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Sample records for 2a pp2a activity

  1. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  2. Inhibitor-1 and -2 of PP2A have preference between PP2A complexes.

    PubMed

    Hino, Hirotsugu; Takaki, Kaori; Mochida, Satoru

    2015-11-13

    Protein phosphatase 2A (PP2A) forms tens of kinds of complexes with different substrate specificity and functions by using various regulatory B subunits. But how these complexes' activities are regulated separately is not well understood. Here we showed unequal enzyme inhibition of each form by two proteinous PP2A inhibitors, I1(PP2A) and I2(PP2A). Immunoprecipitation assay using Xenopus egg extract showed that I1(PP2A) bound B″/PR48, and I2(PP2A) bound B56γ and B″/PR48 among four B subunits analyzed. Thus I1(PP2A) and I2(PP2A) seem to have B-subunit specificity. These results support the hypothesis that PP2A complexes containing common catalytic subunit are individually regulated for their separate functions in vivo. PMID:26449453

  3. 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

    2016-01-01

    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. PMID:27611305

  4. A subset of RAB proteins modulates PP2A phosphatase activity

    PubMed Central

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

    2016-01-01

    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. PMID:27611305

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

    PubMed Central

    Castermans, Dries; Somers, Ils; Kriel, Johan; Louwet, Wendy; Wera, Stefaan; Versele, Matthias; Janssens, Veerle; Thevelein, Johan M

    2012-01-01

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

  6. Inhibition of cdc2 activation by INH/PP2A.

    PubMed Central

    Lee, T H; Turck, C; Kirschner, M W

    1994-01-01

    INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis. Images PMID:8049524

  7. Activation of the Tumor Suppressor PP2A Emerges as a Potential Therapeutic Strategy for Treating Prostate Cancer

    PubMed Central

    Cristóbal, Ion; González-Alonso, Paula; Daoud, Lina; Solano, Esther; Torrejón, Blanca; Manso, Rebeca; Madoz-Gúrpide, Juan; Rojo, Federico; García-Foncillas, Jesús

    2015-01-01

    Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients. PMID:26023836

  8. 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. PMID:26906715

  9. 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

  10. Protein Phosphatase 2A (PP2A) Regulatory Subunits ParA and PabA Orchestrate Septation and Conidiation and Are Essential for PP2A Activity in Aspergillus nidulans

    PubMed Central

    Zhong, Guo-wei; Jiang, Ping; Qiao, Wei-ran; Zhang, Yuan-wei; Wei, Wen-fan

    2014-01-01

    Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans. PMID:25280816

  11. Afferent Arteriolar Dilation to 11,12-EET Analogs Involves PP2A Activity and Ca2+-Activated K+ Channels

    PubMed Central

    Imig, John D.; Dimitropoulou, Christiana; Reddy, D. Sudarshan; White, Richard E.; Falck, John R.

    2008-01-01

    The epoxygenase metabolite, 11,12-epoxyeicosatrienoic acid (11,12-EET), has renal vascular actions. 11,12-EET analogs have been developed to determine the structure activity relationship for 11,12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11,12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically stable 11,12-EET analogs: 11,12-EET-N-methylsulfonimide (11,12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), and 11,12-trans-oxidoeicosa-8(Z)-eonoic acid (11,12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11,12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11,12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11,12-EET analogs were abolished by K+ channel or PP2A inhibition. 11,12-EET analogs activated renal myocyte large-conductance KCa channels. 11,12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11,12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11,12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole. PMID:18260004

  12. PP2A: The Wolf in Sheep’s Clothing?

    PubMed Central

    Kiely, Maeve; Kiely, Patrick A.

    2015-01-01

    Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic subunit (C), a structural subunit (A), and a regulatory/variable B-type subunit. PP2A has a critical role to play in homeostasis where its predominant function is as a phosphatase that regulates the major cell signaling pathways in cells. Changes in the assembly, activity and substrate specificity of the PP2A holoenzyme have a direct role in disease and are a major contributor to the maintenance of the transformed phenotype in cancer. We have learned a lot about how PP2A functions from specific mutations that disrupt the core assembly of PP2A and from viral proteins that target PP2A and inhibit its effect as a phosphatase. This prompted various studies revealing that restoration of PP2A activity benefits some cancer patients. However, our understanding of the mechanism of action of this is limited because of the complex nature of PP2A holoenzyme assembly and because it acts through a wide variety of signaling pathways. Information on PP2A is also conflicting as there are situations whereby inactivation of PP2A induces apoptosis in many cancer cells. In this review we discuss this relationship and we also address many of the pertinent and topical questions that relate to novel therapeutic strategies aimed at altering PP2A activity. PMID:25867001

  13. Fcp1 phosphatase controls Greatwall kinase to promote PP2A-B55 activation and mitotic progression

    PubMed Central

    Della Monica, Rosa; Visconti, Roberta; Cervone, Nando; Serpico, Angela Flavia; Grieco, Domenico

    2015-01-01

    During cell division, progression through mitosis is driven by a protein phosphorylation wave. This wave namely depends on an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1 while activities of major protein phosphatases, like PP1 and PP2A, appear directly or indirectly repressed by Cdk1. However, how Cdk1 inactivation is coordinated with reactivation of major phosphatases at mitosis exit still lacks substantial knowledge. We show here that activation of PP2A-B55, a major mitosis exit phosphatase, required the phosphatase Fcp1 downstream Cdk1 inactivation in human cells. During mitosis exit, Fcp1 bound Greatwall (Gwl), a Cdk1-stimulated kinase that phosphorylates Ensa/ARPP19 and converts these proteins into potent PP2A-B55 inhibitors during mitosis onset, and dephosphorylated it at Cdk1 phosphorylation sites. Fcp1-catalyzed dephosphorylation drastically reduced Gwl kinase activity towards Ensa/ARPP19 promoting PP2A-B55 activation. Thus, Fcp1 coordinates Cdk1 and Gwl inactivation to derepress PP2A-B55, generating a dephosphorylation switch that drives mitosis progression. DOI: http://dx.doi.org/10.7554/eLife.10399.001 PMID:26653855

  14. Potential anti-tumor effects of FTY720 associated with PP2A activation: a brief review.

    PubMed

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

    2016-06-01

    FTY720 (Fingolimod, Gilenya (†) ) is an FDA-approved immunosuppressant currently used in the treatment of multiple sclerosis. However, a large number of studies over the last few years have shown that FTY720 shows potent antitumor properties that suggest its potential usefulness as a novel anticancer agent. Interestingly, the restoration of protein phosphatase 2A (PP2A) activity mediated by FTY720 could play a key role in its antitumor effects. Taking into account that PP2A inactivation is a common event that determines poor outcome in several tumor types, FTY720 could serve as an alternative therapeutic strategy for cancer patients with such alterations. PMID:26950691

  15. 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

  16. 67-kDa laminin receptor-dependent protein phosphatase 2A (PP2A) activation elicits melanoma-specific antitumor activity overcoming drug resistance.

    PubMed

    Tsukamoto, Shuntaro; Huang, Yuhui; Umeda, Daisuke; Yamada, Shuhei; Yamashita, Shuya; Kumazoe, Motofumi; Kim, Yoonhee; Murata, Motoki; Yamada, Koji; Tachibana, Hirofumi

    2014-11-21

    The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment. PMID:25294877

  17. Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

    PubMed

    Oaks, Joshua J; Santhanam, Ramasamy; Walker, Christopher J; Roof, Steve; Harb, Jason G; Ferenchak, Greg; Eisfeld, Ann-Kathrin; Van Brocklyn, James R; Briesewitz, Roger; Saddoughi, Sahar A; Nagata, Kyosuke; Bittman, Robert; Caligiuri, Michael A; Abdel-Wahab, Omar; Levine, Ross; Arlinghaus, Ralph B; Quintas-Cardama, Alfonso; Goldman, John M; Apperley, Jane; Reid, Alistair; Milojkovic, Dragana; Ziolo, Mark T; Marcucci, Guido; Ogretmen, Besim; Neviani, Paolo; Perrotti, Danilo

    2013-09-12

    FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs. PMID:23926298

  18. 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

  19. PR65A Phosphorylation Regulates PP2A Complex Signaling

    PubMed Central

    Kotlo, Kumar; Xing, Yongna; Lather, Sonia; Grillon, Jean Michel; Johnson, Keven; Skidgel, Randal A.; Solaro, R. John; Danziger, Robert S.

    2014-01-01

    Serine-threonine Protein phosphatase 2 A (PP2A), a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac); a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa); and one of at least 18 associated variable regulatory proteins (B subunits) classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314). Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A) and non-phosphorylated (N-PR65A) amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A), elongation factor 2, heat shock protein 60 (HSP60), NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure. PMID:24465463

  20. Protein phosphatase 2A (PP2A) has a potential role in CAPE-induced apoptosis of CCRF-CEM cells via effecting human telomerase reverse transcriptase activity.

    PubMed

    Avci, Cigir Biray; Sahin, Fahri; Gunduz, Cumhur; Selvi, Nur; Aydin, Hikmet Hakan; Oktem, Gulperi; Topcuoglu, Nejat; Saydam, Guray

    2007-12-01

    Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC(50) of 1 muM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC(20) of inhibitors and IC(50) of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC(50) of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE. PMID:17852432

  1. 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

  2. T-type Ca2+ signalling regulates aldosterone-induced CREB activation and cell death through PP2A activation in neonatal cardiomyocytes

    PubMed Central

    Ferron, Laurent; Ruchon, Yann; Renaud, Jean-François; Capuano, Véronique

    2011-01-01

    Aims We have investigated Ca2+ signalling generated by aldosterone-induced T-type current (ICaT), the effects of ICaT in neonatal cardiomyocytes, and a putative role for ICaT in cardiomyocytes during cardiac pathology induced by stenosis in an adult rat. Methods and results Neonatal rat cardiomyocytes treated with aldosterone showed an increase in ICaT density, principally due to the upregulation of the T-type channel Cav3.1 (by 80%). Aldosterone activated cAMP-response element-binding protein (CREB), and this activation was enhanced by blocking ICaT or by inhibiting protein phosphatase 2A (PP2A) activity. Aldosterone induced PP2A activity, an induction that was prevented upon ICaT blockade. ICaT exerted a negative feedback regulation on the transcription of the Cav3.1 gene, and the activation of PP2A by ICaT led to increased levels of the pro-apoptotic markers caspase 9 and Bcl-xS and decreased levels of the anti-apoptotic marker Bcl-2. These findings were corroborated by flow cytometry analysis for apoptosis and necrosis. Similarly, in a rat model of cardiac disease, ICaT re-emergence was associated with a decrease in CREB activation and was correlated with increases in caspase 9 and Bcl-xS and a decrease in Bcl-2 levels. Conclusion Our findings establish PP2A/CREB as targets of ICaT-generated Ca2+ signalling and identify an important role for ICaT in cardiomyocyte cell death. PMID:21123217

  3. 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

  4. 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.

  5. 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.

  6. 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. PMID:27551057

  7. Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase

    PubMed Central

    Duran, Xavier; Pachón, Gisela; Vázquez-Carballo, Ana; Roche, Kelly; Núñez-Roa, Catalina; Garrido-Sánchez, Lourdes; Tinahones, Francisco J.; Vendrell, Joan; Fernández-Veledo, Sonia

    2015-01-01

    Objective Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes. Methods ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR. Results ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG. Conclusions ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner. PMID:26068931

  8. Silencing PP2A inhibitor by lenti-shRNA interference ameliorates neuropathologies and memory deficits in tg2576 mice.

    PubMed

    Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Shi, Hai-Rong; Liu, Xing-Hua; Chai, Gao-Shang; Yao, Xiu-Qing; Zhang, Jia-Yu; Peng, Cai-Xia; Hu, Juan; Li, Xia-Chun; Wang, Qun; Wang, Jian-Zhi

    2013-12-01

    Deficits of protein phosphatase-2A (PP2A) play a crucial role in tau hyperphosphorylation, amyloid overproduction, and synaptic suppression of Alzheimer's disease (AD), in which PP2A is inactivated by the endogenously increased inhibitory protein, namely inhibitor-2 of PP2A (I2(PP2A)). Therefore, in vivo silencing I2(PP2A) may rescue PP2A and mitigate AD neurodegeneration. By infusion of lentivirus-shRNA targeting I2(PP2A) (LV-siI2(PP2A)) into hippocampus and frontal cortex of 11-month-old tg2576 mice, we demonstrated that expression of LV-siI2(PP2A) decreased remarkably the elevated I2(PP2A) in both mRNA and protein levels. Simultaneously, the PP2A activity was restored with the mechanisms involving reduction of the inhibitory binding of I2(PP2A) to PP2A catalytic subunit (PP2AC), repression of the inhibitory Leu309-demethylation and elevation of PP2AC. Silencing I2(PP2A) induced a long-lasting attenuation of amyloidogenesis in tg2576 mice with inhibition of amyloid precursor protein hyperphosphorylation and β-secretase activity, whereas simultaneous inhibition of PP2A abolished the antiamyloidogenic effects of I2(PP2A) silencing. Finally, silencing I2(PP2A) could improve learning and memory of tg2576 mice with preservation of several memory-associated components. Our data reveal that targeting I2(PP2A) can efficiently rescue Aβ toxicities and improve the memory deficits in tg2576 mice, suggesting that I2(PP2A) could be a promising target for potential AD therapies. PMID:23922015

  9. 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

  10. Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

    PubMed Central

    2011-01-01

    Background Striatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between

  11. Cardiomyocyte specific deletion of PP2A causes cardiac hypertrophy

    PubMed Central

    Li, Lei; Fang, Chao; Xu, Di; Xu, Yidan; Fu, Heling; Li, Jianmin

    2016-01-01

    Cardiac hypertrophy is a common pathological alteration in heart disease, which has been reported to be connected with serine/threonine protein phosphatases that control the dephosphorylation of a variety of cardiac proteins. Herein, we generated protein phosphatase type 2A knockout expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the a-myosin heavy chain promoter. Cardiac function of mice was determined by echocardiography. Decrease in PP2A activity leads to increased cardiomyocyte hypertrophy and fibrosis. Loss of PP2ACα leads to the heart failure, including the changes of EF, FS, LV, ANP and BNP. On the molecular level, knockout mice shows increased expression of B55a and B56e at 60 days after tamoxifen injection. Additionally, the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in knockout mice. In conclusion, cardiomyocyte specific deletion of PP2A gene causes the cardiac hypertrophy. We will use the knockout mice to generate a type of cardiomyocyte hypertrophy mouse model with myocardial fibrosis. PMID:27186301

  12. Targeting Inhibitors of the Tumor Suppressor PP2A for the Treatment of Pancreatic Cancer

    PubMed Central

    Farrell, Amy S.; Allen-Petersen, Brittany; Daniel, Colin J.; Wang, Xiaoyan; Wang, Zhiping; Rodriguez, Sarah; Impey, Soren; Oddo, Jessica; Vitek, Michael P.; Lopez, Charles; Christensen, Dale J.; Sheppard, Brett; Sears, Rosalie C.

    2014-01-01

    Pancreatic cancer is a deadly disease that is usually diagnosed in the advanced stages when few effective therapies are available. Given the aggressive clinical course of this disease and lack of good treatment options, the development of new therapeutic agents for the treatment of pancreatic cancer is of the upmost importance. Several pathways shown to contribute to pancreatic cancer progression are negatively regulated by the tumor suppressor, protein phosphatase 2A (PP2A). Here, the endogenous inhibitors of PP2A, SET (also known as I2PP2A) and Cancerous Inhibitor of PP2A (CIP2A), were shown to be overexpressed in human pancreatic cancer, contributing to decreased PP2A activity, and overexpression and stabilization of the oncoprotein c-Myc, a key PP2A target. Knockdown of SET or CIP2A increases PP2A activity, increases c-Myc degradation, and decreases the tumorigenic potential of pancreatic cancer cell lines both in vitro and in vivo. Moreover, treatment with a novel SET inhibitor, OP449, pharmacologically recapitulates the phenotypes and significantly reduces proliferation and tumorigenic potential of several pancreatic cancer cell lines, with an accompanying attenuation of cell growth and survival signaling. Furthermore, primary cells from pancreatic cancer patients were sensitive to OP449 treatment, indicating that PP2A regulated pathways are highly relevant to this deadly disease. PMID:24667985

  13. A novel PP2A enhancer induces caspase-independent apoptosis of MKN28 gastric cancer cells with high MEK activity.

    PubMed

    Tsuchiya, Ayako; Kanno, Takeshi; Shimizu, Tadashi; Nakao, Syuhei; Tanaka, Akito; Tabata, Chiharu; Nakano, Takashi; Nishizaki, Tomoyuki

    2014-05-28

    The newly synthesized phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-PI) significantly enhanced protein phosphatase 2A (PP2A) activity in the cell-free assay. This prompted to assess the antitumor effect of diDCP-LA-PI. diDCP-LA-PI attenuated phosphorylation of mitogen-activated protein kinase (MAPK) kinase (MEK) in Lu65 human lung cancer and MKN28 human gastric cancer cells with high MEK activity. diDCP-LA-PI reduced cell viability in Lu65 and MKN28 cells, but otherwise such effect was not found in 786-O human renal cancer and HUH-7 human hepatoma cells with relatively low MEK activity. For Lu65 and MKN28 cells diDCP-LA-PI increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells, but no significant activation of caspase-3, -8, or -9 was obtained. For MKN28 cells diDCP-LA-PI-induced reduction of MEK phosphorylation and cell viability was prevented by knocking-down PP2Ac. Taken together, these results indicate that diDCP-LA-PI induces caspase-independent apoptosis of Lu65 and MKN28 human cancer cells, for the latter cells by suppressing MEK activity through PP2A-catalyzed dephosphorylation. PMID:24508028

  14. Rapamycin ameliorates cadmium-induced activation of MAPK pathway and neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A.

    PubMed

    Xu, Chong; Wang, Xiaoxue; Zhu, Yu; Dong, Xiaoqing; Liu, Chunxiao; Zhang, Hai; Liu, Lei; Huang, Shile; Chen, Long

    2016-06-01

    Cadmium (Cd) is a highly toxic metal that affects the central nervous system. Recently we have demonstrated that inhibition of mTOR by rapamycin rescues neuronal cells from Cd-poisoning. Here we show that rapamycin inhibited Cd-induced mitochondrial ROS-dependent neuronal apoptosis. Intriguingly, rapamycin remarkably blocked phosphorylation of JNK, Erk1/2 and p38 in neuronal cells induced by Cd, which was strengthened by co-treatment with Mito-TEMPO. Inhibition of JNK and Erk1/2 by SP600125 and U0126, respectively, potentiated rapamycin's prevention from Cd-induced apoptosis. Consistently, over-expression of dominant negative c-Jun or MKK1 also potently improved the inhibitory effect of rapamycin on Cd neurotoxicity. Furthermore, pretreatment with SP600125 or U0126, or expression of dominant negative c-Jun or MKK1 enhanced the inhibitory effects of rapamycin or Mito-TEMPO on Cd-induced ROS. Further investigation found that co-treatment with Mito-TEMPO/rapamycin more effectively rescued cells by preventing Cd inactivation of PP2A than treatment with rapamycin or Mito-TEMPO alone. Over-expression of wild-type PP2A reinforced rapamycin or Mito-TEMPO suppression of activated JNK and Erk1/2 pathways, as well as ROS production and apoptosis in neuronal cells in response to Cd. The findings indicate that rapamycin ameliorates Cd-evoked neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, thereby suppressing activation of JNK and Erk1/2 pathways. Our results underline that rapamycin may have a potential in preventing Cd-induced oxidative stress and neurodegenerative diseases. PMID:26805420

  15. Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

    PubMed Central

    McMahon, K A; Wilson, N J; Marks, D C; Beecroft, T L; Whitty, G A; Hamilton, J A; Csar, X F

    2001-01-01

    M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells. PMID:11513742

  16. Neuroprotective effects of donepezil against Aβ42-induced neuronal toxicity are mediated through not only enhancing PP2A activity but also regulating GSK-3β and nAChRs activity.

    PubMed

    Noh, Min-Young; Koh, Seong H; Kim, Sung-Min; Maurice, Tangui; Ku, Sae-Kwang; Kim, Seung H

    2013-11-01

    The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase-3β (GSK-3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid-beta (Aβ)42-induced neuronal toxicity model of Alzheimer's disease. In Aβ42-induced toxic conditions, each PP2A and GSK-3β activity measured at different times showed time-dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre-treatment showed dose-dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK-3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity would partially contribute to its effects. We investigated neuroprotective mechanisms of donepezil against Aβ42 toxicity: Donepezil increased neuronal viability with reduced p-tau by enhancing PP2A activity. Despite of blocked PP2A activity, donepezil showed additional recovering effect on neuronal viability, which findings led us to assume that additional

  17. Involvement of I2PP2A in the abnormal hyperphosphorylation of tau and its reversal by Memantine.

    PubMed

    Chohan, Muhammad Omar; Khatoon, Sabiha; Iqbal, Inge-Grundke; Iqbal, Khalid

    2006-07-10

    The activity of protein phosphatase (PP)-2A, which regulates tau phosphorylation, is compromised in Alzheimer disease brain. Here we show that the transient transfection of PC12 cells with inhibitor-2 (I2PP2A) of PP2A causes abnormal hyperphosphorylation of tau at Ser396/Ser404 and Ser262/Ser356. This hyperphosphorylation of tau is observed only when a sub-cellular shift of I2PP2A takes place from the nucleus to the cytoplasm and is accompanied by cleavage of I2PP2A into a 20 kDa fragment. Memantine, an un-competitive inhibitor of N-methyl-D-aspartate receptors, inhibits this abnormal phosphorylation of tau and cell death and prevents the I2PP2A-induced inhibition of PP2A activity in vitro. These findings demonstrate novel mechanisms by which I2PP2A regulates the intracellular activity of PP2A and phosphorylation of tau, and by which Memantine modulates PP2A signaling and inhibits neurofibrillary degeneration. PMID:16806196

  18. Low-dose endothelial monocyte-activating polypeptide-II increases permeability of blood-tumor barrier via a PKC-ζ/PP2A-dependent signaling mechanism.

