doped anatase (101) planes. By including van der Waals interactions, we consider the energetics of adsorbed water at anatase surfaces in pristine and copper doped systems. Simulation results indicate that copper dopant at anatase (101) surfaces is most stable in a 2+ oxidation state and a disperse configuration, suggesting the formation of secondary CuO phases is energetically unfavourable. In agreement with previous reports, water at the studied surface is predicted to exhibit molecular adsorption with this tendency slightly enhanced by copper. Results imply that the enhancement of photoactivity at anatase surfaces through Cu doping is more likely to arise from electronic interactions mediated by charge transfer and inter-bandgap states increasing photoexcitation and extending surface-hole lifetimes rather than through the increased density of adsorbed hydroxyl groups.

  11. Activation of serotonin2A receptors in the medial septum-diagonal band of Broca complex enhanced working memory in the hemiparkinsonian rats.

    PubMed

    Li, Li-Bo; Zhang, Li; Sun, Yi-Na; Han, Ling-Na; Wu, Zhong-Heng; Zhang, Qiao-Jun; Liu, Jian

    2015-04-01

    Serotonin2A (5-HT2A) receptors are highly expressed in the medial septum-diagonal band of Broca complex (MS-DB), especially in parvalbumin (PV)-positive neurons linked to hippocampal theta rhythm, which is involved in cognition. Cognitive impairments commonly occur in Parkinson's disease. Here we performed behavioral, electrophysiological, neurochemical and immunohistochemical studies in rats with complete unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) to assess the importance of dopamine (DA) depletion and MS-DB 5-HT2A receptors for working memory. The MFB lesions resulted in working memory impairment and decreases in firing rate and density of MS-DB PV-positive neurons, peak frequency of hippocampal theta rhythm, and DA levels in septohippocampal system and medial prefrontal cortex (mPFC) compared to control rats. Intra-MS-DB injection of high affinity 5-HT2A receptor agonist TCB-2 enhanced working memory, increased firing rate of PV-positive neurons and peak frequency of hippocampal theta rhythm, elevated DA levels in the hippocampus and mPFC, and decreased 5-HT level in the hippocampus in control and lesioned rats. Compared to control rats, the duration of the excitatory effect produced by TCB-2 on the firing rate of PV-positive neurons was markedly shortened in lesioned rats, indicating dysfunction of 5-HT2A receptors. These findings suggest that unilateral lesions of the MFB in rats induced working memory deficit, and activation of MS-DB 5-HT2A receptors enhanced working memory, which may be due to changes in the activity of septohippocampal network and monoamine levels in the hippocampus and mPFC.

  12. Effect of GABAergic ligands on the anxiolytic-like activity of DOI (a 5-HT(2A/2C) agonist) in the four-plate test in mice.

    PubMed

    Massé, Fabienne; Hascoët, Martine; Bourin, Michel

    2007-01-01

    5-HTergic and GABAergic systems are involved in neurobiology of anxiety. Precedent studies have demonstrated that SSRIs possessed an anxiolytic-like effect in the four-plate test (FPT) at doses that did not modify spontaneous locomotor activity. This effect seems to be mediated through the activation of 5-HT(2A) postsynaptic receptors. The purpose of the present study was to examine the implication of GABA system in the anxiolytic-like activity of DOI in the FPT. To achieve this, the co-administration of DOI (5-HT(2A/2C) receptor agonists) with GABA(A) and GABA(B) receptor ligands was evaluated in the FPT. Alprazolam, diazepam and muscimol (for higher dose) potentiated the anxiolytic-like effect of DOI. Bicuculline, picrotoxin and baclofen inhibited the anxiolytic-like effect of DOI. Flumazenil and CGP 35348 had no effect on the anxiolytic-like activity of DOI. These results suggest that the GABA system seems to be strongly implicated in the anxiolytic-like activity of DOI in the FPT.

  13. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

    PubMed

    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  14. Chlorinated Biphenyl Quinones and Phenyl-2,5-benzoquinone Differentially Modify the Catalytic Activity of Human Hydroxysteroid Sulfotransferase hSULT2A1

    PubMed Central

    Qin, Xiaoyan; Lehmler, Hans-Joachim; Teesch, Lynn M.; Robertson, Larry W.; Duffel, Michael W.

    2013-01-01

    Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2′-chlorophenyl-2,5-benzoquinone (2′-Cl-BQ), 4′-chlorophenyl-2,5-benzoquinone (4′-Cl-BQ), 4′-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4′-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1. PMID:24059442

  15. Prostaglandin F2a activates stress response signaling and induces expression of activating transcription factor 3 (ATF3) in bovine large luteal cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pulsatile uterine secretion of prostaglandin F2 alpha (PGF) triggers the regression of the corpus luteum (CL). Recent studies have explored global changes in gene expression in response to PGF that may contribute to structural and functional regression of the CL. Activating transcription facto...