    PubMed

    Li, Zhen; Liu, Yun-Hui; Liu, Xiao-Bai; Xue, Yi-Xue; Wang, Ping; Liu, Li-Bo

    2015-02-15

    Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/β signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function. PMID:25592443

  19. 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. PMID:26062569

  20. Inhibition of PP2A by LIS1 increases HIV-1 gene expression

    PubMed Central

    Epie, Nicolas; Ammosova, Tatyana; Turner, Willie; Nekhai, Sergei

    2006-01-01

    Background Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. Results Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. Conclusion Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A. PMID:17018134

  1. 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 Central

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

    2013-01-01

    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-Thr308 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-Thr308 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-Thr308 phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr308 by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr172 and p70S6K-Thr389, 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. PMID:23788636

  2. 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

  3. PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

    PubMed Central

    2014-01-01

    Background HER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling. Results Long-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L. Conclusions Our data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies. PMID:24958351

  4. Proteome-wide search for PP2A substrates in fission yeast.

    PubMed

    Bernal, Manuel; Zhurinsky, Jacob; Iglesias-Romero, Ana B; Sanchez-Romero, Maria A; Flor-Parra, Ignacio; Tomas-Gallardo, Laura; Perez-Pulido, Antonio J; Jimenez, Juan; Daga, Rafael R

    2014-06-01

    PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates. PMID:24634168

  5. TLR2 ligation induces corticosteroid insensitivity in A549 lung epithelial cells: Anti-inflammatory impact of PP2A activators.

    PubMed

    Rahman, Md Mostafizur; Prabhala, Pavan; Rumzhum, Nowshin N; Patel, Brijeshkumar S; Wickop, Thomas; Hansbro, Philip M; Verrills, Nicole M; Ammit, Alaina J

    2016-09-01

    Corticosteroids are effective anti-inflammatory therapies widely utilized in chronic respiratory diseases. But these medicines can lose their efficacy during respiratory infection resulting in disease exacerbation. Further in vitro research is required to understand how infection worsens lung function control in order to advance therapeutic options to treat infectious exacerbation in the future. In this study, we utilize a cellular model of bacterial exacerbation where we pretreat A549 lung epithelial cells with the synthetic bacterial lipoprotein Pam3CSK4 (a TLR2 ligand) to mimic bacterial infection and tumor necrosis factor α (TNFα) to simulate inflammation. Under these conditions, Pam3CSK4 induces corticosteroid insensitivity; demonstrated by substantially reduced ability of the corticosteroid dexamethasone to repress TNFα-induced interleukin 6 secretion. We then explored the molecular mechanism responsible and found that corticosteroid insensitivity induced by bacterial mimics was not due to altered translocation of the glucocorticoid receptor into the nucleus, nor an impact on the NF-κB pathway. Moreover, Pam3CSK4 did not affect corticosteroid-induced upregulation of anti-inflammatory MAPK deactivating phosphatase-MKP-1. However, Pam3CSK4 can induce oxidative stress and we show that a proportion of the MKP-1 produced in response to corticosteroid in the context of TLR2 ligation was rendered inactive by oxidation. Thus to combat inflammation in the context of bacterial exacerbation we sought to discover effective strategies that bypassed this road-block. We show for the first time that known (FTY720) and novel (theophylline) activators of the phosphatase PP2A can serve as non-steroidal anti-inflammatory alternatives and/or corticosteroid-sparing approaches in respiratory inflammation where corticosteroid insensitivity exists. PMID:27477309

  6. PP2A mediates diosmin p53 activation to block HA22T cell proliferation and tumor growth in xenografted nude mice through PI3K-Akt-MDM2 signaling suppression.

    PubMed

    Dung, Tran Duc; Day, Cecilia Hsuan; Binh, Truong Viet; Lin, Chih-Hsueh; Hsu, Hsi-Hsien; Su, Cheng-Chuan; Lin, Yueh-Min; Tsai, Fuu-Jen; Kuo, Wei-Wen; Chen, Li-Mien; Huang, Chih-Yang

    2012-05-01

    Hepatocellular carcinoma is a common type of cancer with poor prognosis. This study examines the in vitro and in vivo mechanisms of diosmin on human hepato-cellular carcinoma HA22T cell proliferation inhibition. HA22T cells were treated with different diosmin concentrations and analyzed with Western blot analysis, MTT assay, wound healing, flow cytometry, siRNA transfection assays and co-immuno-precipitation assay. The HA22T-implanted xeno-graft nude mice model was applied to confirm the cellular effects. Diosmin showed strong HA22T cell viability inhibition in a dose dependent manner and significantly reduced the cell proliferative proteins as well as inducing cell cycle arrest in the G2/M phase through p53 activation and PI3K-Akt-MDM2 signaling pathway inhibition. However, protein phosphatase 2A (PP2A) siRNA or PP2A inhibitor totally reversed the diosmin effects. The HA22T-implanted nude mice model further confirmed that diosmin inhibited HA22T tumor cell growth and down regulated the PI3K-Akt-MDM2 signaling and cell cycle regulating proteins, as well as activating PP2A and p53 proteins. Our findings indicate that HA22T cell proliferation inhibition and tumor growth suppression by diosmin are mediated through PP2A activation. PMID:22289577

  7. 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. PMID:9612228

  8. 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

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

    PubMed

    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

    2016-05-01

    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

  10. Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

    PubMed Central

    Michelson, S; Turowski, P; Picard, L; Goris, J; Landini, M P; Topilko, A; Hemmings, B; Bessia, C; Garcia, A; Virelizier, J L

    1996-01-01

    Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another. PMID:8627658

  11. PMC, a potent hydrophilic α-tocopherol derivative, inhibits NF-κB activation via PP2A but not IκBα-dependent signals in vascular smooth muscle cells

    PubMed Central

    Hsieh, Cheng-Ying; Hsiao, George; Hsu, Ming-Jen; Wang, Yi-Hsuan; Sheu, Joen-Rong

    2014-01-01

    The hydrophilic α-tocopherol derivative, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro-inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)-γ. Treatment of LPS/IFN-γ-stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 in a concentration-dependent manner. A reduction in LPS/IFN-γ-induced nuclear factor (NF)-κB activation was also observed in PMC-treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN-γ-activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase-1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A-selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC-mediated inhibition of iNOS expression, NF-κB-promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN-γ-stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H2O2, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC-mediated inhibition of NF-κB activity in LPS/IFN-γ-stimulated VSMCs occurs through the ROS-PP2A-p65 signalling cascade, an IKK-IκBα-independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory

  12. PRG-1 Regulates Synaptic Plasticity via Intracellular PP2A/β1-Integrin Signaling.

    PubMed

    Liu, Xingfeng; Huai, Jisen; Endle, Heiko; Schlüter, Leslie; Fan, Wei; Li, Yunbo; Richers, Sebastian; Yurugi, Hajime; Rajalingam, Krishnaraj; Ji, Haichao; Cheng, Hong; Rister, Benjamin; Horta, Guilherme; Baumgart, Jan; Berger, Hendrik; Laube, Gregor; Schmitt, Ulrich; Schmeisser, Michael J; Boeckers, Tobias M; Tenzer, Stefan; Vlachos, Andreas; Deller, Thomas; Nitsch, Robert; Vogt, Johannes

    2016-08-01

    Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation. PMID:27453502

  13. The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

    PubMed

    Guenebeaud, Céline; Goldschneider, David; Castets, Marie; Guix, Catherine; Chazot, Guillaume; Delloye-Bourgeois, Céline; Eisenberg-Lerner, Avital; Shohat, Galit; Zhang, Mingjie; Laudet, Vincent; Kimchi, Adi; Bernet, Agnès; Mehlen, Patrick

    2010-12-22

    The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation. PMID:21172653

  14. The tumor suppressor PP2A Abeta regulates the RalA GTPase.

    PubMed

    Sablina, Anna A; Chen, Wen; Arroyo, Jason D; Corral, Laura; Hector, Melissa; Bulmer, Sara E; DeCaprio, James A; Hahn, William C

    2007-06-01

    The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme family that regulates numerous signaling pathways. Biallelic mutations of the structural PP2A Abeta subunit occur in several types of human tumors; however, the functional consequences of these cancer-associated PP2A Abeta mutations in cell transformation remain undefined. Here we show that suppression of PP2A Abeta expression permits immortalized human cells to achieve a tumorigenic state. Cancer-associated Abeta mutants fail to reverse tumorigenic phenotype induced by PP2A Abeta suppression, indicating that these mutants function as null alleles. Wild-type PP2A Abeta but not cancer-derived Abeta mutants form a complex with the small GTPase RalA. PP2A Abeta-containing complexes dephosphorylate RalA at Ser183 and Ser194, inactivating RalA and abolishing its transforming function. These observations identify PP2A Abeta as a tumor suppressor gene that transforms immortalized human cells by regulating the function of RalA. PMID:17540176

  15. A Conserved Motif Provides Binding Specificity to the PP2A-B56 Phosphatase.

    PubMed

    Hertz, Emil Peter Thrane; Kruse, Thomas; Davey, Norman E; López-Méndez, Blanca; Sigurðsson, Jón Otti; Montoya, Guillermo; Olsen, Jesper V; Nilsson, Jakob

    2016-08-18

    Dynamic protein phosphorylation is a fundamental mechanism regulating biological processes in all organisms. Protein phosphatase 2A (PP2A) is the main source of phosphatase activity in the cell, but the molecular details of substrate recognition are unknown. Here, we report that a conserved surface-exposed pocket on PP2A regulatory B56 subunits binds to a consensus sequence on interacting proteins, which we term the LxxIxE motif. The composition of the motif modulates the affinity for B56, which in turn determines the phosphorylation status of associated substrates. Phosphorylation of amino acid residues within the motif increases B56 binding, allowing integration of kinase and phosphatase activity. We identify conserved LxxIxE motifs in essential proteins throughout the eukaryotic domain of life and in human viruses, suggesting that the motifs are required for basic cellular function. Our study provides a molecular description of PP2A binding specificity with broad implications for understanding signaling in eukaryotes. PMID:27453045

  16. 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

  17. PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.

    PubMed

    Martin, Maud; Geudens, Ilse; Bruyr, Jonathan; Potente, Michael; Bleuart, Anouk; Lebrun, Marielle; Simonis, Nicolas; Deroanne, Christophe; Twizere, Jean-Claude; Soubeyran, Philippe; Peixoto, Paul; Mottet, Denis; Janssens, Veerle; Hofmann, Wolf-Karsten; Claes, Filip; Carmeliet, Peter; Kettmann, Richard; Gerhardt, Holger; Dequiedt, Franck

    2013-09-11

    To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion. PMID:23955003

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

    PubMed

    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

  19. 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

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

    PubMed Central

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

    2012-01-01

    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

  1. PP2A Phosphatase as a Regulator of ROS Signaling in Plants

    PubMed Central

    Rahikainen, Moona; Pascual, Jesús; Alegre, Sara; Durian, Guido; Kangasjärvi, Saijaliisa

    2016-01-01

    Reactive oxygen species (ROS) carry out vital functions in determining appropriate stress reactions in plants, but the molecular mechanisms underlying the sensing, signaling and response to ROS as signaling molecules are not yet fully understood. Recent studies have underscored the role of Protein Phosphatase 2A (PP2A) in ROS-dependent responses involved in light acclimation and pathogenesis responses in Arabidopsis thaliana. Genetic, proteomic and metabolomic studies have demonstrated that trimeric PP2A phosphatases control metabolic changes and cell death elicited by intracellular and extracellular ROS signals. Associated with this, PP2A subunits contribute to transcriptional and post-translational regulation of pro-oxidant and antioxidant enzymes. This review highlights the emerging role of PP2A phosphatases in the regulatory ROS signaling networks in plants. PMID:26950157

  2. PP2A Phosphatase as a Regulator of ROS Signaling in Plants.

    PubMed

    Rahikainen, Moona; Pascual, Jesús; Alegre, Sara; Durian, Guido; Kangasjärvi, Saijaliisa

    2016-01-01

    Reactive oxygen species (ROS) carry out vital functions in determining appropriate stress reactions in plants, but the molecular mechanisms underlying the sensing, signaling and response to ROS as signaling molecules are not yet fully understood. Recent studies have underscored the role of Protein Phosphatase 2A (PP2A) in ROS-dependent responses involved in light acclimation and pathogenesis responses in Arabidopsis thaliana. Genetic, proteomic and metabolomic studies have demonstrated that trimeric PP2A phosphatases control metabolic changes and cell death elicited by intracellular and extracellular ROS signals. Associated with this, PP2A subunits contribute to transcriptional and post-translational regulation of pro-oxidant and antioxidant enzymes. This review highlights the emerging role of PP2A phosphatases in the regulatory ROS signaling networks in plants. PMID:26950157

  3. PP2A delays APC/C-dependent degradation of separase-associated but not free securin

    PubMed Central

    Hellmuth, Susanne; Böttger, Franziska; Pan, Cuiping; Mann, Matthias; Stemmann, Olaf

    2014-01-01

    The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2- or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpoint-mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase. PMID:24781523

  4. 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

    2016-01-01

    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

  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. Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A.

    PubMed

    Zonta, Francesca; Pagano, Mario Angelo; Trentin, Livio; Tibaldi, Elena; Frezzato, Federica; Trimarco, Valentina; Facco, Monica; Zagotto, Giuseppe; Pavan, Valeria; Ribaudo, Giovanni; Bordin, Luciana; Semenzato, Gianpietro; Brunati, Anna Maria

    2015-06-11

    Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients. PMID:25931585

  7. The broken "Off" switch in cancer signaling: PP2A as a regulator of tumorigenesis, drug resistance, and immune surveillance.

    PubMed

    Ruvolo, Peter P

    2016-12-01

    Aberrant activation of signal transduction pathways can transform a normal cell to a malignant one and can impart survival properties that render cancer cells resistant to therapy. A diverse set of cascades have been implicated in various cancers including those mediated by serine/threonine kinases such RAS, PI3K/AKT, and PKC. Signal transduction is a dynamic process involving both "On" and "Off" switches. Activating mutations of RAS or PI3K can be viewed as the switch being stuck in the "On" position resulting in continued signaling by a survival and/or proliferation pathway. On the other hand, inactivation of protein phosphatases such as the PP2A family can be seen as the defective "Off" switch that similarly can activate these pathways. A problem for therapeutic targeting of PP2A is that the enzyme is a hetero-trimer and thus drug targeting involves complex structures. More importantly, since PP2A isoforms generally act as tumor suppressors one would want to activate these enzymes rather than suppress them. The elucidation of the role of cellular inhibitors like SET and CIP2A in cancer suggests that targeting these proteins can have therapeutic efficacy by mechanisms involving PP2A activation. Furthermore, drugs such as FTY-720 can activate PP2A isoforms directly. This review will cover the current state of knowledge of PP2A role as a tumor suppressor in cancer cells and as a mediator of processes that can impact drug resistance and immune surveillance. PMID:27556014

  8. 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

  9. PP2A as a master regulator of the cell cycle

    PubMed Central

    Wlodarchak, Nathan; Xing, Yongna

    2016-01-01

    Protein phosphatase 2A (PP2A) plays a critical multi-faceted role in the regulation of the cell cycle. It is known to dephosphorylate over 300 substrates involved in the cell cycle, regulating almost all major pathways and cell cycle checkpoints. PP2A is involved in such diverse processes by the formation of structurally distinct families of holoenzymes, which are regulated spatially and temporally by specific regulators. Here, we review the involvement of PP2A in the regulation of three cell signaling pathways: wnt, mTOR and MAP kinase, as well as the G1→S transition, DNA synthesis and mitotic initiation. These processes are all crucial for proper cell survival and proliferation and are often deregulated in cancer and other diseases. PMID:26906453

  10. PP2A as a master regulator of the cell cycle.

    PubMed

    Wlodarchak, Nathan; Xing, Yongna

    2016-01-01

    Protein phosphatase 2A (PP2A) plays a critical multi-faceted role in the regulation of the cell cycle. It is known to dephosphorylate over 300 substrates involved in the cell cycle, regulating almost all major pathways and cell cycle checkpoints. PP2A is involved in such diverse processes by the formation of structurally distinct families of holoenzymes, which are regulated spatially and temporally by specific regulators. Here, we review the involvement of PP2A in the regulation of three cell signaling pathways: wnt, mTOR and MAP kinase, as well as the G1→S transition, DNA synthesis and mitotic initiation. These processes are all crucial for proper cell survival and proliferation and are often deregulated in cancer and other diseases. PMID:26906453

  11. Free fatty acid-induced PP2A hyperactivity selectively impairs hepatic insulin action on glucose metabolism.

    PubMed

    Galbo, Thomas; Olsen, Grith Skytte; Quistorff, Bjørn; Nishimura, Erica

    2011-01-01

    In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is "selective", in the sense that the insulin-mediated decrease of glucose production is blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity. PMID:22087313

  12. Protein phosphatases PP2A, PP4 and PP6: mediators and regulators in development and responses to environmental cues.

    PubMed

    Lillo, Cathrine; Kataya, Amr R A; Heidari, Behzad; Creighton, Maria T; Nemie-Feyissa, Dugassa; Ginbot, Zekarias; Jonassen, Else M

    2014-12-01

    The three closely related groups of serine/threonine protein phosphatases PP2A, PP4 and PP6 are conserved throughout eukaryotes. The catalytic subunits are present in trimeric and dimeric complexes with scaffolding and regulatory subunits that control activity and confer substrate specificity to the protein phosphatases. In Arabidopsis, three scaffolding (A subunits) and 17 regulatory (B subunits) proteins form complexes with five PP2A catalytic subunits giving up to 255 possible combinations. Three SAP-domain proteins act as regulatory subunits of PP6. Based on sequence similarities with proteins in yeast and mammals, two putative PP4 regulatory subunits are recognized in Arabidopsis. Recent breakthroughs have been made concerning the functions of some of the PP2A and PP6 regulatory subunits, for example the FASS/TON2 in regulation of the cellular skeleton, B' subunits in brassinosteroid signalling and SAL proteins in regulation of auxin transport. Reverse genetics is starting to reveal also many more physiological functions of other subunits. A system with key regulatory proteins (TAP46, TIP41, PTPA, LCMT1, PME-1) is present in all eukaryotes to stabilize, activate and inactivate the catalytic subunits. In this review, we present the status of knowledge concerning physiological functions of PP2A, PP4 and PP6 in Arabidopsis, and relate these to yeast and mammals. PMID:24810976

  13. PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

    PubMed

    Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

    2016-06-01

    Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes. PMID:27096707

  14. LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential.

    PubMed

    Hong, Christopher S; Ho, Winson; Zhang, Chao; Yang, Chunzhang; Elder, J Bradley; Zhuang, Zhengping

    2015-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that plays a significant role in mitotic progression and cellular responses to DNA damage. While traditionally viewed as a tumor suppressor, inhibition of PP2A has recently come to attention as a novel therapeutic means of driving senescent cancer cells into mitosis and promoting cell death via mitotic catastrophe. These findings have been corroborated in numerous studies utilizing naturally produced compounds that selectively inhibit PP2A. To overcome the known human toxicities associated with these compounds, a water-soluble small molecule inhibitor, LB100, was recently developed to competitively inhibit the PP2A protein. This review summarizes the pre-clinical studies to date that have demonstrated the anti-cancer activity of LB100 via its chemo- and radio-sensitizing properties. These studies demonstrate the tremendous therapeutic potential of LB100 in a variety of cancer types. The results of an ongoing phase 1 trial are eagerly anticipated. PMID:25897893

  15. LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

    PubMed Central

    Hong, Christopher S; Ho, Winson; Zhang, Chao; Yang, Chunzhang; Elder, J Bradley; Zhuang, Zhengping

    2015-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that plays a significant role in mitotic progression and cellular responses to DNA damage. While traditionally viewed as a tumor suppressor, inhibition of PP2A has recently come to attention as a novel therapeutic means of driving senescent cancer cells into mitosis and promoting cell death via mitotic catastrophe. These findings have been corroborated in numerous studies utilizing naturally produced compounds that selectively inhibit PP2A. To overcome the known human toxicities associated with these compounds, a water-soluble small molecule inhibitor, LB100, was recently developed to competitively inhibit the PP2A protein. This review summarizes the pre-clinical studies to date that have demonstrated the anti-cancer activity of LB100 via its chemo- and radio-sensitizing properties. These studies demonstrate the tremendous therapeutic potential of LB100 in a variety of cancer types. The results of an ongoing phase 1 trial are eagerly anticipated. PMID:25897893

  16. Capsaicin inhibits the Wnt/β-catenin signaling pathway by down-regulating PP2A.

    PubMed

    Park, Dong-Seok; Yoon, Gang-Ho; Lee, Hyun-Shik; Choi, Sun-Cheol

    2016-09-01

    Xenopus embryo serves as an ideal model for teratogenesis assays to examine the effects of any substances on the cellular processes critical for early development and adult tissue homeostasis. In our chemical library screening with frog embryo, capsaicin was found to repress the Wnt/β-catenin signaling. Depending on the stages at which embryos became exposed to capsaicin, it could disrupt formation of dorsal or posterior body axis of embryo, which is associated with inhibition of maternal or zygotic Wnt signal in early development. In agreement with these phenotypes, capsaicin suppressed the expression of Wnt target genes such as Siamois and Chordin in the organizer region of embryo and in Wnt signals-stimulated tissue explants. In addition, the cellular level of β-catenin, a key component of Wnt pathway, was down-regulated in capsaicin-treated embryonic cells. Unlike wild-type β-catenin, its non-phosphorylatable mutant in which serine and threonine residues phosphorylated by GSK3 are substituted with alanine was not destabilized by capsaicin, indicative of the effect of this chemical on the phosphorylation status of β-catenin. In support of this, capsaicin up-regulated the level of GSK3- or CK1-phosphorylated β-catenin, concomitantly lowering that of its de-phosphorylated version. Notably, capsaicin augmented the phosphorylation of a phosphatase, PP2A at tyrosine 307, suggesting its repression of the enzymatic activity of the phosphatase. Furthermore, capsaicin still enhanced β-catenin phosphorylation in cells treated with a GSK3 inhibitor, LiCl but not in those treated with a phosphatase inhibitor, okadaic acid. Together, these results indicate that capsaicin inhibits the patterning of the dorso-ventral and anterior-posterior body axes of embryo by repressing PP2A and thereby down-regulating the Wnt/β-catenin signaling. PMID:27318088

  17. Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy

    PubMed Central

    Arrouss, Issam; Nemati, Fariba; Roncal, Fernando; Wislez, Marie; Dorgham, Karim; Vallerand, David; Rabbe, Nathalie; Karboul, Narjesse; Carlotti, Françoise; Bravo, Jeronimo; Mazier, Dominique

    2013-01-01

    Purpose PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. Experimental Design We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitonealy administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. Results We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. Conclusion Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression. PMID:23637769

  18. Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis[W

    PubMed Central

    Beneteau, Julie; Renard, Denis; Marché, Laurent; Douville, Elise; Lavenant, Laurence; Rahbé, Yvan; Dupont, Didier; Vilaine, Françoise; Dinant, Sylvie

    2010-01-01

    Phloem Protein2 (PP2) is a component of the phloem protein bodies found in sieve elements. We describe here the lectin properties of the Arabidopsis (Arabidopsis thaliana) PP2-A1. Using a recombinant protein produced in Escherichia coli, we demonstrated binding to N-acetylglucosamine oligomers. Glycan array screening showed that PP2-A1 also bound to high-mannose N-glycans and 9-acyl-N-acetylneuraminic sialic acid. Fluorescence spectroscopy-based titration experiments revealed that PP2-A1 had two classes of binding site for N,N′,N″-triacetylchitotriose, a low-affinity site and a high-affinity site, promoting the formation of protein dimers. A search for structural similarities revealed that PP2-A1 aligned with the Cbm4 and Cbm22-2 carbohydrate-binding modules, leading to the prediction of a β-strand structure for its conserved domain. We investigated whether PP2-A1 interacted with phloem sap glycoproteins by first characterizing abundant Arabidopsis phloem sap proteins by liquid chromatography-tandem mass spectrometry. Then we demonstrated that PP2-A1 bound to several phloem sap proteins and that this binding was not completely abolished by glycosidase treatment. As many plant lectins have insecticidal activity, we also assessed the effect of PP2-A1 on weight gain and survival in aphids. Unlike other mannose-binding lectins, when added to an artificial diet, recombinant PP2-A1 had no insecticidal properties against Acyrthosiphon pisum and Myzus persicae. However, at mid-range concentrations, the protein affected weight gain in insect nymphs. These results indicate the presence in PP2-A1 of several carbohydrate-binding sites, with potentially different functions in the trafficking of endogenous proteins or in interactions with phloem-feeding insects. PMID:20442276

  19. The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action.

    PubMed

    Xing, Hongyan; Vanderford, Nathan L; Sarge, Kevin D

    2008-11-01

    To maintain phenotypes of cell lineages, cells must 'remember' which genes were active before mitosis entry and transmit this information to their daughter cells so that expression patterns can be faithfully re-established in G1. This phenomenon is called gene bookmarking. However, during mitosis transcription ceases, most sequence-specific proteins dissociate from DNA and the chromatin is tightly compacted, making it difficult to understand how gene activity 'memory' is maintained through this stage of the cell cycle. A feature of gene bookmarking is that in mitotic cells, the promoters of formerly active genes lack compaction, but how compaction of these regions is inhibited is unknown. Here we show that during mitosis, TATA-binding protein (TBP), which remains bound to DNA during mitosis, recruits PP2A. TBP also interacts with condensin to allow efficient dephosphorylation and inactivation of condensin near these promoters to inhibit their compaction. Further, ChIP-on-chip data show that TBP is bound to many chromosomal sites during mitosis, and is higher in transcribed regions but low in regions containing pseudogenes and genes whose expression is tissue-restricted. These results suggest that TBP is involved not only in gene transcription during interphase but also in preserving the memory of gene activity through mitosis to daughter cells. PMID:18931662

  20. 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. PMID:25038454

  1. 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

  2. Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

    SciTech Connect

    Chuang, Jian-Ying; Hung, Jan-Jong

    2011-04-15

    Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

  3. Two highly-related regulatory subunits of PP2A exert opposite effects on TGF-β/Activin/Nodal signalling

    PubMed Central

    Batut, Julie; Schmierer, Bernhard; Cao, Jing; Raftery, Laurel A.; Hill, Caroline S.; Howell, Michael

    2016-01-01

    Summary We identify Bα (PPP2R2A) and Bδ (PPP2R2D), two highly-related members of the B family of regulatory subunits of the protein phosphatase PP2A, as important modulators of TGF-β/Activin/Nodal signalling, which affect the pathway in opposite ways. Knockdown of Bα in Xenopus embryos or mammalian tissue culture cells suppresses TGF-β/Activin/Nodal-dependent responses, whereas knockdown of Bδ enhances these responses. Moreover, in Drosophila, overexpression of Smad2 rescues a severe wing phenotype caused by overexpression of the single Drosophila PP2A B subunit, Twins. We show that in vertebrates Bα enhances TGF-β/Activin/Nodal signalling by stabilising the basal levels of type I receptor, whereas Bδ negatively modulates these pathways by restricting receptor activity. Thus, these highly-related members of the same subfamily of PP2A regulatory subunits differentially regulate TGF-β/Activin/Nodal signalling to elicit opposing biological outcomes. PMID:18697906

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

    PubMed

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

    2016-04-15

    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 byPPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn(2+)-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. Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates*

    PubMed Central

    Kirchhefer, Uwe; Brekle, Christiane; Eskandar, John; Isensee, Gunnar; Kučerová, Dana; Müller, Frank U.; Pinet, Florence; Schulte, Jan S.; Seidl, Matthias D.; Boknik, Peter

    2014-01-01

    Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser16 was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca2+ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation. PMID:25320082

  6. Lapatinib inhibits CIP2A/PP2A/p-Akt signaling and induces apoptosis in triple negative breast cancer cells

    PubMed Central

    Liu, Chun-Yu; Hu, Ming-Hung; Hsu, Chia-Jung; Huang, Chun-Teng; Wang, Duen-Shian; Tsai, Wen-Chun; Chen, Yi-Ting; Lee, Chia-Han; Chu, Pei-Yi; Hsu, Chia-Chi; Chen, Ming-Huang; Shiau, Chung-Wai; Tseng, Ling-Ming; Chen, Kuen-Feng

    2016-01-01

    We tested the efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and epidermal growth factor receptor (EGFR) pathways, in a panel of triple-negative breast cancer (TNBC) cells, and examined the drug mechanism. Lapatinib showed an anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis and inhibited CIP2A and p-Akt in a dose and time-dependent manner in the three TNBC cell lines. Overexpression of CIP2A reduced lapatinib-induced apoptosis in MDA-MB-468 cells. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition). Moreover, lapatinib-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, lapatinib indirectly decreased CIP2A transcription by disturbing the binding of Elk1 to the CIP2A promoter. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and suppressed CIP2A as well as p-Akt in these xenografted tumors. In summary, inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. In addition to being a dual tyrosine kinase inhibitor of HER2 and EGFR, lapatinib also inhibits CIP2A/PP2A/p-Akt signaling in TNBC cells. PMID:26824320

  7. Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2.

    PubMed

    Xu, Hui; Ginsburg, Kenneth S; Hall, Duane D; Zimmermann, Maike; Stein, Ivar S; Zhang, Mingxu; Tandan, Samvit; Hill, Joseph A; Horne, Mary C; Bers, Donald; Hell, Johannes W

    2010-12-01

    The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the β(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex. PP2A counteracts increase in Ca(v)1.2 channel activity by PKA and other protein kinases, whereas PP2B can either augment or decrease Ca(v)1.2 currents in cardiomyocytes depending on the precise experimental conditions. We found that PP2A binds to two regions in the C-terminus of the central, pore-forming α(1) subunit of Ca(v)1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residue 1971. Injection of a peptide that contained residues 1965-1971 and displaced PP2A but not PP2B from endogenous Ca(v)1.2 increased basal and isoproterenol-stimulated L-type Ca(2+) currents in acutely isolated cardiomyocytes. Together with our biochemical data, these physiological results indicate that anchoring of PP2A at this site of Ca(v)1.2 in the heart negatively regulates cardiac L-type currents, likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases. PMID:21053940

  8. 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. PMID:27106144

  9. Maintenance of CSF arrest: A role for Cdc2 and PP2A-mediated regulation of Emi2

    PubMed Central

    Wu, Qiju; Guo, Yanxiang; Yamada, Ayumi; Perry, Jennifer A.; Wang, Michael Z.; Araki, Marito; Freel, Christopher D.; Tung, Jeffrey J.; Tang, Wanli; Margolis, Seth S.; Jackson, Peter K.; Yamano, Hiroyuki; Asano, Maki; Kornbluth, Sally

    2009-01-01

    Summary Background Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell cycle arrest by inhibiting the anaphase promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. Results We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor, Emi2. Emi2 bound to the core APC and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2 mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. Conclusions Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest. PMID:17276914

  10. Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

    PubMed Central

    Okumura, Eiichi; Morita, Atsushi; Wakai, Mizuho; Mochida, Satoru; Hara, Masatoshi

    2014-01-01

    Entry into M phase is governed by cyclin B–Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B–Cdk1–dependent inhibition of the protein phosphatase 2A (PP2A)–B55, which antagonizes cyclin B–Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B–Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B–Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B–Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B–Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles. PMID:24616226

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26053095

  12. Regulation of Beclin 1 Protein Phosphorylation and Autophagy by Protein Phosphatase 2A (PP2A) and Death-associated Protein Kinase 3 (DAPK3).

    PubMed

    Fujiwara, Nobuyuki; Usui, Tatsuya; Ohama, Takashi; Sato, Koichi

    2016-05-13

    Autophagy is an evolutionarily conserved intracellular degradation system that is involved in cell survival and activated in various diseases, including cancer. Beclin 1 is a central scaffold protein that assembles components for promoting or inhibiting autophagy. Association of Beclin 1 with its interacting proteins is regulated by the phosphorylation of Beclin 1 by various Ser/Thr kinases, but the Ser/Thr phosphatases that regulate these phosphorylation events remain unknown. Here we identify Ser-90 in Beclin 1 as a regulatory site whose phosphorylation is markedly enhanced in cells treated with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). Beclin 1 Ser-90 phosphorylation is induced in skeletal muscle tissues isolated from starved mice. The Beclin 1 S90A mutant blocked starvation-induced autophagy. We found association of PP2A B55α with Beclin 1, which dissociate by starvation. We also found that death-associated protein kinase 3 directly phosphorylates Beclin 1 Ser-90. We propose that physiological regulation of Beclin 1 Ser-90 phosphorylation by PP2A and death-associated protein kinase 3 controls autophagy. PMID:26994142

  13. Discovery of a small molecule targeting SET-PP2A interaction to overcome BCR-ABLT315I mutation of chronic myeloid leukemia

    PubMed Central

    Zheng, Yan; Liu, Nan; Dai, Wen; Wang, Yang; Wang, Zongqiang; Yang, Yong; Chen, Yijun

    2015-01-01

    Despite the great success in using tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML), the frequent development of multi-drug resistance, particularly the T315I mutation of BCR-ABL, remains a challenging issue. Enhancement of protein phosphatase 2A (PP2A) activity by dissociating its endogenous inhibitor SET is an effective approach to combat TKI-based resistance. Here, we report the identification of a novel 2-phenyloxypyrimidine compound TGI1002 to specifically disrupt SET-PP2A interaction. By binding to SET, TGI1002 inhibits SET-PP2A interaction and increases PP2A activity. In addition, knocking-down SET expression decreases tumor cell sensitivity to TGI1002. TGI1002 treatments also markedly increase dephosphorylation of BCR-ABL. Moreover, TGI1002 significantly inhibits tumor growth and prolongs survival of xenografted mice implanted with BaF3-p210T315I cells. These findings demonstrate that TGI1002 is a novel SET inhibitor with important therapeutic potential for the treatment of drug-resistant CML. PMID:25900240

  14. Quantitative proteomics reveals novel protein interaction partners of PP2A catalytic subunit in pancreatic β-cells.

    PubMed

    Zhang, Xiangmin; Damacharla, Divyasri; Ma, Danjun; Qi, Yue; Tagett, Rebecca; Draghici, Sorin; Kowluru, Anjaneyulu; Yi, Zhengping

    2016-03-15

    Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases. We hypothesize that PP2A regulates signaling cascades in pancreatic β-cells in the context of glucose-stimulated insulin secretion (GSIS). Using co-immunoprecipitation (co-IP) and tandem mass spectrometry, we globally identified the protein interaction partners of the PP2A catalytic subunit (PP2Ac) in insulin-secreting pancreatic β-cells. Among the 514 identified PP2Ac interaction partners, 476 were novel. This represents the first global view of PP2Ac protein-protein interactions caused by hyperglycemic conditions. Additionally, numerous PP2Ac partners were found involved in a variety of signaling pathways in the β-cell function, such as insulin secretion. Our data suggest that PP2A interacts with various signaling proteins necessary for physiological insulin secretion as well as signaling proteins known to regulate cell dysfunction and apoptosis in the pancreatic β-cells. PMID:26780722

  15. 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

  16. PP2A(Cdc55)'s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function.

    PubMed

    Kerr, Gary W; Wong, Jin Huei; Arumugam, Prakash

    2016-01-01

    PP2A(Cdc55) is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2A(Cdc55) activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2A(Cdc55) prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2A(Cdc55)'s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function. PMID:27455870

  17. 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.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26792519

  19. 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

    2016-02-01

    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. PMID:26792519

  20. PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

    PubMed Central

    Espert, Antonio; Uluocak, Pelin; Bastos, Ricardo Nunes; Mangat, Davinderpreet; Graab, Philipp

    2014-01-01

    The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC. PMID:25246613

  1. Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways.

    PubMed

    Meng, Guanmin; Wang, Wei; Chai, Kequn; Yang, Suwen; Li, Fangqiong; Jiang, Kai

    2015-03-01

    Lung cancer is the leading cause of cancer death worldwide. Recently, two plant-derived drugs triptolide (TP) and hydroxycamptothecin (HCPT) both have shown broad-spectrum anticancer activities. Our previous study documented that combination treatment with these two drugs acted more effectively than mono-therapy, however, the molecular basis underlying the synergistic cytotoxicity remains poorly understood. In this study, we aimed to clarify the molecular mechanism of TP/HCPT anticancer effect in A549 lung adenocarcinoma cells, by investigating the involvement of phosphatase 2A (PP2A) and PP2A-regulated mitogen-activated protein kinases (MAPKs) and Akt signaling pathways. The results showed that TP and HCPT synergistically exerted cytotoxicity in the growth of A549 cells. Combinatorial TP/HCPT treatment significantly enhanced the activation of caspase-3 and -9, Bax/Bcl-2 ratio, release of cytochrome c from mitochondrial and subsequent apoptosis. While the Akt survival pathway was inhibited, ERK and p38 MAPKs were dramatically activated. Furthermore, the activity of PP2A was significantly augmented. Regulation of p38, ERK and Akt by PP2A was demonstrated, by using a specific PP2A inhibitor okadaic acid (OA). Finally, pharmacological inhibitors OA, SB203580, SP600125 and PD98059 confirm the role of PP2A and its substrates ERK, p38 MAPK and Akt in mediating TP/HCPT-induced apoptosis. Taken together, this study provides the first evidence for a synergistic TP/HCPT anticancer activity in A549 cells and also supports a critical role of PP2A and PP2A-regulated signaling pathways, providing new insight into the mode of action of TP/HCPT in cancer therapy. PMID:25573072

  2. Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways

    PubMed Central

    MENG, GUANMIN; WANG, WEI; CHAI, KEQUN; YANG, SUWEN; LI, FANGQIONG; JIANG, KAI

    2015-01-01

    Lung cancer is the leading cause of cancer death worldwide. Recently, two plant-derived drugs triptolide (TP) and hydroxycamptothecin (HCPT) both have shown broad-spectrum anticancer activities. Our previous study documented that combination treatment with these two drugs acted more effectively than mono-therapy, however, the molecular basis underlying the synergistic cytotoxicity remains poorly understood. In this study, we aimed to clarify the molecular mechanism of TP/HCPT anticancer effect in A549 lung adenocarcinoma cells, by investigating the involvement of phosphatase 2A (PP2A) and PP2A-regulated mitogen-activated protein kinases (MAPKs) and Akt signaling pathways. The results showed that TP and HCPT synergistically exerted cytotoxicity in the growth of A549 cells. Combinatorial TP/HCPT treatment significantly enhanced the activation of caspase-3 and -9, Bax/Bcl-2 ratio, release of cytochrome c from mitochondrial and subsequent apoptosis. While the Akt survival pathway was inhibited, ERK and p38 MAPKs were dramatically activated. Furthermore, the activity of PP2A was significantly augmented. Regulation of p38, ERK and Akt by PP2A was demonstrated, by using a specific PP2A inhibitor okadaic acid (OA). Finally, pharmacological inhibitors OA, SB203580, SP600125 and PD98059 confirm the role of PP2A and its substrates ERK, p38 MAPK and Akt in mediating TP/HCPT-induced apoptosis. Taken together, this study provides the first evidence for a synergistic TP/HCPT anti-cancer activity in A549 cells and also supports a critical role of PP2A and PP2A-regulated signaling pathways, providing new insight into the mode of action of TP/HCPT in cancer therapy. PMID:25573072

  3. PP2A inhibition with LB100 enhances cisplatin cytotoxicity and overcomes cisplatin resistance in medulloblastoma cells

    PubMed Central

    Maric, Dragan; Amable, Lauren; Hall, Matthew D.; Feldman, Gerald M.; Ray-Chaudhury, Abhik; Lizak, Martin J.; Vera, Juan-Carlos; Robison, R. Aaron; Zhuang, Zhengping; Heiss, John D.

    2016-01-01

    The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 μM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model. PMID:26799670

  4. The B55α-containing PP2A holoenzyme dephosphorylates FOXO1 in islet β-cells under oxidative stress

    PubMed Central

    Yan, Ling; Guo, Shuangli; Brault, Marie; Harmon, Jamie; Robertson, R. Paul; Hamid, Rizwan; Stein, Roland; Yang, Elizabeth

    2016-01-01

    The FOXO1 (forkhead box O1) transcription factor influences many key cellular processes, including those important in metabolism, proliferation and cell death. Reversible phosphorylation of FOXO1 at Thr24 and Ser256 regulates its subcellular localization, with phosphorylation promoting cytoplasmic localization, whereas dephosphorylation triggers nuclear import and transcriptional activation. In the present study, we used biochemical and molecular approaches to isolate and link the serine/threonine PP2A (protein phosphatase 2A) holoenzyme containing the B55α regulatory subunit, with nuclear import of FOXO1 in pancreatic islet β-cells under oxidative stress, a condition associated with cellular dysfunction in Type 2 diabetes. The mechanism of FOXO1 dephosphorylation and nuclear translocation was investigated in pancreatic islet INS-1 and βTC-3 cell lines subjected to oxidative stress. A combined chemical cross-linking and MS strategy revealed the association of FOXO1 with a PP2A holoenzyme composed of the catalytic C, structural A and B55α regulatory subunits. Knockdown of B55α in INS-1 cells reduced FOXO1 dephosphorylation, inhibited FOXO1 nuclear translocation and attenuated oxidative stress-induced cell death. Furthermore, both B55α and nuclear FOXO1 levels were increased under hyperglycaemic conditions in db/db mouse islets, an animal model of Type 2 diabetes. We conclude that B55α-containing PP2A is a key regulator of FOXO1 activity in vivo. PMID:22417654

  5. The PP2A Regulatory Subunit Tap46, a Component of the TOR Signaling Pathway, Modulates Growth and Metabolism in Plants[W

    PubMed Central

    Ahn, Chang Sook; Han, Jeong-A; Lee, Ho-Seok; Lee, Semi; Pai, Hyun-Sook

    2011-01-01

    Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway. PMID:21216945

  6. Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells

    PubMed Central

    Jeon, Kye-Im; Jono, Hirofumi; Miller, Clint L.; Cai, Yujun; Lim, Soyeon; Liu, Xuan; Gao, Pingjin; Abe, Jun-Ichi; Li, Jian-Dong; Yan, Chen

    2010-01-01

    The phenotypic change of vascular smooth muscle cells (VSMCs), from a “contractile” phenotype to “synthetic” phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca2+-calmodulin stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating β-catenin/TCF signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated β-catenin/TCF signaling by down-regulating β-catenin protein. The effect of PDE1 inhibition on β-catenin protein reduction is exerted via promoting GSK3β activation, β-catenin phosphorylation, and subsequent β-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase PP2A B56γ subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3β and β-catenin/TCF signaling. Further more, the effect of PDE1 inhibition on β-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs PP2A B56γ, phospho-GSK3β, and phospho-β-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear β-catenin protein stability through the nuclear PP2A-GSK3β-β-catenin signaling axis. Taken together these findings provide direct evidence for the first time that PP2A B56γ is a critical mediator for PDE1A in the regulation of β-catenin signaling in proliferating VSMCs. PMID:21078118

  7. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

    PubMed

    Guillonneau, Maëva; Paris, François; Dutoit, Soizic; Estephan, Hala; Bénéteau, Elise; Huot, Jacques; Corre, Isabelle

    2016-08-01

    Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response. PMID:27142525

  8. Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

    PubMed Central

    Palii, Stela S.; Cui, Yuxia; Innes, Cynthia L.; Paules, Richard S.