  16. Design, synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives.

    PubMed

    Guillon, Jean; Cohen, Anita; Gueddouda, Nassima Meriem; Das, Rabindra Nath; Moreau, Stéphane; Ronga, Luisa; Savrimoutou, Solène; Basmaciyan, Louise; Monnier, Alix; Monget, Myriam; Rubio, Sandra; Garnerin, Timothée; Azas, Nadine; Mergny, Jean-Louis; Mullié, Catherine; Sonnet, Pascal

    2017-12-01

    Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results showed good antimalarial activity with IC50 in the μM range. In attempting to investigate the large broad-spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotoxicity of these molecules was assessed on the human HepG2 cell line. Structure-activity relationships of these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric G-quadruplexes through a FRET melting assay by our new compounds.

  17. Effects of serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibition plus 5-HT(2A) receptor antagonism on the firing activity of norepinephrine neurons.

    PubMed

    Szabo, Steven T; Blier, Pierre

    2002-09-01

    YM992 [(S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride] is a selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor (SSRI) and a potent 5-HT(2A) antagonist. The aim of the present study was to assess, using in vivo extracellular unitary recordings, the effect of acute and sustained administration of YM992 (40 mg kg(-1) day(-1) s.c., using osmotic minipumps) on the spontaneous firing activity of locus coeruleus (LC) norepinephrine (NE) neurons. Acute intravenous injection of YM992 (4 mg kg(-1)) significantly decreased NE neuron firing activity by 29% and blocked the inhibitory effect of a subsequent injection of the 5-HT(2) agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride]. A 2-day treatment with YM992 decreased the firing rate of NE neurons by 66%, whereas a partial recovery was observed after a 7-day treatment and a complete one after a 21-day treatment. Following the injection of the alpha(2)-adrenoceptor antagonist idazoxan (1 mg kg(-1) i.v.), NE neuron firing was equalized in controls and 2-day YM992-treated rats. This put into evidence an increased degree of activation of alpha(2)-adrenergic autoreceptors in the treated rats. The suppressant effect of the alpha(2)-adrenoceptor agonist clonidine was significantly decreased in long-term YM992-treated rats. The recovery of LC firing activity after long-term YM992 administration could thus be explained by a decreased sensitivity of alpha(2)-adrenergic autoreceptors. Sustained SSRI administration leads to a gradual reduction of the firing activity of NE neurons during long-term administration, whereas YM992 produced opposite effects. The exact basis for the increased synaptic availability of NE by YM992 remains to be elucidated. This NE activity, resulting from 5-HT reuptake inhibition plus 5-HT(2A) receptor antagonism, might confer additional benefits in affective and anxiety disorders.

  18. Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

    PubMed

    Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2017-02-07

    Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type)-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) and hNphs2.Cre mice to Phb2(fl/fl) mice. The podocyte-specific Phb2 knockout by Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.

  19. Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation.

    PubMed

    Jerónimo-Santos, André; Fonseca-Gomes, João; Guimarães, Diogo Andrade; Tanqueiro, Sara Ramalho; Ramalho, Rita Mira; Ribeiro, Joaquim Alexandre; Sebastião, Ana Maria; Diógenes, Maria José

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A2A receptors (A2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A2AR influence upon BDNF-mediated neuroprotection against Aβ25-35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A2AR activation or inhibition. Moreover neither activation nor inhibition of A2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A2AR.

  20. Activation of 5-HT2a receptors in the basolateral amygdala promotes defeat-induced anxiety and the acquisition of conditioned defeat in Syrian hamsters.