    2013-01-01

    Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G₂/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G₂/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G₂/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G₂/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments. PMID:23462183

  9. Sgo1 recruits PP2A to chromosomes to ensure sister chromatid bi-orientation during mitosis

    PubMed Central

    Eshleman, Heather D.; Morgan, David O.

    2014-01-01

    ABSTRACT Sister chromatid bi-orientation on the mitotic spindle is essential for proper chromosome segregation. Defects in bi-orientation are sensed and corrected to prevent chromosome mis-segregation and aneuploidy. This response depends on the adaptor protein Sgo1, which associates with pericentromeric chromatin in mitosis. The mechanisms underlying Sgo1 function and regulation are unclear. Here, we show that Sgo1 is an anaphase-promoting complex/cyclosome (APC/C) substrate in budding yeast (Saccharomyces cerevisiae), and that its mitotic destruction depends on an unusual D-box-related sequence motif near its C-terminus. We find that the removal of Sgo1 from chromosomes before anaphase is not dependent on its destruction, but rather on other mechanisms responsive to tension between sister chromatids. Additionally, we find that Sgo1 recruits the protein phosphatase 2A (PP2A) isoform containing Rts1 to the pericentromeric region prior to bi-orientation, and that artificial recruitment of Rts1 to this region of a single chromosome is sufficient to perform the function of Sgo1 on that chromosome. We conclude that in early mitosis, Sgo1 associates transiently with pericentromeric chromatin to promote bi-orientation, in large part by recruiting the Rts1 isoform of PP2A. PMID:25236599

  10. 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

  11. Phosphorylation/dephosphorylation of human SULT4A1: role of Erk1 and PP2A.

    PubMed

    Mitchell, Deanne J; Butcher, Neville J; Minchin, Rodney F

    2011-01-01

    SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr(11) residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr(11) by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bβ was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase. PMID:20920535

  12. Inhibition of Protein Phosphatase 2A (PP2A) Prevents Mcl-1 Protein Dephosphorylation at the Thr-163/Ser-159 Phosphodegron, Dramatically Reducing Expression in Mcl-1-amplified Lymphoma Cells*

    PubMed Central

    Nifoussi, Shanna K.; Ratcliffe, Nora R.; Ornstein, Deborah L.; Kasof, Gary; Strack, Stefan; Craig, Ruth W.

    2014-01-01

    Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells. PMID:24939844

  13. Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis

    PubMed Central

    Margolis, Seth S.; Perry, Jennifer A.; Forester, Craig M.; Nutt, Leta K.; Guo, Yanxiang; Jardim, Melanie J.; Thomenius, Michael J.; Freel, Christopher D.; Darbandi, Rashid; Ahn, Jung-Hyuck; Arroyo, Jason D.; Wang, Xiao-Fan; Shenolikar, Shirish; Nairn, Angus C.; Dunphy, William G.; Hahn, William C.; Virshup, David M.; Kornbluth, Sally

    2009-01-01

    Summary DNA-responsive checkpoints prevent cell cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. S287 phosphorylation is a major locus of G2/M checkpoint control, though several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and S287 dephosphorylation. We show here that DNA-responsive checkpoints activate PP2A/B56δ phosphatase complexes to dephosphorylate Cdc25 at a site (T138) whose phosphorylation is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Rather, our data suggest that creation of a 14-3-3 “sink”, consisting of phosphorylated 14-3-3-binding intermediate filament proteins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56δ as a central checkpoint effector, and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis. PMID:17110335

  14. Ceramide Mediates Vascular Dysfunction in Diet-Induced Obesity by PP2A-Mediated Dephosphorylation of the eNOS-Akt Complex

    PubMed Central

    Zhang, Quan-Jiang; Holland, William L.; Wilson, Lloyd; Tanner, Jason M.; Kearns, Devin; Cahoon, Judd M.; Pettey, Dix; Losee, Jason; Duncan, Bradlee; Gale, Derrick; Kowalski, Christopher A.; Deeter, Nicholas; Nichols, Alexandrea; Deesing, Michole; Arrant, Colton; Ruan, Ting; Boehme, Christoph; McCamey, Dane R.; Rou, Janvida; Ambal, Kapil; Narra, Krishna K.; Summers, Scott A.; Abel, E. Dale; Symons, J. David

    2012-01-01

    Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction. PMID:22586587

  15. 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

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

    PubMed

    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

  17. PP2A-mediated regulation of Ras signaling in G2 is essential for stable quiescence and normal G1 length

    PubMed Central

    Naetar, Nana; Soundarapandian, Velmurugan; Litovchick, Larisa; Goguen, Kelsey L.; Sablina, Anna A.; Bowman-Colin, Christian; Sicinski, Piotr; Hahn, William C.; DeCaprio, James A.; Livingston, David M.

    2014-01-01

    SUMMARY Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point, R. Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase, PP2A, results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. Following PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling-modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and, thereby, achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established. PMID:24857551

  18. PP2A-mediated regulation of Ras signaling in G2 is essential for stable quiescence and normal G1 length.

    PubMed

    Naetar, Nana; Soundarapandian, Velmurugan; Litovchick, Larisa; Goguen, Kelsey L; Sablina, Anna A; Bowman-Colin, Christian; Sicinski, Piotr; Hahn, William C; DeCaprio, James A; Livingston, David M

    2014-06-19

    Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point (R). Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase PP2A results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. After PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and thereby achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established. PMID:24857551

  19. Epithelial membrane protein 2 regulates sphingosylphosphorylcholine-induced keratin 8 phosphorylation and reorganization: Changes of PP2A expression by interaction with alpha4 and caveolin-1 in lung cancer cells.

    PubMed

    Lee, Eun Ji; Park, Mi Kyung; Kim, Hyun Ji; Kim, Eun Ji; Kang, Gyeoung-Jin; Byun, Hyun Jung; Lee, Chang Hoon

    2016-06-01

    Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear. We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells. Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK. PMID:26876307

  20. Cloning and Characterization of TaPP2AbB"-α, a Member of the PP2A Regulatory Subunit in Wheat

    PubMed Central

    Liu, Dan; Li, Ang; Mao, Xinguo; Jing, Ruilian

    2014-01-01

    Protein phosphatase 2A (PP2A), a major Serine/Threonine protein phosphatase, consists of three subunits; a highly conserved structural subunit A, a catalytic subunit C, and a highly variable regulatory subunit B which determines the substrate specificity. Although the functional mechanism of PP2A in signaling transduction in Arabidopsis is known, their physiological roles in wheat remain to be characterized. In this study, we identified a novel regulatory subunit B, TaPP2AbB"-α, in wheat (Triticum aestivum L.). Subcellular localization indicated that TaPP2AbB"-α is located in the cell membrane, cytoplasm and nucleus. It interacts with both TaPP2Aa and TaPP2Ac. Expression pattern analyses revealed that TaPP2AbB"-α is strongly expressed in roots, and responds to NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA) stresses at the transcription level. Transgenic Arabidopsis plants overexpressing TaPP2AbB"-α developed more lateral roots, especially when treated with mannitol or NaCl. These results suggest that TaPP2AbB"-α, in conjunction with the other two PP2A subunits, is involved in multi-stress response, and positively regulates lateral root development under osmotic stress. PMID:24709994

  1. Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae.

    PubMed

    Petty, Emily L; Lafon, Anne; Tomlinson, Shannon L; Mendelsohn, Bryce A; Pillus, Lorraine

    2016-08-01

    Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability. PMID:27317677

  2. Vitamin B12 deficiency reduces proliferation and promotes differentiation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE

    PubMed Central

    Battaglia-Hsu, Shyue-fang; Akchiche, Nassila; Noel, Nicole; Alberto, Jean-Marc; Jeannesson, Elise; Orozco-Barrios, Carlos Enrique; Martinez-Fong, Daniel; Daval, Jean-Luc; Guéant, Jean-Louis

    2009-01-01

    Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and S-adenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75NTR. The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75NTR-regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-α converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE. PMID:19959661

  3. 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

  4. par-1, atypical pkc, and PP2A/B55 sur-6 are implicated in the regulation of exocyst-mediated membrane trafficking in Caenorhabditis elegans.

    PubMed

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

    2014-01-01

    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

  5. Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells.

    PubMed

    Zhao, Qingxia; Zhao, Ming; Parris, Amanda B; Xing, Ying; Yang, Xiaohe

    2016-09-01

    Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents. PMID:27574003

  6. Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells

    PubMed Central

    Zhao, Qingxia; Zhao, Ming; Parris, Amanda B.; Xing, Ying; Yang, Xiaohe

    2016-01-01

    Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents.

  7. Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1

    PubMed Central

    Nie, Minghua; Prudden, John; Schaffer, Lana; Head, Steven; Boddy, Michael N.

    2016-01-01

    Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors. PMID:27398807

  8. Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1.

    PubMed

    Nie, Minghua; Arner, Emily; Prudden, John; Schaffer, Lana; Head, Steven; Boddy, Michael N

    2016-07-01

    Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors. PMID:27398807

  9. Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway.

    PubMed

    Caraballo, Juan Carlos; Yshii, Cecilia; Butti, Maria L; Westphal, Whitney; Borcherding, Jennifer A; Allamargot, Chantal; Comellas, Alejandro P

    2011-04-01

    During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC. PMID:21257729

  10. Basal protein phosphatase 2A activity restrains cytokine expression: role for MAPKs and tristetraprolin.

    PubMed

    Rahman, Md Mostafizur; Rumzhum, Nowshin N; Morris, Jonathan C; Clark, Andrew R; Verrills, Nicole M; Ammit, Alaina J

    2015-01-01

    PP2A is a master controller of multiple inflammatory signaling pathways. It is a target in asthma; however the molecular mechanisms by which PP2A controls inflammation warrant further investigation. In A549 lung epithelial cells in vitro we show that inhibition of basal PP2A activity by okadaic acid (OA) releases restraint on MAPKs and thereby increases MAPK-mediated pro-asthmatic cytokines, including IL-6 and IL-8. Notably, PP2A inhibition also impacts on the anti-inflammatory protein - tristetraprolin (TTP), a destabilizing RNA binding protein regulated at multiple levels by p38 MAPK. Although PP2A inhibition increases TTP mRNA expression, resultant TTP protein builds up in the hyperphosphorylated inactive form. Thus, when PP2A activity is repressed, pro-inflammatory cytokines increase and anti-inflammatory proteins are rendered inactive. Importantly, these effects can be reversed by the PP2A activators FTY720 and AAL(s), or more specifically by overexpression of the PP2A catalytic subunit (PP2A-C). Moreover, PP2A plays an important role in cytokine expression in cells stimulated with TNFα; as inhibition of PP2A with OA or PP2A-C siRNA results in significant increases in cytokine production. Collectively, these data reveal the molecular mechanisms of PP2A regulation and highlight the potential of boosting the power of endogenous phosphatases as novel anti-inflammatory strategies to combat asthmatic inflammation. PMID:25985190

  11. Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion.

    PubMed

    Martín-Ávila, Alejandro; Medina-Tamayo, Jaciel; Ibarra-Sánchez, Alfredo; Vázquez-Victorio, Genaro; Castillo-Arellano, Jorge Iván; Hernández-Mondragón, Alma Cristal; Rivera, Juan; Madera-Salcedo, Iris K; Blank, Ulrich; Macías-Silva, Marina; González-Espinosa, Claudia

    2016-06-15

    Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3(+) carriers. PMID:27183589

  12. Mitochondrial reactive oxygen species perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of PP2A in lowdose irradiated human fibroblasts

    PubMed Central

    Shimura, Tsutomu; Sasatani, Megumi; Kamiya, Kenji; Kawai, Hidehiko; Inaba, Yohei; Kunugita, Naoki

    2016-01-01

    Here we investigated the cellular response of normal human fibroblasts to repeated exposure to low-dose radiation. In contrast to acute single radiation, low-dose fractionated radiation (FR) with 0.01 Gy/fraction or 0.05 Gy/fraction for 31 days increased in mitochondrial mass, decreased cellular levels of the antioxidant glutathione and caused persistent accumulation of mitochondrial reactive oxygen species (ROS). Excess ROS promoted oxidative inactivation of protein phosphatase PP2A which in turn led to disruption of normal negative feed-back control of AKT/cyclin D1 signaling in cells treated with long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 causes growth retardation, cellular senescence and genome instability in low-dose irradiated cells. Thus, loss of redox control and subsequently elevated levels of ROS perturb signal transduction as a result of oxidative stress. Our study highlights a specific role of mitochondrial ROS in perturbation of AKT/cyclin D1 cell cycle signaling after low-dose long-term FR. The antioxidants N-acetyl-L-cysteine, TEMPO and mitochondrial-targeted antioxidant Mito-TEMPO provided protection against the harmful cell cycle perturbations induced by low-dose long-term FR. PMID:26657292

  13. 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. PMID:25972378

  14. Overexpression of AtPTPA in Arabidopsis increases protein phosphatase 2A activity by promoting holoenzyme formation and ABA negatively affects holoenzyme formation

    PubMed Central

    Chen, Jian; Zhu, Xunlu; Shen, Guoxin; Zhang, Hong

    2015-01-01

    AtPTPA is a critical regulator for the holoenzyme assembling of protein phosphatase 2A (PP2A) in Arabidopsis. Characterization of AtPTPA improves our understanding of the function and regulation of PP2A in eukaryotes. Further analysis of AtPTPA-overexpressing plants indicates that AtPTPA increases PP2A activity by promoting PP2A's AC dimer formation, thereby holoenzyme assembling. Plant hormone abscisic acid (ABA) reduces PP2A enzyme activity by negatively affects PP2A's AC dimer formation. Therefore, AtPTPA is a positive factor that promotes PP2A holoenzyme assembly, and ABA is a negative factor that prevents PP2A holoenzyme assembly. PMID:26633567

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

    PubMed

    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; Symons, J David

    2015-11-01

    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

  16. Cancerous inhibitor of PP2A is targeted by natural compound celastrol for degradation in non-small-cell lung cancer

    PubMed Central

    Liu, Zi; Ma, Liang; Wen, Zhe-Sheng; Hu, Zheng; Wu, Fu-Qun; Li, Wei; Liu, Jinsong; Zhou, Guang-Biao

    2014-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed and inversely associated with prognosis in lung and many other human cancers. It modulates phospho-Akt and stabilizes c-Myc, and is required for cell proliferation and malignant transformation, indicating that CIP2A may play an important role in carcinogenesis. We reported here that a small compound celastrol could induce a rapid degradation of CIP2A, through the ubiquitin–proteasome pathway with the carboxyl terminus of Hsp70-interacting protein (CHIP) as the E3 ligase. Celastrol directly bound CIP2A protein and promoted CIP2A–CHIP interaction, leading to subsequent degradation of CIP2A in non-small-cell lung cancer cells. Furthermore, celastrol effectively inhibited cell proliferation and induced apoptosis in non-small-cell lung cancer cells, whereas CIP2A silencing enhanced these effects. Celastrol also suppressed tumor growth in xenograft murine models. In addition, celastrol potentiated the inhibitory effect of cytotoxic agent cisplatin on lung cancer cells in vitro and in vivo via inhibition of CIP2A–Akt pathway. These data indicate that celastrol is a CIP2A-targeting agent that may have therapeutic potentials in lung cancer. PMID:24293411

  17. PP2, a potent inhibitor of Src family kinases, protects against hippocampal CA1 pyramidal cell death after transient global brain ischemia.

    PubMed

    Hou, Xiao-Yu; Liu, Yong; Zhang, Guang-Yi

    2007-06-15

    It has been indicated that Src family protein tyrosine kinases (SrcPTKs) potentiate N-methyl-D-aspartate (NMDA) receptor function by phosphorylating NR2A subunits and that postsynaptic density protein 95 (PSD-95) facilitates this regulation. In this paper, we define the role of SrcPTKs in delayed neuronal damage following transient brain ischemia and explore the underlying mechanisms involved in this event. Transient global brain ischemia was induced by the four-vessel occlusion method. A specific Src family kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) and a PP2 negative control PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) were infused into rat cerebroventricule 30 min before occlusion. Hematoxylin and eosine staining showed that the number of surviving pyramidal neurons in rat hippocampal CA1 subfield increased markedly in PP2-treated rats comparing to PP3-treated groups after 5 days of reperfusion following ischemia. Additionally, immunoprecipitation and immunoblot analysis revealed that preadministration of PP2, but not PP3, attenuated not only the increased tyrosine phosphorylation of NR2A but also the enhanced interactions among Src, NR2A and PSD-95 induced by ischemia/reperfusion. In conclusion, SrcPTKs promote binding of the kinases and their substrate NR2A attributed to the scaffolding effect of PSD-95 during transient brain ischemia and reperfusion, which are responsible for the elevation of NR2A tyrosine phosphorylation and consequent delayed neuronal cell death. PMID:17556100

  18. The repair of gamma-radiation-induced DNA damage is inhibited by microcystin-LR, the PP1 and PP2A phosphatase inhibitor.

    PubMed

    Lankoff, A; Bialczyk, J; Dziga, D; Carmichael, W W; Gradzka, I; Lisowska, H; Kuszewski, T; Gozdz, S; Piorun, I; Wojcik, A

    2006-01-01

    The genotoxic activity of microcystin-LR (MC-LR) is a matter of debate. MC-LR is known to be a phosphatase inhibitor and it may be expected that it is involved in the regulation of the activity of DNA-dependent protein kinase (DNA-PK), the key enzyme involved in the repair of radiation-induced DNA damage. We studied the effect of MC-LR on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. A dose of 0.5 microg/ml of MC-LR was chosen because it induced very little early apoptosis which gives no false positive results in the comet assay. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 h and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition the frequencies of chromosomal aberrations were analysed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage and lead to enhanced frequencies of chromosomal aberrations including dicentric chromosomes. The results of a split-dose experiment, where cells were exposed to two 1.5 Gy doses of radiation separated by 3 h with or without MC-LR, confirmed that the toxin increased the frequency of dicentric chromosomes. We also determined the effect of MC-LR and ionizing radiation on the frequency of gamma-H2AX foci. The pre-treatment with MC-LR resulted in reduced numbers of gamma-H2AX foci in irradiated cells. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma MO59J and MO59K cells. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analysed by the comet assay. A strong inhibitory effect was observed in the MO59K but not in the MO59J cells. These results indicate that DNA-PK might be involved in DNA repair inhibition by MC-LR. PMID:16434448

  19. Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

    PubMed

    Little, Sean C; Curran, Jerry; Makara, Michael A; Kline, Crystal F; Ho, Hsiang-Ting; Xu, Zhaobin; Wu, Xiangqiong; Polina, Iuliia; Musa, Hassan; Meadows, Allison M; Carnes, Cynthia A; Biesiadecki, Brandon J; Davis, Jonathan P; Weisleder, Noah; Györke, Sandor; Wehrens, Xander H; Hund, Thomas J; Mohler, Peter J

    2015-07-21

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart. PMID:26198358

  20. An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator.

    PubMed Central

    Longin, Sari; Jordens, Jan; Martens, Ellen; Stevens, Ilse; Janssens, Veerle; Rondelez, Evelien; De Baere, Ivo; Derua, Rita; Waelkens, Etienne; Goris, Jozef; Van Hoof, Christine

    2004-01-01

    We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A. PMID:14748741

  1. Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)*

    PubMed Central

    Huang, Bofu; Yang, Chih-Sheng; Wojton, Jeffrey; Huang, Nai-Jia; Chen, Chen; Soderblom, Erik J.; Zhang, Liguo; Kornbluth, Sally

    2014-01-01

    High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr393/Ser395) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway. PMID:25378403

  2. Protein phosphatase 2A activity is required for functional adherent junctions in endothelial cells.

    PubMed

    Kása, Anita; Czikora, István; Verin, Alexander D; Gergely, Pál; Csortos, Csilla

    2013-09-01

    Reversible Ser/Thr phosphorylation of cytoskeletal and adherent junction (AJ) proteins has a critical role in the regulation of endothelial cell (EC) barrier function. We have demonstrated earlier that protein phosphatase 2A (PP2A) activity is important in EC barrier integrity. In the present work, macro- and microvascular EC were examined and we provided further evidence on the significance of PP2A in the maintenance of EC cytoskeleton and barrier function with special focus on the Bα (regulatory) subunit of PP2A. Immunofluorescent staining revealed that the inhibition of PP2A results in changes in the organization of EC cytoskeleton as microtubule dissolution and actin re-arrangement were detected. Depletion of Bα regulatory subunit of PP2A had similar effect on the cytoskeleton structure of the cells. Furthermore, transendothelial electric resistance measurements demonstrated significantly slower barrier recovery of Bα depleted EC after thrombin treatment. AJ proteins, VE-cadherin and β-catenin, were detected along with Bα in pull-down assay. Also, the inhibition of PP2A (by okadaic acid or fostriecin) or depletion of Bα caused β-catenin translocation from the membrane to the cytoplasm in parallel with its phosphorylation on Ser552. In conclusion, our data suggest that the A/Bα/C holoenzyme form of PP2A is essential in EC barrier integrity both in micro- and macrovascular EC. PMID:23721711

  3. Protein phosphatase 2A is associated in an inactive state with microtubules through 2A1-specific interaction with tubulin.

    PubMed Central

    Hiraga, A; Tamura, S

    2000-01-01

    Protein phosphatase (PP) 2A1, a trimer composed of A-, B- and C-subunits in the PP2A family, has been regarded as a principal form localizing at microtubules (MT), but PP2A2, the dimer of A- and C-subunits, has not. Substantiating the claim, the present work shows that the PP2A1 but not PP2A2, both isolated from bovine extract, largely associated with the purified preparation of MT. Furthermore, PP2A1 was found to bind purifiedtubulin polymerized by taxol. The presence of MT associated proteins with purified tubulin hardly affected the binding of PP2A1 to the tubulin. In addition, PP2A1 activity towards glycogen phosphorylase, a probably unphysiological but good substrate, was similarly inhibited by MT proteins and purified tubulin, which accounts for > or =85% of MT proteins, with their IC(50) of about 0.15 mg/ml. In contrast, the inhibition of PP2A2 was about 40% with 1 mg/ml MT proteins and 20% with 0.8 mg/ml tubulin, consistent with its weak association with MT. Therefore, the association with and resultant inhibition by MT proteins of PP2A1 is largely effected by the binding of PP2A1 to tubulin molecule. Moreover, PP2A1 isolated from MT has higher affinity for polymerized MT proteins than has PP2A1 from the postmicrotubule supernatant. The MT PP2A1 has also higher sensitivity to the inhibition by tubulin and MT proteins than has the supernatant PP2A1 (IC(50): 0.1-0.2 mg/ml vs. 0.3-0.6 mg/ml), demonstrating the importance of its association with polymerized tubulin. PMID:10677363

  4. Role of protein phosphatase 2A in the regulation of mitogen-activated protein kinase activity in ventricular cardiomyocytes.

    PubMed

    Braconi Quintaje, S; Church, D J; Rebsamen, M; Valloton, M B; Hemmings, B A; Lang, U

    1996-04-25

    Incubation of cultured, neonatal rat ventricular cardiomyocytes with 100 nM phorbol 12-myristate-13-acetate (PMA) induced a transient suppression of PP2A activity at 5 min, an effect that was reversed after 15 min of exposure to PMA. This inactivation was correlated with a transient increase in the phosphorylation level of the catalytic subunit of PP2A (193 +/- 38% of control levels at 5 min). Simultaneously to the transient inactivation of PP2A, we observed a rapid and reversible phosphorylation of 42-kDa MAP kinase (474 +/- 65% of control levels at 5 min, and 316 +/- 44% at 15 min) in cardiomyocytes treated with PMA. This transient phosphorylation was accompanied by a transient increase in cytosolic MAP kinase activity (209 +/- 17% of control values at 5 min and 125 +/- 7% at 15 min). Okadaic acid (1 microM ) completely blocked the decrease in the phosphorylation level and activity of MAP kinase occurring after 5 min of exposure to PMA. These data demonstrate that PP2A inactivation and MAP kinase activation are very strongly correlated in cardiomyocytes, indicating that PP2A plays a negative modulatory role in the regulation of MAP kinase activity. PMID:8629997

  5. 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

  6. 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

  7. 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.

  8. Protein phosphatase 2A dysfunction in Alzheimer’s disease

    PubMed Central

    Sontag, Jean-Marie; Sontag, Estelle

    2014-01-01

    Protein phosphatase 2A (PP2A) is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD) field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. “PP2A” dysfunction has been linked to tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/Bα holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/Bα isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/Bα-tau protein interactions likely contribute to tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/Bα, enhanced phosphorylation of tau and amyloid precursor protein, tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie “PP2A” dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes. PMID:24653673

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

    PubMed

    Côme, Christophe; 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; Hrabě de Angelis, Martin; 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

  10. 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

  11. The Protein Phosphatase 2A Regulatory Subunit B56γ Mediates Suppression of T Cell Receptor (TCR)-induced Nuclear Factor-κB (NF-κB) Activity*

    PubMed Central

    Breuer, Rebecca; Becker, Michael S.; Brechmann, Markus; Mock, Thomas; Arnold, Rüdiger; Krammer, Peter H.

    2014-01-01

    NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB. PMID:24719332

  12. Protein Phosphatase 2A Regulates Interleukin-2 Receptor Complex Formation and JAK3/STAT5 Activation*

    PubMed Central

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

    2010-01-01

    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-2Rβ, but not IL-2Rγ. 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-2Rβ. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rβ, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rβ by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rγ 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. PMID:19923221

  13. Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex.

    PubMed

    Lee, In Hye; Lim, Hee Jung; Yoon, Suhyeon; Seong, Je Kyung; Bae, Duk Soo; Rhee, Sue Goo; Bae, Yun Soo

    2008-03-01

    We have previously reported that central repeated units (CRUs) of Ahnak act as a scaffolding protein networking phospholipase Cgamma and protein kinase C (PKC). Here, we demonstrate that an Ahnak derivative consisting of four central repeated units binds and activates PKC-alpha in a phosphatidylserine/1,2-dioleoyl-sn-glycerol-independent manner. Moreover, NIH3T3 cells expressing the 4 CRUs of Ahnak showed enhanced c-Raf, MEK, and Erk phosphorylation in response to phorbol 12-myristate 13-acetate (PMA) compared with parental cells. To evaluate the effect of loss-of-function of Ahnak in cell signaling, we investigated PKC activation and Raf phosphorylation in embryonic fibroblast cells (MEFs) of the Ahnak knock-out (Ahnak(-/-)) mouse. Membrane translocation of PKC-alpha and phosphorylation of Raf in response to PMA or platelet-derived growth factor were decreased in Ahnak null MEF cells compared with wild type MEFs. Several lines of evidence suggest that PKC-alpha activity is regulated through association with protein phosphatase 2A (PP2A). A co-immunoprecipitation assay indicated that the association of PKC-alpha with PP2A was disrupted in NIH3T3 cells expressing 4 CRUs of Ahnak in response to PMA. Consistently, Ahnak null MEF cells stimulated by PMA showed enhanced PKC-PP2A complex formation, and add-back expression of Ahnak into Ahnak null MEF cells abolished the PKC-PP2A complex formation in response to PMA. These data indicate that Ahnak potentiates PKC activation through inhibiting the interaction of PKC with PP2A. PMID:18174170

  14. Protein Phosphatase 2A as a Therapeutic Target in Acute Myeloid Leukemia

    PubMed Central

    Arriazu, Elena; Pippa, Raffaella; Odero, María D.

    2016-01-01

    Acute myeloid leukemia (AML) is a heterogeneous malignant disorder of hematopoietic progenitor cells in which several genetic and epigenetic aberrations have been described. Despite progressive advances in our understanding of the molecular biology of this disease, the outcome for most patients is poor. It is, therefore, necessary to develop more effective treatment strategies. Genetic aberrations affecting kinases have been widely studied in AML; however, the role of phosphatases remains underexplored. Inactivation of the tumor-suppressor protein phosphatase 2A (PP2A) is frequent in AML patients, making it a promising target for therapy. There are several PP2A inactivating mechanisms reported in this disease. Deregulation or specific post-translational modifications of PP2A subunits have been identified as a cause of PP2A malfunction, which lead to deregulation of proliferation or apoptosis pathways, depending on the subunit affected. Likewise, overexpression of either SET or cancerous inhibitor of protein phosphatase 2A, endogenous inhibitors of PP2A, is a recurrent event in AML that impairs PP2A activity, contributing to leukemogenesis progression. Interestingly, the anticancer activity of several PP2A-activating drugs (PADs) depends on interaction/sequestration of SET. Preclinical studies show that pharmacological restoration of PP2A activity by PADs effectively antagonizes leukemogenesis, and that these drugs have synergistic cytotoxic effects with conventional chemotherapy and kinase inhibitors, opening new possibilities for personalized treatment in AML patients, especially in cases with SET-dependent inactivation of PP2A. Here, we review the role of PP2A as a druggable tumor suppressor in AML. PMID:27092295

  15. 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.

  16. 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.

  17. Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2.

    PubMed

    Athanasopoulos, Panagiotis S; Jacob, Wright; Neumann, Sebastian; Kutsch, Miriam; Wolters, Dirk; Tan, Eng K; Bichler, Zoë; Herrmann, Christian; Heumann, Rolf

    2016-06-01

    Mutations in the gene coding for the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) are the leading cause of genetically inherited Parkinson's disease (PD). Two of the common found mutations are the R1441C and G2019S. In this study we identified protein phosphatase 2A (PP2A) as an interacting partner of LRRK2. We were able to demonstrate that the Ras of complex protein (ROC) domain is sufficient to interact with the three subunits of PP2A in human neuroblastoma SH-SY5Y cells and in HeLa cells. The alpha subunit of PP2A is interacting with LRRK2 in the perinuclear region of HeLa cells. Silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic R1441C-LRRK2 mutant expressed in neuroblastoma SH-SY5Y cells. A similar enhancement of apoptotic nuclei was observed by downregulation of the catalytic subunit of PP2A in cultured cortical cells derived from neurons overexpressing the pathogenic mutant G2019S-LRRK2. Conversely, pharmacological activation of PP2A by sodium selenate showed a partial neuroprotection from R1441C-LRRK2-induced cellular degeneration. All these data suggest that PP2A is a new interacting partner of LRRK2 and reveal the importance of PP2A as a potential therapeutic target in PD. PMID:26894577

  18. 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.

  19. Restricted Protein Phosphatase 2A Targeting by Merkel Cell Polyomavirus Small T Antigen

    PubMed Central

    Kwun, Hyun Jin; Shuda, Masahiro; Camacho, Carlos J.; Gamper, Armin M.; Thant, Mamie; Chang, Yuan

    2015-01-01

    ABSTRACT Merkel cell polyomavirus (MCV) is a newly discovered human cancer virus encoding a small T (sT) oncoprotein. We performed MCV sT FLAG-affinity purification followed by mass spectroscopy (MS) analysis, which identified several protein phosphatases (PP), including PP2A A and C subunits and PP4C, as potential cellular interacting proteins. PP2A targeting is critical for the transforming properties of nonhuman polyomaviruses, such as simian virus 40 (SV40), but is not required for MCV sT-induced rodent cell transformation. We compared similarities and differences in PP2A binding between MCV and SV40 sT. While SV40 sT coimmunopurified with subunits PP2A Aα and PP2A C, MCV sT coimmunopurified with PP2A Aα, PP2A Aβ, and PP2A C. Scanning alanine mutagenesis at 29 sites across the MCV sT protein revealed that PP2A-binding domains lie on the opposite molecular surface from a previously described large T stabilization domain (LSD) loop that binds E3 ligases, such as Fbw7. MCV sT-PP2A interactions can be functionally distinguished by mutagenesis from MCV sT LSD-dependent 4E-BP1 hyperphosphorylation and viral DNA replication enhancement. MCV sT has a restricted range for PP2A B subunit substitution, inhibiting only the assembly of B56α into the phosphatase holoenzyme. In contrast, SV40 sT inhibits the assembly of B55α, B56α and B56ε into PP2A. We conclude that MCV sT is required for Merkel cell carcinoma growth, but its in vitro transforming activity depends on LSD interactions rather than PP2A targeting. IMPORTANCE Merkel cell polyomavirus is a newly discovered human cancer virus that promotes cancer, in part, through expression of its small T (sT) oncoprotein. Animal polyomavirus sT oncoproteins have been found to cause experimental tumors by blocking the activities of a group of phosphatases called protein phosphatase 2A (PP2A). Our structural analysis reveals that MCV sT also displaces the B subunit of PP2A to inhibit PP2A activity. MCV sT, however, only

  20. Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.

    PubMed Central

    Wilson, N J; Moss, S T; Csar, X F; Ward, A C; Hamilton, J A

    1999-01-01

    Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. PMID:10215588

  1. Structural and Biochemical Insights into the Regulation of Protein Phosphatase 2A by Small t Antigen of SV40

    SciTech Connect

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

    2007-01-01

    The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.

  2. cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation and activity of the cAMP-specific phosphodiesterase PDE4D3.

    PubMed

    Dodge-Kafka, Kimberly L; Bauman, Andrea; Mayer, Nicole; Henson, Edward; Heredia, Lorena; Ahn, Jung; McAvoy, Thomas; Nairn, Angus C; Kapiloff, Michael S

    2010-04-01

    The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56delta. Like PDE4D3, B56delta is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56delta enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56delta mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56delta phosphorylation. In general, PKA.PP2A.mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. PMID:20106966

  3. CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

    PubMed Central

    Puustinen, Pietri; Rytter, Anna; Mortensen, Monika; Kohonen, Pekka; Moreira, José M.

    2014-01-01

    mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation. PMID:24590173

  4. Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam.

    PubMed

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

    2014-01-21

    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 Tyr(307) 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 Tyr(307) 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 Tyr(307). 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 Tyr(307) by Src. PMID:24395787

  5. 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

  6. Cytoplasmic SET induces tau hyperphosphorylation through a decrease of methylated phosphatase 2A

    PubMed Central

    2014-01-01

    Background The neuronal cytoplasmic localization of SET, an inhibitor of the phosphatase 2A (PP2A), results in tau hyperphosphorylation in the brains of Alzheimer patients through mechanisms that are still not well defined. Results We used primary neurons and mouse brain slices to show that SET is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. The localization of SET in the cytoplasm, either by the translocation of endogenous SET or by internalization of the recombinant full-length SET protein, induced tau hyperphosphorylation. Cytoplasmic recombinant full-length SET in mouse brain slices induced a decrease of PP2A activity through a decrease of methylated PP2A levels. The levels of methylated PP2A were negatively correlated with tau hyperphosphorylation at Ser-202 but not with the abnormal phosphorylation of tau at Ser-422. Conclusions The presence of full-length SET in the neuronal cytoplasm is sufficient to impair PP2A methylation and activity, leading to tau hyperphosphorylation. In addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. The translocation of SET to the neuronal cytoplasm, the low activity of PP2A, and tau hyperphosphorylation are associated in the brains of Alzheimer patients. Our data show a link between the translocation of SET in the cytoplasm and the decrease of methylated PP2A levels leading to a decrease of PP2A activity and tau hyperphosphorylation. This chain of events may contribute to the pathogenesis of Alzheimer disease. PMID:24981783

  7. 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.

  8. Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5

    PubMed Central

    Gu, Ying; Barzegar, Mansoureh; Chen, Xin; Wu, Yang; Shang, Chaowei; Mahdavian, Elahe; Salvatore, Brian A.; Jiang, Shanxiang; Huang, Shile

    2015-01-01

    Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death. PMID:26517353

  9. cAMP-stimulated Protein Phosphatase 2A Activity Associated with Muscle A Kinase-anchoring Protein (mAKAP) Signaling Complexes Inhibits the Phosphorylation and Activity of the cAMP-specific Phosphodiesterase PDE4D3*

    PubMed Central

    Dodge-Kafka, Kimberly L.; Bauman, Andrea; Mayer, Nicole; Henson, Edward; Heredia, Lorena; Ahn, Jung; McAvoy, Thomas; Nairn, Angus C.; Kapiloff, Michael S.

    2010-01-01

    The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085–2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56δ. Like PDE4D3, B56δ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56δ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56δ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56δ phosphorylation. In general, PKA·PP2A·mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. PMID:20106966

  10. 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

  11. 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

    2008-01-01

    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. PMID:17975112

  12. Theophylline Represses IL-8 Secretion from Airway Smooth Muscle Cells Independently of Phosphodiesterase Inhibition. Novel Role as a Protein Phosphatase 2A Activator.

    PubMed

    Patel, Brijeshkumar S; Rahman, Md Mostafizur; Rumzhum, Nowshin N; Oliver, Brian G; Verrills, Nicole M; Ammit, Alaina J

    2016-06-01

    Theophylline is an old drug experiencing a renaissance owing to its beneficial antiinflammatory effects in chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Multiple modes of antiinflammatory action have been reported, including inhibition of the enzymes that degrade cAMP-phosphodiesterase (PDE). Using primary cultures of airway smooth muscle (ASM) cells, we recently revealed that PDE4 inhibitors can potentiate the antiinflammatory action of β2-agonists by augmenting cAMP-dependent expression of the phosphatase that deactivates mitogen-activated protein kinase (MAPK)-MAPK phosphatase (MKP)-1. Therefore, the aim of this study was to address whether theophylline repressed cytokine production in a similar, PDE-dependent, MKP-1-mediated manner. Notably, theophylline did not potentiate cAMP release from ASM cells treated with the long-acting β2-agonist formoterol. Moreover, theophylline (0.1-10 μM) did not increase formoterol-induced MKP-1 messenger RNA expression nor protein up-regulation, consistent with the lack of cAMP generation. However, theophylline (at 10 μM) was antiinflammatory and repressed secretion of the neutrophil chemoattractant cytokine IL-8, which is produced in response to TNF-α. Because theophylline's effects were independent of PDE4 inhibition or antiinflammatory MKP-1, we then wished to elucidate the novel mechanisms responsible. We investigated the impact of theophylline on protein phosphatase (PP) 2A, a master controller of multiple inflammatory signaling pathways, and show that theophylline increases TNF-α-induced PP2A activity in ASM cells. Confirmatory results were obtained in A549 lung epithelial cells. PP2A activators have beneficial effects in ex vivo and in vivo models of respiratory disease. Thus, our study is the first to link theophylline with PP2A activation as a novel mechanism to control respiratory inflammation. PMID:26574643

  13. 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

  14. Phosphatase 2A and polo kinase, two antagonistic regulators of cdc25 activation and MPF auto-amplification.

    PubMed

    Karaïskou, A; Jessus, C; Brassac, T; Ozon, R

    1999-11-01

    The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. We have studied the Cdc2/Cdc25 feed-back loop using cell-free systems derived from Xenopus prophase-arrested oocyte. Our findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase, Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independently of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process. PMID:10523510

  15. Polymerized collagen inhibits fibroblast proliferation via a mechanism involving the formation of a beta1 integrin-protein phosphatase 2A-tuberous sclerosis complex 2 complex that suppresses S6K1 activity.

    PubMed

    Xia, Hong; Nho, Richard; Kleidon, Jill; Kahm, Judy; Henke, Craig A

    2008-07-18

    Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition. PMID:18487611

  16. Protein Phosphatase 2A Inhibition with LB100 Enhances Radiation-Induced Mitotic Catastrophe and Tumor Growth Delay in Glioblastoma.

    PubMed

    Gordon, Ira K; Lu, Jie; Graves, Christian A; Huntoon, Kristin; Frerich, Jason M; Hanson, Ryan H; Wang, Xiaoping; Hong, Christopher S; Ho, Winson; Feldman, Michael J; Ikejiri, Barbara; Bisht, Kheem; Chen, Xiaoyuan S; Tandle, Anita; Yang, Chunzhang; Arscott, W Tristram; Ye, Donald; Heiss, John D; Lonser, Russell R; Camphausen, Kevin; Zhuang, Zhengping

    2015-07-01

    Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity. PMID:25939762

  17. Sodium selenate, a protein phosphatase 2A activator, mitigates hyperphosphorylated tau and improves repeated mild traumatic brain injury outcomes.

    PubMed

    Tan, Xin L; Wright, David K; Liu, Shijie; Hovens, Christopher; O'Brien, Terence J; Shultz, Sandy R

    2016-09-01

    Mild traumatic brain injuries may result in cumulative brain damage and neurodegenerative disease. To date, there is no pharmaceutical intervention known to prevent these consequences. Hyperphosphorylated tau has been associated in this process, and protein phosphatase 2A 55 kDa regulatory B subunit (PP2A/PR55) - the major tau phosphatase - is decreased after a brain insult. Sodium selenate up-regulates PP2A/PR55 and dephosphorylates tau, and may hold promise as a treatment in the mild brain injury setting. Here we investigated sodium selenate treatment in rats given repeated mild traumatic brain injuries. Rats were given three mild fluid percussion injuries or three sham-injuries, and treated with sodium selenate (1 mg/kg/day) or saline-vehicle for three months before undergoing behavioral testing, MRI, and post-mortem analysis of brain tissue. Repeated mild traumatic brain injuries increased the phosphorylation of tau and decreased PP2A/PR55, whilst inducing brain atrophy and cognitive and sensorimotor deficits. Sodium selenate treatment increased PP2A/PR55, and decreased tau phosphorylation, brain damage, and cognitive and motor impairments in rats given repeated mild traumatic brain injuries. Our findings implicate PP2A/PR55 and tau as important mechanisms in the pathophysiological aftermath of repeated mild brain traumas, and support sodium selenate as a novel and translatable treatment for these common injuries. PMID:27163189

  18. Structural basis of protein phosphatase 2A stable latency

    PubMed Central

    Jiang, Li; Stanevich, Vitali; Satyshur, Kenneth A; Kong, Mei; Watkins, Guy R.; Wadzinski, Brian E.; Sengupta, Rituparna; Xing, Yongna

    2013-01-01

    The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac–α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes. PMID:23591866

  19. Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α

    PubMed Central

    Flegg, Cameron P.; Sharma, Manisha; Medina-Palazon, Cahora; Jamieson, Cara; Galea, Melanie; Brocardo, Mariana G.; Mills, Kate; Henderson, Beric R.