    PubMed

    Clinard, Catherine T; Bader, Lauren R; Sullivan, Molly A; Cooper, Matthew A

    2015-03-01

    Conditioned defeat is a model in Syrian hamsters (Mesocricetus auratus) in which normal territorial aggression is replaced by increased submissive and defensive behavior following acute social defeat. The conditioned defeat response involves both a fear-related memory for a specific opponent as well as anxiety-like behavior indicated by avoidance of novel conspecifics. We have previously shown that systemic injection of a 5-HT2a receptor antagonist reduces the acquisition of conditioned defeat. Because neural activity in the basolateral amygdala (BLA) is critical for the acquisition of conditioned defeat and BLA 5-HT2a receptors can modulate anxiety but have a limited effect on emotional memories, we investigated whether 5-HT2a receptor modulation alters defeat-induced anxiety but not defeat-related memories. We injected the 5-HT2a receptor antagonist MDL 11,939 (0 mM, 1.7 mM or 17 mM) or the 5-HT2a receptor agonist TCB-2 (0 mM, 8 mM or 80 mM) into the BLA prior to social defeat. We found that injection of MDL 11,939 into the BLA impaired acquisition of the conditioned defeat response and blocked defeat-induced anxiety in the open field, but did not significantly impair avoidance of former opponents in the Y-maze. Furthermore, we found that injection of TCB-2 into the BLA increased the acquisition of conditioned defeat and increased anxiety-like behavior in the open field, but did not alter avoidance of former opponents. Our data suggest that 5-HT2a receptor signaling in the BLA is both necessary and sufficient for the development of conditioned defeat, likely via modulation of defeat-induced anxiety.

  1. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

    PubMed Central

    Wong, Po Yee; Ma, Hoi Tang; Lee, Hyun-jung; Poon, Randy Y. C.

    2016-01-01

    The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation. PMID:26923777

  2. Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells

    PubMed Central

    Sidhar, Himakshi; Giri, Ranjit K.

    2017-01-01

    Brain expressed X-linked (Bex) genes are newer group of pro-apoptotic genes. Role of any Bex gene in neuroblastoma and Bex4 and Bex6 in any cancer is completely unknown. Re-expression of all endogenous Bex genes by any nutraceutical is also unknown. Therefore, we investigated the induction of all endogenous Bex genes and associated mechanisms by curcumin using N2a, an aggressive neuroblastoma cell line. Curcumin induced all endogenous Bex genes prior to apoptosis in N2a cells in a dose- and time-dependent manner. Wortmannin (PI-3Kinases inhibitor), SP600125 (JNK inhibitor) and pifithrin-α (p53 inhibitor) abrogated curcumin-mediated induction of Bex genes. Inhibition of curcumin-mediated induction of Bex genes by pifithrin-α also inhibited N2a cells apoptosis suggesting, a direct role of Bex genes in N2a cells apoptosis and involvement of p53 in Bex genes induction. Curcumin treatment activated p53 through hyperphosphorylation at serine 15 before Bex genes induction indicating Bex genes are novel downstream targets of p53. Collectively, curcumin, a safe nutraceutical has the potential to induce all endogenous Bex genes to harness their anti-cancer properties in neuroblastoma cells. Re-expression of Bex genes by curcumin acts as tumor suppressors and may provide alternate strategy to treat neuroblastomas and other cancers with silenced Bex genes. PMID:28145533

  3. Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells.

    PubMed

    Sidhar, Himakshi; Giri, Ranjit K

    2017-02-01

    Brain expressed X-linked (Bex) genes are newer group of pro-apoptotic genes. Role of any Bex gene in neuroblastoma and Bex4 and Bex6 in any cancer is completely unknown. Re-expression of all endogenous Bex genes by any nutraceutical is also unknown. Therefore, we investigated the induction of all endogenous Bex genes and associated mechanisms by curcumin using N2a, an aggressive neuroblastoma cell line. Curcumin induced all endogenous Bex genes prior to apoptosis in N2a cells in a dose- and time-dependent manner. Wortmannin (PI-3Kinases inhibitor), SP600125 (JNK inhibitor) and pifithrin-α (p53 inhibitor) abrogated curcumin-mediated induction of Bex genes. Inhibition of curcumin-mediated induction of Bex genes by pifithrin-α also inhibited N2a cells apoptosis suggesting, a direct role of Bex genes in N2a cells apoptosis and involvement of p53 in Bex genes induction. Curcumin treatment activated p53 through hyperphosphorylation at serine 15 before Bex genes induction indicating Bex genes are novel downstream targets of p53. Collectively, curcumin, a safe nutraceutical has the potential to induce all endogenous Bex genes to harness their anti-cancer properties in neuroblastoma cells. Re-expression of Bex genes by curcumin acts as tumor suppressors and may provide alternate strategy to treat neuroblastomas and other cancers with silenced Bex genes.

  4. Cerebral metabolic responses to 5-HT2A/C receptor activation in mice with genetically modified serotonin transporter (SERT) expression.