    2010-01-01

    Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. PMID:20378546

  20. Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.

    PubMed Central

    Takai, A; Ohno, Y; Yasumoto, T; Mieskes, G

    1992-01-01

    As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses. PMID:1329723

  1. Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A.

    PubMed

    Maton, Gilliane; Lorca, Thierry; Girault, Jean-Antoine; Ozon, René; Jessus, Catherine

    2005-06-01

    The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, we demonstrate that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. We therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase. PMID:15923661

  2. 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

  3. Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease*

    PubMed Central

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

    2013-01-01

    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, I2PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I2PP2A 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 I2PP2A 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-I2PP2A, except when I2PP2A 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 I2PP2A, 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-I2PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target. PMID:23640887

  4. 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.

  5. 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

  6. Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae.

    PubMed

    Sugajska, E; Swiatek, W; Zabrocki, P; Geyskens, I; Thevelein, J M; Zolnierowicz, S; Wera, S

    2001-05-01

    The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients. PMID:11401708

  7. Microcystin-LR promotes proliferation by activating Akt/S6K1 pathway and disordering apoptosis and cell cycle associated proteins phosphorylation in HL7702 cells.

    PubMed

    Liu, Jinghui; Wang, Hao; Wang, Beilei; Chen, Tao; Wang, Xiaofeng; Huang, Pu; Xu, Lihong; Guo, Zonglou

    2016-01-01

    Our previous studies had shown that MC-LR inhibited PP2A activity and hyperphosphorylated PP2A substrates at 24 h exposure in HL7702 cells. Although the cytoskeleton was rearranged, the cellular effects were not observed. The purpose of the present study with HL7702 cell exposed to MC-LR for 1-72 h was to further uncover the adverse effects of MC-LR comprehensively. The results showed that there were no obvious difference in apoptosis rate and cell-cycle distribution but the cell proliferation was changed since 36 h exposure while the uptake of MC-LR and its binding to PP2A/C kept unchanged since 1h exposure. PP2A activity had not manifested continued decline compare to 24h exposure and PP2A regulator α4 was found to release its associated PP2A/C since 1h exposure. The increasing of p-Akt-T308, p-Akt-S473, p-S6K1, p-S6, and p-4E-BP1 since 1h MC-LR exposure indicated that Akt/S6K1 cascade had been activated as early as 1h MC-LR treatment. And, PI3K/Akt inhibitor (LY294002) blocked MC-LR-induced Akt/S6K1 activation and proliferation. Besides, MC-LR also led to hyperphosphorylation of c-Myc, c-Jun, Bcl-2 and Bad and activation of Cdk1. Our study indicated that MC-LR exposure promoted HL7702 cell proliferation and the main mechanism was the activation of Akt/S6K1 cascade. Meanwhile, hyperphosphorylation of Bcl-2, Bad, c-Myc and c-Jun might also be involved. And, the inhibition of PP2A was the major reason for these molecular changes. PMID:26506538

  8. Telomerase activation as a repair response to radiation-induced DNA damage in Y79 retinoblastoma cells.

    PubMed

    Akiyama, Masaharu; Ozaki, Kohji; Kawano, Takeshi; Yamada, Osamu; Kawauchi, Kiyotaka; Ida, Hiroyuki; Yamada, Hisashi

    2013-10-28

    The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation. PMID:23850566

  9. Novel FTY720-Based Compounds Stimulate Neurotrophin Expression and Phosphatase Activity in Dopaminergic Cells

    PubMed Central

    2014-01-01

    α-Synuclein is a chaperone-like protein implicated in Parkinson’s disease (PD). Among α-synuclein’s normal functions is an ability to bind to and stimulate the activity of the protein phosphatase 2A (PP2A) catalytic subunit in vitro and in vivo. PP2A activity is impaired in PD and in dementia with Lewy Bodies in brain regions harboring α-synuclein aggregates. Using PP2A as the readout, we measured PP2A activity in response to α-synuclein, ceramides, and FTY720, and then on the basis of those results, we created new FTY720 compounds. We then measured the effects of those compounds in dopaminergic cells. In addition to stimulating PP2A, all three compounds stimulated the expression of brain derived neurotrophic factor and protected MN9D cells against tumor-necrosis-factor-α-associated cell death. FTY720-C2 appears to be more potent while FTY720-Mitoxy targets mitochondria. Importantly, FTY720 is already FDA approved for treating multiple sclerosis and is used clinically worldwide. Our findings suggest that FTY720 and our new FTY720-based compounds have considerable potential for treating synucleinopathies such as PD. PMID:25050165

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

    PubMed

    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

  11. 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

  12. 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.

  13. Acute administration of L-dopa induces changes in methylation metabolites, reduced protein phosphatase 2A methylation and hyperphosphorylation of Tau protein in mouse brain

    PubMed Central

    Bottiglieri, Teodoro; Arning, Erland; Wasek, Brandi; Nunbhakdi-Craig, Viyada; Sontag, Jean-Marie; Sontag, Estelle

    2012-01-01

    Folate deficiency and hypomethylation have been implicated in a number of age-related neurodegenerative disorders including dementia and Parkinson’s disease (PD). Levodopa (L-dopa) therapy in PD patients has been shown to cause an increase in plasma total homocysteine (tHcy) as well as depleting cellular concentrations of the methyl donor, S-adenosylmethionine (SAM), and increasing the demethylated product S-adenosylhomocysteine (SAH). Modulation of the cellular SAM/SAH ratio can influence activity of methyltransferase enzymes including leucine carboxyl methyltransferase (LCMT1), that specifically methylates Ser/Thr protein phosphatase 2A (PP2A), a major Tau phosphatase. Here we show in human SH-SY5Y cells and dopaminergic neurons, and in wild type mice that L-dopa results in a reduced SAM/SAH ratio that is associated with hypomethylation of PP2A and increased phosphorylation of Tau (p-Tau) at the Alzheimer disease-like PHF-1 phosphoepitope. The effect of L-dopa on PP2A and p-Tau was exacerbated in cells exposed to folate deficiency. In the folate deficient mouse model, L-dopa resulted in a marked depletion of SAM and increase in SAH in various brain regions with parallel down regulation of PP2A methylation and increased Tau phosphorylation. L-dopa also enhanced demethylated PP2A amounts in the liver. These findings reveal a novel mechanism involving methylation-dependent pathways in which L-dopa induces PP2A hypomethylation and increases Tau phosphorylation, which may be potentially detrimental to neuronal cells. PMID:22764226

  14. Theoretical research into anticancer activity of diterpenes isolated from the paraiban flora.

    PubMed

    Ishiki, Hamilton; Junior, Francisco J B Mendonça; Santos, Paula F; Tavares, Josean F; Tavares, Josean F; Silva, Marcelo S; Scotti, Marcus T

    2014-07-01

    Many studies of the scientific literature discuss the anticancer activity of diterpenes inhibiting the Akt/IKK/NF-kappaB pro-survival signaling cascade, mainly by the activation of serine/threonine phosphatase PP2A. The aim of this work was to evaluate and compare the anticancer potential of three atisane, three kaurane and three trachylobane diterpenes extracted from the roots of Xylopia langsdorffiana. Thus, we investigated the reactivity (H-L(GAP) parameter), HOMO atmosphere favorable to neutralize the radical reactivity, and the docking of compounds with PP2A. With all approaches, this theoretical study showed that atisane diterpenes have favorable characteristics for antitumor activity, like electron donating ability and greater hydrophilic interactions with the enzyme, by inhibition of Akt/IKK/NF-kappaB, and activation of PP2A. PMID:25230491

  15. Cucurbitacin B reverses multidrug resistance by targeting CIP2A to reactivate protein phosphatase 2A in MCF-7/adriamycin cells.

    PubMed

    Cai, Fen; Zhang, Liang; Xiao, Xiangling; Duan, Chao; Huang, Qiuyue; Fan, Chunsheng; Li, Jian; Liu, Xuewen; Li, Shan; Liu, Ying

    2016-08-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in various tumors. A previous study found that CIP2A expression is associated with doxorubicin (Dox) resistance. In the present study, we investigated whether cucurbitacin B (CuB), a natural anticancer compound found in Cucurbitaceae, reversed multidrug resistance (MDR) and downregulated CIP2A expression in MCF-7/Adriamycin (MCF-7/Adr) cells, a human breast multidrug-resistant cancer cell line. CuB treatment significantly suppressed MCF-7/Adr cell proliferation, and reversed Dox resistance. CuB treatment also induced caspase-dependent apoptosis, decreased phosphorylation of Akt (pAkt). The suppression of pAkt was mediated through CuB-induced activation of protein phosphatase 2A (PP2A). Furthermore, CuB activated PP2A through the suppression of CIP2A. Silencing CIP2A enhanced CuB-induced growth inhibition, apoptosis and MDR inhibition in MCF-7/Adr cells. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A promotes the reversal of MDR induced by CuB. PMID:27350399

  16. PME-1 protects ERK pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma

    PubMed Central

    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

    2010-01-01

    ERK/MAPK 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 (PP2A) 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 PKC. In malignant glioblastoma, 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 important functional role for PME-1 in the disease progression of human astrocytic gliomas. PMID:19293187

  17. Rescue of GABAB and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice.

    PubMed

    Lecca, Salvatore; Pelosi, Assunta; Tchenio, Anna; Moutkine, Imane; Lujan, Rafael; Hervé, Denis; Mameli, Manuel

    2016-03-01

    The lateral habenula (LHb) encodes aversive signals, and its aberrant activity contributes to depression-like symptoms. However, a limited understanding of the cellular mechanisms underlying LHb hyperactivity has precluded the development of pharmacological strategies to ameliorate depression-like phenotypes. Here we report that an aversive experience in mice, such as foot-shock exposure (FsE), induces LHb neuronal hyperactivity and depression-like symptoms. This occurs along with increased protein phosphatase 2A (PP2A) activity, a known regulator of GABAB receptor (GABABR) and G protein-gated inwardly rectifying potassium (GIRK) channel surface expression. Accordingly, FsE triggers GABAB1 and GIRK2 internalization, leading to rapid and persistent weakening of GABAB-activated GIRK-mediated (GABAB-GIRK) currents. Pharmacological inhibition of PP2A restores both GABAB-GIRK function and neuronal excitability. As a consequence, PP2A inhibition ameliorates depression-like symptoms after FsE and in a learned-helplessness model of depression. Thus, GABAB-GIRK plasticity in the LHb represents a cellular substrate for aversive experience. Furthermore, its reversal by PP2A inhibition may provide a novel therapeutic approach to alleviate symptoms of depression in disorders that are characterized by LHb hyperactivity. PMID:26808347

  18. Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.

    PubMed Central

    Clarke, P R; Hoffmann, I; Draetta, G; Karsenti, E

    1993-01-01

    We have examined the roles of type-1 (PP-1) and type-2A (PP-2A) protein-serine/threonine phosphatases in the mechanism of activation of p34cdc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP-2A by okadaic acid but not by specific inhibition of PP-1 by inhibitor-2. Activation of the kinase can be blocked by addition of the purified catalytic subunit of PP-2A at a twofold excess over the activity in the extract. The catalytic subunit of PP-1 can also block kinase activation, but very high levels of activity are required. Activation of p34cdc2/cyclin B protein kinase requires dephosphorylation of p34cdc2 on Tyr15. This reaction is catalysed by cdc25-C phosphatase that is itself activated by phosphorylation. We show that, in interphase extracts, inhibition of PP-2A by okadaic acid completely blocks cdc25-C dephosphorylation, whereas inhibition of PP-1 by specific inhibitors has no effect. This indicates that a type-2A protein phosphatase negatively regulates p34cdc2/cyclin B protein kinase activation primarily by maintaining cdc25-C phosphatase in a dephosphorylated, low activity state. In extracts containing active p34cdc2/cyclin B protein kinase, dephosphorylation of cdc25-C is inhibited, whereas the activity of PP-2A (and PP-1) towards other substrates is unaffected. We propose that this specific inhibition of cdc25-C dephosphorylation is part of a positive feedback loop that also involves direct phosphorylation and activation of cdc25-C by p34cdc2/cyclin B. Dephosphorylation of cdc25-C is also inhibited when cyclin A-dependent protein kinase is active, and this may explain the potentiation of p34cdc2/cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc2/cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited or when stably phosphorylated cdc25-C is added, but not when PP-1 is specifically inhibited. This suggests

  19. 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.

  20. The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits.

    PubMed Central

    Zolnierowicz, S; Van Hoof, C; Andjelković, N; Cron, P; Stevens, I; Merlevede, W; Goris, J; Hemmings, B A

    1996-01-01

    Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein micro-sequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription-PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either alpha or beta isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the epsilon isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates. PMID:8694763

  1. Activation of carbohydrate response element binding protein (ChREBP) by ethanol

    PubMed Central

    Liangpunsakul, Suthat; Ross, Ruth A.; Crabb, David W.

    2012-01-01

    Carbohydrate response element binding protein (ChREBP) is a transcription factor involved in hepatic lipogenesis. Its function is in part under the control of AMP-activated protein kinase (AMPK) and protein phosphatase 2A (PP2A). Given known effects of ethanol on AMPK and PP2A, it is plausible that ethanol might enhance fatty acid synthesis by increasing the activity of ChREBP. We hypothesized that another potential pathway of ethanol-induced hepatic steatosis is mediated by activation of ChREBP. Methods The effects of ethanol on ChREBP were assessed in hepatoma cells and in C57BL/6J mice fed with the Lieber-DeCarli diet. Results When the cells were exposed to ethanol (50 mM) for 24 hrs, the activity of a liver pyruvate kinase (LPK) promoter-luciferase reporter was increased by ~4-fold. Ethanol feeding of mice resulted in the translocation of ChREBP from cytosol to the nucleus. PP2A activity was increased in the liver of ethanol-fed mice by 22%. We found no difference in the levels of hepatic Xu-5-P between ethanol-fed mice and controls. Transfection of a constitutively active AMPK expression plasmid suppressed the basal activity of the LPK luciferase reporter and abolished the effect of ethanol on the reporter activity. However, transfection of rat hepatoma cells with a dominant negative AMPK expression plasmid induced basal LPK luciferase activity by only ~20%. The effect of ethanol on ChREBP was attenuated in the presence of okadaic acid, an inhibitor of PP2A. Conclusions The effects of ethanol on AMPK and PP2A may result in activation of ChREBP, providing another potential mechanism for ethanol-induced hepatic steatosis. However, additional okadaic acid-insensitive effects appear to be important as well. PMID:23266705

  2. Novel effects of FTY720 on perinuclear reorganization of keratin network induced by sphingosylphosphorylcholine: Involvement of protein phosphatase 2A and G-protein-coupled receptor-12.

    PubMed

    Park, Mi Kyung; Park, Soyeun; Kim, Hyun Ji; Kim, Eun Ji; Kim, So Yeon; Kang, Gyeoung Jin; Byun, Hyun Jung; Kim, Sang Hee; Lee, Ho; Lee, Chang Hoon

    2016-03-15

    Sphingosylphosphorylcholine (SPC) evokes perinuclear reorganization of keratin 8 (K8) filaments and regulates the viscoelasticity of metastatic cancer cells leading to enhanced migration. Few studies have addressed the compounds modulating the viscoelasticity of metastatic cancer cells. We studied the effects of sphingosine (SPH), sphingosine 1-phosphate (S1P), FTY720 and FTY720-phosphate (FTY720P) on SPC-induced K8 phosphorylation and reorganization using Western blot and confocal microscopy, and also evaluated the elasticity of PANC-1 cells by atomic force microscopy. FTY720, FTY720P, SPH, and S1P concentration-dependently inhibited SPC-evoked phosphorylation and reorganization of K8, and migration of PANC-1 cells. SPC triggered reduction and narrow distribution of elastic constant K and conversely, FTY720 blocked them. A common upstream regulator of JNK and ERK, protein phosphatase 2A (PP2A) expression was reduced by SPC, but was restored by FTY720 and FTY72P. Butyryl forskolin, a PP2A activator, suppressed SPC-induced K8 phosphorylation and okadaic acid, a PP2A inhibitor, induced K8 phosphorylation. Gene silencing of PP2A also led to K8 phosphorylation, reorganization and migration. We also investigated the involvement of GPR12, a high-affinity SPC receptor, in SPC-evoked keratin phosphorylation and reorganization. GPR12 siRNA suppressed the SPC-triggered phosphorylation and reorganization of K8. GPR12 overexpression stimulated keratin phosphorylation and reorganization even without SPC. FTY720 and FTY720P suppressed the GPR12-induced phosphorylation and reorganization of K8. The collective data indicates that FTY720 and FTY720P suppress SPC-induced phosphorylation and reorganization of K8 in PANC-1 cells by restoring the expression of PP2A via GPR12. These findings might be helpful in the development of compounds that modulate the viscoelasticity of metastatic cancer cells and various SPC actions. PMID:26872988

  3. Induction of p53-Independent Apoptosis by the Adenovirus E4orf4 Protein Requires Binding to the Bα Subunit of Protein Phosphatase 2A

    PubMed Central

    Marcellus, Richard C.; Chan, Helen; Paquette, Denis; Thirlwell, Sarah; Boivin, Dominique; Branton, Philip E.

    2000-01-01

    Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Bα subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Bα-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Bα subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing. PMID:10933694

  4. Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    PubMed Central

    Mo, Shu-Ting; Chiang, Shang-Ju; Lai, Tai-Yu; Cheng, Yu-Ling; Chung, Cheng-En; Kuo, Spencer C. H.; Reece, Kelie M.; Chen, Yung-Cheng; Chang, Nan-Shan; Wadzinski, Brian E.; Chiang, Chi-Wu

    2014-01-01

    Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells. PMID:25536081

  5. Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts.

    PubMed

    Huang, Chong-xin; Lv, Bo; Wang, Yue

    2015-01-01

    Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis. PMID:26538836

  6. Protein Phosphatase 2A Mediates Oxidative Stress Induced Apoptosis in Osteoblasts

    PubMed Central

    Huang, Chong-xin; Lv, Bo; Wang, Yue

    2015-01-01

    Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis. PMID:26538836

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

    PubMed Central

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

    2013-01-01

    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. PMID:24297885

  8. Regulation of autophagy by coordinated action of mTORC1 and protein phosphatase 2A.

    PubMed

    Wong, Pui-Mun; Feng, Yan; Wang, Junru; Shi, Rong; Jiang, Xuejun

    2015-01-01

    Autophagy is a cellular catabolic process critical for cell viability and homoeostasis. Inhibition of mammalian target of rapamycin (mTOR) complex-1 (mTORC1) activates autophagy. A puzzling observation is that amino acid starvation triggers more rapid autophagy than pharmacological inhibition of mTORC1, although they both block mTORC1 activity with similar kinetics. Here we find that in addition to mTORC1 inactivation, starvation also causes an increase in phosphatase activity towards ULK1, an mTORC1 substrate whose dephosphorylation is required for autophagy induction. We identify the starvation-stimulated phosphatase for ULK1 as the PP2A-B55α complex. Treatment of cells with starvation but not mTORC1 inhibitors triggers dissociation of PP2A from its inhibitor Alpha4. Furthermore, pancreatic ductal adenocarcinoma cells, whose growth depends on high basal autophagy, possess stronger basal phosphatase activity towards ULK1 and require ULK1 for sustained anchorage-independent growth. Taken together, concurrent mTORC1 inactivation and PP2A-B55α stimulation fuel ULK1-dependent autophagy. PMID:26310906

  9. 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.

  10. Retinoic Acid Protects Cardiomyocytes from High Glucose-Induced Apoptosis via Inhibition of Sustained Activation of NF-κB Signaling

    PubMed Central

    Nizamutdinova, Irina T.; Guleria, Rakeshwar S.; Singh, Amar B.; Kendall, Jonathan A.; Baker, Kenneth M.; Pan, Jing

    2012-01-01

    We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6 and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets. PMID:22718360

  11. B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

    PubMed Central

    Maertens, Goedele N.

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  12. B'-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase.

    PubMed

    Maertens, Goedele N

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  13. Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

    PubMed Central

    Chang, Heng-Kwei

    2015-01-01

    Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

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

    PubMed

    Cristóbal, Ion; Rojo, Federico; Madoz-Gúrpide, Juan; García-Foncillas, Jesús

    2016-06-15

    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

  15. The role of protein phosphatase 2A in regulating Wnt signaling and apoptosis

    NASA Astrophysics Data System (ADS)

    Li, Xinghai

    Protein phosphatase 2A (PP2A) is a major serine/threonine-specific phosphatase and regulates a significant array of cellular events. This dissertation primarily describes the novel role of PP2A in Wnt signaling and apoptosis. First, PP2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus. PP2A is required for β- catenin degradation in vitro. A PP2A heterotrimer containing A, C, and B56 subunits was co- immunoprecipitated with axin. A, C, and B56 subunits each have ventralizing ability in Xenopus embryos. B56 was epistatically positioned downstream of GSK3β and axin but upstream of β-catenin. Second, B56-targeted PP2A is required for survival and protects from apoptosis in Drosophila. Loss of A, C, or B56 subunits by RNA interference (RNAi) induced apoptosis in S2 cells, which requires the presence of specific caspases. Epistasis analysis placed B56-targeted PP2A functionally upstream of Apaf-1, Reaper and Hid, and p53. Loss of B56-targeted PP2A in Drosophila embryos by RNAi resulted in abortion of embryo development and this phenotype was rescued by co-RNAi of Drice. Third, two conserved domains in B subunits mediate binding to the A subunit of PP2A. B subunits have no detectable sequence homology among different families. In vitro expression of a series of B56α fragments identified two distinct domains that independently bound to the A subunit. Sequence alignment of these A subunit-binding domains recognized conserved residues in B/PR55 and B'/PR72 family members that serve a similar function. Fourth, to examine whether the B56β gene within 11q12 is a tumor suppressor mutated in neuroblastoma, the DNA and RNA samples from neuroblastoma patients and cell lines were analyzed and no mutations were identified in the coding regions of the B56β gene. Finally, to identify novel regulatory subunits of PP2A in S. cerevisiae , biochemical approaches for purifying PP2A-associated novel regulators were undertaken. Although the A and C subunit complex in the

  16. Resistin, a fat-derived secretory factor, promotes metastasis of MDA-MB-231 human breast cancer cells through ERM activation

    PubMed Central

    Lee, Jung Ok; Kim, Nami; Lee, Hye Jeong; Lee, Yong Woo; Kim, Su Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2016-01-01

    Resistin, an adipocyte-secreted factor, is known to be elevated in breast cancer patients. However, the molecular mechanism by which resistin acts is not fully understood. The aim of this study was to investigate whether resistin could stimulate invasion and migration of breast cancer cells. Here, we report that resistin stimulated invasion and migration of breast cancer cells as well as phosphorylation of c-Src. Inhibition of c-Src blocked resistin-induced breast cancer cell invasion. Resistin increased intracellular calcium concentration, and chelation of intracellular calcium blocked resistin-mediated activation of Src. Resistin also induced phosphorylation of protein phosphatase 2A (PP2A). Inhibition of c-Src blocked resistin-mediated PP2A phosphorylation. In addition, resistin increased phosphorylation of PKCα. Inhibition of PP2A enhanced resistin-induced PKCα phosphorylation, demonstrating that PP2A activity is critical for PKCα phosphorylation. Resistin also increased phosphorylation of ezrin, radixin, and moesin (ERM). Additionally, ezrin interacted with PKCα, and resistin promoted co-localization of ezrin and PKCα. Either inhibition of c-Src and PKCα or knock-down of ezrin blocked resistin-induced breast cancer cells invasion. Moreover, resistin increased expression of vimentin, a key molecule for cancer cell invasion. Knock-down of ezrin abrogated resistin-induced vimentin expression. These results suggest that resistin play as a critical regulator of breast cancer metastasis. PMID:26729407

  17. 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

  18. 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

    2016-03-01

    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

  19. 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. PMID:26080028

  20. Knockdown of microRNA-195 contributes to protein phosphatase-2A inactivation in rats with chronic brain hypoperfusion.

    PubMed

    Liu, Cheng-Di; Wang, Qin; Zong, De-Kang; Pei, Shuang-Chao; Yan, Yan; Yan, Mei-Ling; Sun, Lin-Lin; Hao, Yang-Yang; Mao, Meng; Xing, Wen-Jing; Ren, Huan; Ai, Jing

    2016-09-01

    Reduction of protein phosphatase-2A (PP2A) activity is a common clinical feature of Alzheimer's disease and vascular dementia. In this study, we observed that chronic brain hypoperfusion induced by bilateral common carotid artery occlusion of rats led to PP2A inactivation based on the increase in tyrosine-307 phosphorylation and leucine-309 demethylation of PP2AC and the depression in PP2ABα. Knockdown of miR-195 using overexpression of its antisense molecule oligonucleotide (pre-AMO-miR-195) delivered by a lentivirus (lenti-pre-AMO-miR-195) increased tyrosine-307 phosphorylation and decreased both PP2ABα expression and leucine-309 methylation; these effects were prevented by the overexpression of miR-195 using lenti-pre-miR-195 and controlled by an increase in methylesterase (PME-1) and a decrease in leucine carboxyl methyltransferase-1. In vitro studies demonstrated that miR-195 regulated PME-1 expression by binding to the Ppme1 gene 3'-untranslated region (3'UTR) domain. Masking the miR-195 binding sites in the amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 genes prevented miR-195-induced leucine carboxyl methyltransferase-1 elevation. We concluded that the miR-195 downregulation in chronic brain hypoperfusion involved PP2A inactivity, which was mediated by the post-transcriptional regulation PME-1, APP, and β-site APP cleaving enzyme 1 expression. PMID:27459928

  1. Curcumin treatment recovery the decrease of protein phosphatase 2A subunit B induced by focal cerebral ischemia in Sprague-Dawley rats

    PubMed Central

    Shah, Fawad-Ali; Park, Dong-Ju; Gim, Sang-Ah

    2015-01-01

    Curcumin provides various biological effects through its anti-inflammatory and antioxidant properties. Moreover, curcumin exerts a neuroprotective effect against ischemic condition-induced brain damage. Protein phosphatase 2A (PP2A) is a ubiquitous serine and threonine phosphatase with various cell functions and broad substrate specificity. Especially PP2A subunit B plays an important role in nervous system. This study investigated whether curcumin regulates PP2A subunit B expression in focal cerebral ischemia. Cerebral ischemia was induced surgically by middle cerebral artery occlusion (MCAO). Adult male rats were injected with either vehicle or curcumin (50 mg/kg) 1 h after MCAO and cerebral cortex tissues were isolated 24 h after MCAO. A proteomics study, reverse transverse-PCR and Western blot analyses were performed to examine PP2A subunit B expression levels. We identified a reduction in PP2A subunit B expression in MCAO-operated animals using a proteomic approach. However, curcumin treatment prevented injury-induced reductions in PP2A subunit B levels. Reverse transverse-PCR and Western blot analyses confirmed that curcumin treatment attenuated the injury-induced reduction in PP2A subunit B levels. These findings can suggest that the possibility that curcumin maintains levels of PP2A subunit B in response to cerebral ischemia, which likely contributes to the neuroprotective function of curcumin in cerebral ischemic injury. PMID:26472966

  2. 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.

  3. Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A.

    PubMed

    Dagda, Ruben K; Barwacz, Chris A; Cribbs, J Thomas; Strack, Stefan

    2005-07-22

    Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B'') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme. PMID:15923182

  4. Effects of light and the regulatory B-subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae) infestation

    PubMed Central

    Rasool, Brwa; Karpinska, Barbara; Konert, Grzegorz; Durian, Guido; Denessiouk, Konstantin; Kangasjärvi, Saijaliisa; Foyer, Christine H.

    2014-01-01

    The interactions between biotic and abiotic stress signaling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP)2A regulatory subunit B'γ (gamma; pp2a-b'γ) or B'ζ (zeta; pp2a-b'ζ1-1 and pp2a-b'ζ 1-2) and in gamma zeta double mutants (pp2a-b'γζ) lacking both subunits. All the mutants except for pp2a-b'ζ 1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b'γ mutant relative to the wild type but not in the pp2a-b'γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b'γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of B-subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonization, depending on the prevailing abiotic stress environment. PMID:25191331

  5. 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

  6. 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

  7. 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 Responses.

    PubMed

    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

    2015-09-01

    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

  8. Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in Chlamydomonas reinhardtii

    PubMed Central

    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. PMID:24086163

  9. Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair

    PubMed Central

    Wei, Dongping; Parsels, Leslie A.; Karnak, David; Davis, Mary A.; Parsels, Joshua D.; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S.; Sun, Yi; Morgan, Meredith A.

    2013-01-01

    Purpose To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we performed an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. Experimental Design We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacological (LB100, a small molecule entering Phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. Results PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P<0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γH2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. Conclusions Collectively, our data demonstrate that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer. PMID:23780887

  10. 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.

  11. Magnetic activity of the star Corot-Exo-2a

    NASA Astrophysics Data System (ADS)

    Savanov, I. S.

    2010-05-01

    Continuous photometric observations of the young active solar-type star Corot-Exo-2a using the “Corot” space telescope obtained over 142 days were used to analyze the star’s surface temperature inhomogeneities and to monitor their continuous evolution. This analysis was based on the iPH code, which reconstructs the distribution of temperature inhomogeneities on the surface of a star based on its light curve in a two-temperature approximation. We identified five time intervals in the positions of active areas, with corresponding flip-flop events, interpreted as activity periods. Their durations were between 55 and 15 days. The time scale for the active-longitude flip-flops of Corot-Exo-2a is a few tens of days, rather than years, as for other stars studied earlier. We detected motions of the active longitudes, possibly indicating differential rotation of the star. The phenomenon of flip-flops in the positions of active longitudes has a complex character. This is the first case apart fromthe Sun where we are able to follow the appearance and development of temperature inhomogeneities on a stellar surface in such detail. We determined typical timescales for variations of the activity parameter of the star in the ranges 17-20, 28-32, 33-38, and 51-55 days, which characterize changes of the brightness variation amplitude, the spotted surface area, positions of active areas, and brightness variations.

  12. Structure of the Protein Phosphatase 2A Holoenzyme

    SciTech Connect

    Xu,Y.; Xing, Y.; Chen, Y.; Chao, Y.; Lin, Z.; Fan, E.; Yu, J.; Strack, S.; Jeffrey, P.; Shi, Y.

    2006-01-01

    Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

  13. 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

  14. Roles of phosphotase 2A in nociceptive signal processing

    PubMed Central

    2013-01-01

    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. PMID:24010880

  15. Low α2β1 Integrin Function Enhances the Proliferation of Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis by Activation of the β-Catenin Pathway

    PubMed Central

    Xia, Hong; Seeman, Jeremy; Hong, Jian; Hergert, Polla; Bodem, Vidya; Jessurun, Jose; Smith, Karen; Nho, Richard; Kahm, Judy; Gaillard, Philippe; Henke, Craig

    2013-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and incurable fibroproliferative disorder characterized by unrelenting proliferation of fibroblasts and their deposition of collagen within alveoli, resulting in permanently scarred, nonfunctional airspaces. Normally, polymerized collagen suppresses fibroblast proliferation and serves as a physiological restraint to limit fibroproliferation after tissue injury. The IPF fibroblast, however, is a pathologically altered cell that has acquired the capacity to elude the proliferation-suppressive effects of polymerized collagen. The mechanism for this phenomenon remains incompletely understood. Here, we demonstrate that expression of α2β1 integrin, a major collagen receptor, is pathologically low in IPF fibroblasts interacting with polymerized collagen. Low integrin expression in IPF fibroblasts is associated with a failure to induce PP2A phosphatase activity, resulting in abnormally high levels of phosphorylated (inactive) GSK-3β and high levels of active β-catenin in the nucleus. Knockdown of β-catenin in IPF fibroblasts inhibits their ability to proliferate on collagen. Interdiction of α2β1 integrin in control fibroblasts reproduces the IPF phenotype and leads to the inability of these cells to activate PP2A, resulting in high levels of phosphorylated GSK-3β and active β-catenin and in enhanced proliferation on collagen. Our findings indicate that the IPF fibroblast phenotype is characterized by low α2β1 integrin expression, resulting in a failure of integrin to activate PP2A phosphatase, which permits inappropriate activation of the β-catenin pathway. PMID:22642910

  16. 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.

  17. Oridonin inhibits gefitinib-resistant lung cancer cells by suppressing EGFR/ERK/MMP-12 and CIP2A/Akt signaling pathways.

    PubMed

    Xiao, Xiangling; He, Zhongwei; Cao, Wei; Cai, Fen; Zhang, Liang; Huang, Qiuyue; Fan, Chunsheng; Duan, Chao; Wang, Xiaobo; Wang, Jiu; Liu, Ying

    2016-06-01

    Oridonin (Ori), a diterpenoid compound extracted from traditional medicinal herbs, elicits antitumor effects on many cancer types. However, whether Ori can be used in gefitinib-resistant non-small cell lung cancer (NSCLC) cells remains unclear. This study investigated the antitumor activity and underlying mechanisms of Ori. Results demonstrated that this compound dose-dependently inhibited the proliferation, invasion, and migration of the gefitinib-resistant NSCLC cells in vitro. Ori also significantly downregulated the phosphorylation of EGFR, ERK, Akt, expression levels of matrix metalloproteinase-12 (MMP-12), and the cancerous inhibitor of protein phosphatase 2A (CIP2A). In addition, Ori upregulated protein phosphatase 2A (PP2A) activity of gefitinib-resistant NSCLC cells. Ori combined with docetaxel synergistically inhibited these cells. Ori also inhibited tumor growth in murine models. Immunohistochemistry results further revealed that Ori downregulated phospho-EGFR, MMP-12, and CIP2A in vivo. These findings indicated that Ori can inhibit the proliferation, invasion, and migration of gefitinib-resistant NSCLC cells by suppressing EGFR/ERK/MMP-12 and CIP2A/PP2A/Akt signaling pathways. Thus, Ori may be a novel effective candidate to treat gefitinib-resistant NSCLC. PMID:27082429

  18. The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

    PubMed Central

    Brownlee, Christopher W.; Klebba, Joey E.; Buster, Daniel W.

    2011-01-01

    Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. In this paper, we show that PP2A (Protein Phosphatase 2ATwins) counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A’s regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. We demonstrate that ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification. PMID:21987638

  19. 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. PMID:27177932

  20. Deregulation of Protein Phosphatase 2A and Hyperphosphorylation of τ Protein Following Onset of Diabetes in NOD Mice

    PubMed Central

    Papon, Marie-Amélie; El Khoury, Noura B.; Marcouiller, François; Julien, Carl; Morin, Françoise; Bretteville, Alexis; Petry, Franck R.; Gaudreau, Simon; Amrani, Abdelaziz; Mathews, Paul M.; Hébert, Sébastien S.; Planel, Emmanuel

    2013-01-01

    The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated τ protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects τ pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on τ phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display τ hyperphosphorylation. τ phosphorylation at τ-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, τ was hyperphosphorylated at the τ-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and τ hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated τ phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like τ hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology. PMID:22961084

  1. 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. PMID:26548718

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

    PubMed

    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

  4. 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

  5. Force-inhibiting effect of Ser/Thr protein phosphatase 2A inhibitors on bovine ciliary muscle.

    PubMed

    Ishida, Minori; Takeya, Kosuke; Miyazu, Motoi; Yoshida, Akitoshi; Takai, Akira

    2015-01-01

    Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca2+-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca2+-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction. PMID:26727726

  6. Rapamycin enhances eIF4E phosphorylation by activating MAP kinase-interacting kinase 2a (Mnk2a).

    PubMed

    Stead, Rebecca L; Proud, Christopher G

    2013-08-19

    Eukaryotic initiation factor eIF4E and its phosphorylation play key roles in cell transformation and tumorigenesis. eIF4E is phosphorylated by the Mnks (MAP (mitogen-activated protein) kinase-interacting kinases). Rapamycin increases eIF4E phosphorylation in cancer cells, potentially limiting their anti-cancer effects. Here we show that the rapamycin-induced increase in eIF4E phosphorylation reflects increased activity of Mnk2 but not Mnk1. This activation requires a novel phosphorylation site in Mnk2a, Ser437. Our findings have potentially important implications for the use of rapamycin and its analogues in cancer therapy, suggesting that inhibitors of mTOR and Mnk (or Mnk2) may be more efficacious than rapalogs alone. PMID:23831578

  7. LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-β phosphorylation

    PubMed Central

    Menden, Heather; Tate, Everett; Hogg, Neil

    2013-01-01

    Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E+ (marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-β (IKK-β) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-β phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-β associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-β phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD. PMID:23333803

  8. CIP2A regulates cancer metabolism and CREB phosphorylation in non-small cell lung cancer.

    PubMed

    Peng, Bo; Lei, Ningjing; Chai, Yurong; Chan, Edward K L; Zhang, Jian-Ying

    2015-01-01

    The cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized endogenous inhibitor of the phosphatase activity of protein phosphatase 2A (PP2A), which extends the half-life of oncogenic protein c-myc and promotes in vivo tumor growth. The function of CIP2A in cancer progression is still poorly understood. To uncover the underlying mechanism of CIP2A-mediated cell proliferation, we implemented a two-dimensional electrophoresis (2DE)-based proteomic approach to examine lung cancer cell H1299 with and without CIP2A. We found 47 proteins differentially expressed where 19 proteins were upregulated and 28 proteins were downregulated. These were categorized into functional groups such as metabolism (25%), transcriptional and translational control (23%), and the signaling pathway and protein degradation (20%). On one hand, we validate our proteomic work by measuring the metabolic change. The knockdown of CIP2A decreased the expression of LDH-A as well as the enzymatic activity, accompanying with a decreased lactate production, an increased NADH/NAD+ ratio and ROS production. On the other hand, we found that CIP2A may regulate CREB activity through bioinformatics analysis. Our following experiments showed that, CIP2A positively regulated the phosphorylation of CREB in response to the serum treatment. Therefore, our proteomic study suggested that CIP2A mediates cancer progression through the metabolic pathway and intracellular signaling cascade. PMID:25325377

  9. Ceramide-Activated Phosphatase Mediates Fatty Acid–Induced Endothelial VEGF Resistance and Impaired Angiogenesis

    PubMed Central

    Mehra, Vishal C.; Jackson, Elias; Zhang, Xian M.; Jiang, Xian-Cheng; Dobrucki, Lawrence W.; Yu, Jun; Bernatchez, Pascal; Sinusas, Albert J.; Shulman, Gerald I.; Sessa, William C.; Yarovinsky, Timur O.; Bender, Jeffrey R.

    2014-01-01

    Endothelial dysfunction, including endothelial hyporesponsiveness to prototypical angiogenic growth factors and eNOS agonists, underlies vascular pathology in many dysmetabolic states. We investigated effects of a saturated free fatty acid, palmitic acid (PA), on endothelial cell responses to VEGF. PA-pretreated endothelial cells had markedly diminished Akt, eNOS, and ERK activation responses to VEGF, despite normal VEGFR2 phosphorylation. PA inhibited VEGF-induced angiogenic cord formation in Matrigel, and PA-treated endothelial cells accumulated early species (C16) ceramide. The serine palmitoyltransferase inhibitor myriocin reversed these defects. Protein phosphatase 2A (PP2A) became more eNOS-associated in PA-treated cells; the PP2A inhibitor okadaic acid reversed PA-induced signaling defects. Mice fed a diet high in saturated fat for 2 to 3 weeks had impaired i) aortic Akt and eNOS phosphorylation to infused VEGF, ii) ear angiogenic responses to intradermal adenoviral-VEGF injection, and iii) vascular flow recovery to hindlimb ischemia as indicated by laser Doppler and αVβ3 SPECT imaging. High-fat feeding did not impair VEGF-induced signaling or angiogenic responses in mice with reduced serine palmitoyltransferase expression. Thus, de novo ceramide synthesis is required for these detrimental PA effects. The findings demonstrate an endothelial VEGF resistance mechanism conferred by PA, which comprises ceramide-induced, PP2A-mediated dephosphorylation of critical activation sites on enzymes central to vascular homeostasis and angiogenesis. This study defines potential molecular targets for preservation of endothelial function in metabolic syndrome. PMID:24606881

  10. B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

    PubMed Central

    Houge, Gunnar; Haesen, Dorien; Vissers, Lisenka E.L.M.; Mehta, Sarju; Parker, Michael J.; Wright, Michael; Vogt, Julie; McKee, Shane; Tolmie, John L.; Cordeiro, Nuno; Kleefstra, Tjitske; Willemsen, Marjolein H.; Reijnders, Margot R.F.; Berland, Siren; Hayman, Eli; Lahat, Eli; Brilstra, Eva H.; van Gassen, Koen L.I.; Zonneveld-Huijssoon, Evelien; de Bie, Charlotte I.; Hoischen, Alexander; Eichler, Evan E.; Holdhus, Rita; Steen, Vidar M.; Døskeland, Stein Ove; Hurles, Matthew E.; FitzPatrick, David R.; Janssens, Veerle

    2015-01-01

    Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding–deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3β, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance. PMID:26168268

  11. 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

  12. Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

    PubMed Central

    Kan, Tsung-Wai; Chalkley, Gillian E.; Sap, Karen; Bezstarosti, Karel; Demmers, Jeroen A.; Ozgur, Zeliha; van Ijcken, Wilfred F. J.; Verrijzer, C. Peter

    2013-01-01

    Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis. PMID:24086141

  13. A Novel Effect of MARCKS Phosphorylation by Activated PKC: The Dephosphorylation of Its Serine 25 in Chick Neuroblasts

    PubMed Central

    Toledo, Andrea; Zolessi, Flavio R.; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS. PMID:23634231

  14. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

    PubMed

    Toledo, Andrea; Zolessi, Flavio R; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS. PMID:23634231

  15. P21-activated kinase 2 (PAK2) regulates glucose uptake and insulin sensitivity in neuronal cells.

    PubMed

    Varshney, Pallavi; Dey, Chinmoy Sankar

    2016-07-01

    P21-activated kinases (PAKs) are recently reported as important players of insulin signaling and glucose homeostasis in tissues like muscle, pancreas and liver. However, their role in neuronal insulin signaling is still unknown. Present study reports the involvement of PAK2 in neuronal insulin signaling, glucose uptake and insulin resistance. Irrespective of insulin sensitivity, insulin stimulation decreased PAK2 activity. PAK2 downregulation displayed marked enhancement of GLUT4 translocation with increase in glucose uptake whereas PAK2 over-expression showed its reduction. Treatment with Akti-1/2 and wortmannin suggested that Akt and PI3K are mediators of insulin effect on PAK2 and glucose uptake. Rac1 inhibition demonstrated decreased PAK2 activity while inhibition of PP2A resulted in increased PAK2 activity, with corresponding changes in glucose uptake. Taken together, present study demonstrates an inhibitory role of insulin signaling (via PI3K-Akt) and PP2A on PAK2 activity and establishes PAK2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity. PMID:27040307

  16. Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation.