    PubMed

    Dawson, Neil; Ferrington, Linda; Lesch, Klaus-Peter; Kelly, Paul A T

    2011-01-01

    Variation in the human serotonin transporter gene (hSERT; 5-HTT) resulting in a life-long alteration in SERT function influences anxiety and the risk of developing affective disorders. The mechanisms underlying the influence of the hSERT gene on these phenotypes remain unclear but may involve altered 5-HT receptor function. Here we characterise the cerebral metabolic response to 5-HT(2A/C) receptor activation in two transgenic mouse models of altered SERT function, SERT knock-out (SERT KO) and hSERT over-expressing (hSERT OE) mice, to test the hypothesis that genetically mediated variability in SERT expression alters 5-HT(2A/C) function. We found that a constitutive increase in SERT expression (hSERT OE) enhanced, whereas a constitutive decrease in SERT expression (SERT KO) attenuated, 5-HT(2A/C) function. Therefore, altered 5-HT(2A/C) receptor functioning in response to hSERT gene variation may contribute to its influence on affective phenotypes.

  5. Influence of S100A6 on CacyBP/SIP Phosphorylation and Elk-1 Transcriptional Activity in Neuroblastoma NB2a Cells.

    PubMed

    Wasik, Urszula; Kadziolka, Beata; Kilanczyk, Ewa; Filipek, Anna

    2016-01-01

    In this work, we have found that casein kinase II (CKII) phosphorylates the CacyBP/SIP protein under in vitro conditions and have mapped the phosphorylation site to threonine 184. Moreover, we present evidence that S100A6, a CacyBP/SIP interacting protein, inhibits this phosphorylation in the presence of Ca(2+). CacyBP/SIP phosphorylation by CKII was also observed in neuroblastoma NB2a cells. Interestingly, we have found that the effect of DRB, a CKII inhibitor, on CacyBP/SIP phosphorylation state is similar to that of S100A6 overexpression. Phosphorylation at threonine 184 seems to have an effect on CacyBP/SIP phosphatase activity since the T184E phosphorylation mimic mutant overexpressed in NB2a cells has lower phosphatase activity toward p-ERK1/2 when compared to the non-phosphorylable T184A mutant or to the wild-type protein. In conclusion, our data suggest that S100A6 and Ca(2+), through inhibiting CacyBP/SIP phosphorylation on threonine 184, are important regulators of CacyBP/SIP phosphatase activity and of ERK1/2-Elk-1 signaling pathway.

  6. CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a,b−/− mice favoring autoimmune cholangitis

    PubMed Central

    Concepcion, Axel R.; Salas, January T.; Sáez, Elena; Sarvide, Sarai; Ferrer, Alex; Portu, Ainhoa; Uriarte, Iker; Hervás-Stubbs, Sandra; Oude Elferink, Ronald P.J.; Prieto, Jesús; Medina, Juan F.

    2015-01-01

    Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl−/HCO3− anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b−/− mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b−/− mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8+ T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2′-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8+ T cells from aged knockouts. Conclusion: Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis. PMID:26396175

  7. The nucleotide-binding domains of sulfonylurea receptor 2A and 2B play different functional roles in nicorandil-induced activation of ATP-sensitive K+ channels.

    PubMed

    Yamada, Mitsuhiko; Kurachi, Yoshihisa

    2004-05-01

    Nicorandil activates ATP-sensitive K(+) channels composed of Kir6.2 and either sulfonylurea receptor (SUR) 2A or 2B. Although SUR2A and SUR2B differ only in their C-terminal 42 amino acids (C42) and possess identical drug receptors and nucleotide-binding domains (NBDs), nicorandil more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Here, we analyzed the roles of NBDs in these channels' response to nicorandil with the inside-out configuration of the patch-clamp method. Binding and hydrolysis of nucleotides by NBDs were impaired by mutations in the Walker A motif of NBD1 (K708A) and NBD2 (K1349A) and in the Walker B motif of NBD2 (D1470N). Experiments were done with internal ATP (1 mM). In SUR2A/Kir6.2 channels, the K708A mutation abolished, and the K1349A but not D1470N mutation reduced the sensitivity to nicorandil. ADP (100 microM) significantly increased the wild-type channels' sensitivity to nicorandil, which was abolished by the K1349A or D1470N mutation. Thus, the SUR2A/Kir6.2 channels' response to nicorandil critically depends on ATP-NBD1 interaction and is facilitated by interactions of ATP or ADP with NBD2. In SUR2B/Kir6.2 channels, either the K708A or K1349A mutation partially suppressed the response to nicorandil, and double mutations abolished it. The D1470N mutation also significantly impaired the response. ADP did not sensitize the channels. Thus, NBD2 hydrolyzes ATP, and NBD1 and NBD2 equally contribute to the response by interacting with ATP and ADP, accounting for the higher nicorandil sensitivity of SUR2B/Kir6.2 than SUR2A/Kir6.2 channels in the presence of ATP alone. Thus, C42 modulates the interaction of both NBDs with intracellular nucleotides.

  8. Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings.

    PubMed Central

    Correia-de-Sá, P.; Ribeiro, J. A.

    1994-01-01

    1. The effec