    PubMed

    Golder, Francis J; Ranganathan, Lavanya; Satriotomo, Irawan; Hoffman, Michael; Lovett-Barr, Mary Rachael; Watters, Jyoti J; Baker-Herman, Tracy L; Mitchell, Gordon S

    2008-02-27

    Acute intermittent hypoxia elicits a form of spinal, brain-derived neurotrophic factor (BDNF)-dependent respiratory plasticity known as phrenic long-term facilitation. Ligands that activate G(s)-protein-coupled receptors, such as the adenosine 2a receptor, mimic the effects of neurotrophins in vitro by transactivating their high-affinity receptor tyrosine kinases, the Trk receptors. Thus, we hypothesized that A2a receptor agonists would elicit phrenic long-term facilitation by mimicking the effects of BDNF on TrkB receptors. Here we demonstrate that spinal A2a receptor agonists transactivate TrkB receptors in the rat cervical spinal cord near phrenic motoneurons, thus inducing long-lasting (hours) phrenic motor facilitation. A2a receptor activation increased phosphorylation and new synthesis of an immature TrkB protein, induced TrkB signaling through Akt, and strengthened synaptic pathways to phrenic motoneurons. RNA interference targeting TrkB mRNA demonstrated that new TrkB protein synthesis is necessary for A2a-induced phrenic motor facilitation. A2a receptor activation also increased breathing in unanesthetized rats, and improved breathing in rats with cervical spinal injuries. Thus, small, highly permeable drugs (such as adenosine receptor agonists) that transactivate TrkB receptors may provide an effective therapeutic strategy in the treatment of patients with ventilatory control disorders, such as obstructive sleep apnea, or respiratory insufficiency after spinal injury or during neurodegenerative diseases. PMID:18305238

  17. 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

  18. 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.

  19. Synthesis and biological activity of pyrimido [1, 2-a] quinoline moiety and its 2-substituted derivatives

    NASA Astrophysics Data System (ADS)

    Jadhav, A. G.; Halikar, N. K.

    2013-04-01

    2-Amino-3-cyano quinoline (1) and bis (methylthio) methylene malononitrile (2) were refluxed in N,N-dimethyl formamide (DMF) in presence of catalytic amount of anhydrous potassium carbonate to afforded 3, 11-dicyano-4-imino-2-methylthio -4H-pyrimido [1, 2-a] quinoline (3). The latter were further reacted with different substituted aniline, phenol, hetryl amine and compound containing active methyl group. Afforded to 3, 11-dicyano-4-imino -4H-pyrimido [1, 2-a] quinoline and their 2-substuited derivatives (4a-7c). All these newly synthesized compounds were characterized by elemental analysis and spectral data, and screened for their antimicrobial activities.

  20. New imidazo[1,2-a]pyridines carrying active pharmacophores: synthesis and anticonvulsant studies.

    PubMed

    Ulloora, Shrikanth; Shabaraya, Ramakrishna; Aamir, Syed; Adhikari, Airody Vasudeva

    2013-03-01

    Five new series of imidazo[1,2-a]pyridines carrying biologically active pyrazoline (4a-e), cyanopyridone (5a, b), cyanopyridine (6a-f), 2-aminopyrimidine (7a-f) and pyrimidine-2-thione (8a-d) systems were designed and synthesized as prominent anticonvulsant agents. The target compounds were screened for their in vivo anticonvulsant activity following maximal electroshock (MES) and subcutaneous pentylene tetrazole (scPTZ) methods at a small test dose of 10 mg/kg. Further, Rotarod toxicity method was used to study the toxicity profile of selected compounds. Compounds 4b, 5a, 5b, 6a, 7e and 8d possessing 4-fluorophenyl substituent at 2nd position of imidazo[1,2-a]pyridine ring displayed potent anticonvulsant activity without displaying any toxicity. Enhanced activity profile was observed for new compounds in PTZ method over MES method. PMID:23352511

  1. The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

    PubMed Central

    Luo, Man-Li; Gong, Chang; Chen, Chun-Hau; Hu, Hai; Huang, Pengyu; Zheng, Min; Yao, Yandan; Wei, Shuo; Wulf, Gerburg; Lieberman, Judy; Zhou, Xiao Zhen; Song, Erwei; Lu, Kun Ping

    2015-01-01

    SUMMARY Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs). Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets. PMID:25818297

  2. Antiulcer agents. 3. Structure-activity-toxicity relationships of substituted imidazo[1,2-a]pyridines and a related imidazo[1,2-a]pyrazine.

    PubMed

    Kaminski, J J; Perkins, D G; Frantz, J D; Solomon, D M; Elliott, A J; Chiu, P J; Long, J F

    1987-11-01

    Investigation of the interrelationship between structure, antiulcer activity, and toxicology screening data derived from a series of compounds selected from structure-activity studies directed toward identifying a successor to 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine, Sch 28080 (1), has identified 3-(cyanomethyl)-2,7-dimethyl-8-(phenylmethoxy)imidazo[1,2 -a]pyridine (5), 3-amino-2-methyl-8-(2-phenylethyl)imidazo[1,2-a]pyridine (6), and 3-amino-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyrazine (7). These analogues exhibit a combination of antisecretory and cytoprotective activity in animal models, while eliminating the adverse effects of the prototype 1. One of these, 3-amino-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyrazine, Sch 32651 (7), has a profile meeting all criteria. PMID:3669012

  3. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.

    PubMed

    Donnelly, M L; Hughes, L E; Luke, G; Mendoza, H; ten Dam, E; Gani, D; Ryan, M D

    2001-05-01

    The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements. PMID:11297677

  4. BAP1/ASXL1 recruitment and activation for H2A deubiquitination.

    PubMed

    Sahtoe, Danny D; van Dijk, Willem J; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K

    2016-01-01

    The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor. PMID:26739236

  5. BAP1/ASXL1 recruitment and activation for H2A deubiquitination

    PubMed Central

    Sahtoe, Danny D.; van Dijk, Willem J.; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

    2016-01-01

    The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor. PMID:26739236

  6. UBXN2A regulates nicotinic receptor degradation by modulating the E3 ligase activity of CHIP.

    PubMed

    Teng, Yanfen; Rezvani, Khosrow; De Biasi, Mariella

    2015-10-15

    Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP's client substrates. PMID:26265139

  7. 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

  8. Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1.

    PubMed

    Meiri, David; Marshall, Christopher B; Mokady, Daphna; LaRose, Jose; Mullin, Michael; Gingras, Anne-Claude; Ikura, Mitsuhiko; Rottapel, Robert

    2014-01-01

    The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gβγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gβγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation. PMID:25209408

  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. Structural Domains Underlying the Activation of Acid-Sensing Ion Channel 2a

    PubMed Central

    Schuhmacher, Laura-Nadine; Srivats, Shyam; Smith, Ewan St. John

    2015-01-01

    The acid-sensing ion channels (ASICs) are a family of ion channels expressed throughout the mammalian nervous system. The principal activator of ASICs is extracellular protons, and ASICs have been demonstrated to play a significant role in many physiologic and pathophysiologic processes, including synaptic transmission, nociception, and fear. However, not all ASICs are proton-sensitive: ASIC2a is activated by acid, whereas its splice variant ASIC2b is not. We made a series of chimeric ASIC2 proteins, and using whole-cell electrophysiology we have identified the minimal region of the ASIC2a extracellular domain that is required for ASIC2 proton activation: the first 87 amino acids after transmembrane domain 1. We next examined the function of different domains within the ASIC2b N-terminus and identified a region proximal to the first transmembrane domain that confers tachyphylaxis upon ASIC2a. We have thus identified domains of ASIC2 that are crucial to channel function and may be important for the function of other members of the ASIC family. PMID:25583083

  11. Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

    PubMed Central

    Evans, DRH.; Stark, MJR.

    1997-01-01

    Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene. PMID:9071579

  12. High CYP2A6 Enzyme Activity as Measured by a Caffeine Test and Unique Distribution of CYP2A6 Variant Alleles in Ethiopian Population

    PubMed Central

    Djordjevic, Natasa; Carrillo, Juan Antonio; Makonnen, Eyasu; Bertilsson, Leif; Ingelman-Sundberg, Magnus

    2014-01-01

    Abstract CYP2A6 metabolizes clinically relevant drugs, including antiretroviral and antimalarial drugs of major public health importance for the African populations. CYP2A6 genotype–phenotype relationship in African populations, and implications of geographic differences on enzyme activity, remain to be investigated. We evaluated the influence of CYP2A6 genotype, geographical differences, gender, and cigarette smoking on enzyme activity, using caffeine as a probe in 100 healthy unrelated Ethiopians living in Ethiopia, and 72 living in Sweden. CYP2A6 phenotype was estimated by urinary 1,7-dimethyluric acid (17U)/1,7-dimethylxanthine or paraxanthine (17X) ratio. The frequencies of CYP2A6*1B, *1D, *2, *4, *9, and *1x2 in Ethiopians were 31.3, 29.4, 0.6, 0.6, 2.8, and 0.3%, respectively. The overall mean±SD for log 17U/17X was 0.12±0.24 and coefficient of variation 199%. No significant difference in the mean log 17U/17X ratio between Ethiopians living in Sweden versus Ethiopia was observed. Analysis of variance revealed CYP2A6 genotype (p=0.04, F=2.01) but not geographical differences, sex, or cigarette smoking as predictors of CYP2A6 activity. Importantly, the median (interquartile range) of 17U/17X ratio in Ethiopians 1.35 (0.99 to 1.84) was 3- and 11-fold higher than the previously reported value in Swedes 0.52 (0.27 to 1.00) and Koreans 0.13 (0.0 to 0.35), respectively (Djordjevic et al., 2013). Taken together, we report here the relevance of CYP2A6 genotype for enzyme activity in this Ethiopian sample, as well as high CYP2A6 activity and unique distribution of the CYP2A6 variant alleles in Ethiopians as compared other populations described hitherto. Because Omics biomarker research is rapidly accelerating in Africa, CYP2A6 pharmacogenetics and clinical pharmacology observations reported herein for the Ethiopian populations have clinical and biological importance to plan for future rational therapeutics efforts in the African continent as well as therapeutics

  13. Preladenant, a selective A(2A) receptor antagonist, is active in primate models of movement disorders.

    PubMed

    Hodgson, Robert A; Bedard, Paul J; Varty, Geoffrey B; Kazdoba, Tatiana M; Di Paolo, Therese; Grzelak, Michael E; Pond, Annamarie J; Hadjtahar, Abdallah; Belanger, Nancy; Gregoire, Laurent; Dare, Aurelie; Neustadt, Bernard R; Stamford, Andrew W; Hunter, John C

    2010-10-01

    Parkinson's Disease (PD) and Extrapyramidal Syndrome (EPS) are movement disorders that result from degeneration of the dopaminergic input to the striatum and chronic inhibition of striatal dopamine D(2) receptors by antipsychotics, respectively. Adenosine A(2A) receptors are selectively localized in the basal ganglia, primarily in the striatopallidal ("indirect") pathway, where they appear to operate in concert with D(2) receptors and have been suggested to drive striatopallidal output balance. In cases of dopaminergic hypofunction, A(2A) receptor activation contributes to the overdrive of the indirect pathway. A(2A) receptor antagonists, therefore, have the potential to restore this inhibitor imbalance. Consequently, A(2A) receptor antagonists have therapeutic potential in diseases of dopaminergic hypofunction such as PD and EPS. Targeting the A(2A) receptor may also be a way to avoid the issues associated with direct dopamine agonists. Recently, preladenant was identified as a potent and highly selective A(2A) receptor antagonist, and has produced a significant improvement in motor function in rodent models of PD. Here we investigate the effects of preladenant in two primate movement disorder models. In MPTP-treated cynomolgus monkeys, preladenant (1 or 3 mg/kg; PO) improved motor ability and did not evoke any dopaminergic-mediated dyskinetic or motor complications. In Cebus apella monkeys with a history of chronic haloperidol treatment, preladenant (0.3-3.0 mg/kg; PO) delayed the onset of EPS symptoms evoked by an acute haloperidol challenge. Collectively, these data support the use of preladenant for the treatment of PD and antipsychotic-induced movement disorders. PMID:20655910

  14. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    PubMed

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  15. 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.

  16. Deubiquitinase USP2a Sustains Interferons Antiviral Activity by Restricting Ubiquitination of Activated STAT1 in the Nucleus

    PubMed Central

    Liu, Jin; Yuan, Yukang; Cheng, Qiao; Zuo, Yibo; Qian, Liping; Guo, Tingting; Qian, Guanghui; Li, Lemin; Ge, Jun; Dai, Jianfeng; Xiong, Sidong; Zheng, Hui

    2016-01-01

    STAT1 is a critical transcription factor for regulating host antiviral defenses. STAT1 activation is largely dependent on phosphorylation at tyrosine 701 site of STAT1 (pY701-STAT1). Understanding how pY701-STAT1 is regulated by intracellular signaling remains a major challenge. Here we find that pY701-STAT1 is the major form of ubiquitinated-STAT1 induced by interferons (IFNs). While total STAT1 remains relatively stable during the early stages of IFNs signaling, pY701-STAT1 can be rapidly downregulated by the ubiquitin-proteasome system. Moreover, ubiquitinated pY701-STAT1 is located predominantly in the nucleus, and inhibiting nuclear import of pY701-STAT1 significantly blocks ubiquitination and downregulation of pY701-STAT1. Furthermore, we reveal that the deubiquitinase USP2a translocates into the nucleus and binds to pY701-STAT1, and inhibits K48-linked ubiquitination and degradation of pY701-STAT1. Importantly, USP2a sustains IFNs-induced pY701-STAT1 levels, and enhances all three classes of IFNs- mediated signaling and antiviral activity. To our knowledge, this is the first identified deubiquitinase that targets activated pY701-STAT1. These findings uncover a positive mechanism by which IFNs execute efficient antiviral signaling and function, and may provide potential targets for improving IFNs-based antiviral therapy. PMID:27434509

  17. 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. PMID:23552268

  18. 5-HT2A receptor activation is necessary for CO2-induced arousal.

    PubMed

    Buchanan, Gordon F; Smith, Haleigh R; MacAskill, Amanda; Richerson, George B

    2015-07-01

    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-HT(2A) receptors dose-dependently blocked CO2-induced arousal. The 5-HT(2C) 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 (Lmx1b(f/f/p)) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT(2A), but not 5-HT(2C), receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT(2A) 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

  19. 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

  20. Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides.

    PubMed

    Shin, Joon-Hee; Kim, Jung-Eun; Malapi-Wight, Martha; Choi, Yoon-E; Shaw, Brian D; Shim, Won-Bo

    2013-06-01

    Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development. PMID:23452277

  1. 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

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

    PubMed

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

    2014-07-15

    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 and 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

  3. Inflammatory Lung Injury After Cardiopulmonary Bypass is Attenuated by Adenosine A2A Receptor Activation

    PubMed Central

    Lisle, Turner C; Gazoni, Leo M; Fernandez, Lucas G; Sharma, Ashish K; Bellizzi, Andrew M; Schifflett, Grant D; Laubach, Victor E; Kron, Irving L

    2008-01-01

    Objectives Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A2A receptor (A2AR) activation attenuates lung ischemia-reperfusion injury, however the effect of A2AR activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific A2AR activation by ATL313 would attenuate inflammatory lung injury following cardiopulmonary bypass. Methods Adult male Sprague-Dawley rats were randomly divided into three groups: 1) SHAM group (underwent cannulation+heparinization only); 2) CONTROL group (underwent 90-minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; 3) ATL group (underwent 90-minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution). Results There was significantly less pulmonary edema and lung injury in the ATL group compared to the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-γ and myeloperoxidase levels compared to the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly decreased in the ATL group compared to the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of pro-inflammatory cytokines. Conclusions The addition of a potent A2AR agonist to the normal priming solution prior to the initiation of CPB significantly protects the lung from the inflammatory effects of CPB and reduces the amount of lung injury. A2AR agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with CPB. Ultra-mini Abstract Pharmacologic activation of the adenosine A2A receptor during cardiopulmonary bypass resulted in

  4. Synthesis and antiproliferative activity evaluation of steroidal imidazo[1,2-a]pyridines.

    PubMed

    Rassokhina, Irina V; Volkova, Yulia A; Kozlov, Andrey S; Scherbakov, Alexander M; Andreeva, Olga E; Shirinian, Valerik Z; Zavarzin, Igor V

    2016-09-01

    An elegant approach to unknown steroidal imidazo[1,2-a]pyridine hybrids is disclosed. Unique derivatives of androstene and estrane series containing imidazo[1,2-a]pyridine motifs were prepared from 17-ethynyl steroids in good yields via copper-catalyzed cascade aminomethylation/cycloisomerization with imines. The synthesized compounds were screened for cytotoxicity against human breast (MCF-7, MDA-MB-231, HBL-100, MDA-MB-453) and prostate (LNCaP-LN3, PC-3, DU 145) cancer cell lines. The majority of tested compounds showed activities at μM level in breast cancer cells. The hormone-responsive breast cancer cells MCF-7 were more sensitive to novel compounds than ERα-negative cells; in particular, compounds 6a,b exhibited promising cytotoxicity against this cell line with the IC50 values in the range of 3-4μM. Furthermore, compound 4a showed remarkable effects as a selective ERα receptor modulator. PMID:27263438

  5. H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA

    PubMed Central

    Larochelle, Marc; Gaudreau, Luc

    2003-01-01

    H2A.Z has been shown to regulate transcription in yeast, and that function resides in its C-terminal region as the reciprocal portion of H2A cannot substitute for the latter. We show that fusion of a transcriptional activating region to the C-terminal region of H2A, which is substituted for that of H2A.Z, can allow the chimera to fulfil the special role of H2A.Z in positive gene regulation, as well as complement growth deficiencies of htz1Δ cells. We further show that the ‘transcription’ function of H2A.Z is linked to its ability to preferentially localize to certain intergenic DNA regions. Our results suggest that H2A.Z modulates functional interactions with transcription regulatory components, and thus increases its localization to promoters where it helps poise chromatin for gene activation. PMID:12941702

  6. Adenosine A2A Receptor Activation Prevents Wear Particle-Induced Osteolysis

    PubMed Central

    Mediero, Aránzazu; Frenkel, Sally R.; Wilder, Tuere; He, Wenjie; Mazumder, Amitabha; Cronstein, Bruce N.

    2012-01-01

    Prosthesis loosening, associated with wear-particle–induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A2A receptors (A2AR) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A2AR agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear-particle–induced bone resorption. C57Bl/6 and A2A knockout (A2ARKO) mice received ultrahigh-molecular weight polyethylene particles (UHMWPE) and were treated daily with either saline or the A2AR agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A2AKO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone-resorption markers receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), cathepsin K, CD163, and osteopontin were reduced following CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin 1β (IL-1β) and TNFα was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A2AR agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery. PMID:22623741

  7. 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.

  8. Optogenetic Activation of Adenosine A2A Receptor Signaling in the Dorsomedial Striatopallidal Neurons Suppresses Goal-Directed Behavior.

    PubMed

    Li, Yan; He, Yan; Chen, Mozi; Pu, Zhilan; Chen, Li; Li, Ping; Li, Bo; Li, Haiyan; Huang, Zhi-Li; Li, Zhihui; Chen, Jiang-Fan

    2016-03-01

    The striatum has an essential role in neural control of instrumental behaviors by reinforcement learning. Adenosine A(2A) receptors (A(2A)Rs) are highly enriched in the striatopallidal neurons and are implicated in instrumental behavior control. However, the temporal importance of the A(2A)R signaling in relation to the reward and specific contributions of the striatopallidal A(2A)Rs in the dorsolateral striatum (DLS) and the dorsomedial striatum (DMS) to the control of instrumental learning are not defined. Here, we addressed temporal relationship and sufficiency of transient activation of optoA(2A)R signaling precisely at the time of the reward to the control of instrumental learning, using our newly developed rhodopsin-A2AR chimeras (optoA(2A)R). We demonstrated that transient light activation of optoA(2A)R signaling in the striatopallidal neurons in 'time-locked' manner with the reward delivery (but not random optoA(2A)R activation) was sufficient to change the animal's sensitivity to outcome devaluation without affecting the acquisition or extinction phases of instrumental learning. We further demonstrated that optogenetic activation of striatopallidal A(2A)R signaling in the DMS suppressed goal-directed behaviors, as focally genetic knockdown of striatopallidal A(2A)Rs in the DMS enhanced goal-directed behavior by the devaluation test. By contrast, optogenetic activation or focal AAV-Cre-mediated knockdown of striatopallidal A(2A)R in the DLS had relatively limited effects on instrumental learning. Thus, the striatopallidal A(2A)R signaling in the DMS exerts inhibitory and predominant control of goal-directed behavior by acting precisely at the time of reward, and may represent a therapeutic target to reverse abnormal habit formation that is associated with compulsive obsessive disorder and drug addiction. PMID:26216520

  9. 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

  10. 77 FR 65715 - Agency Information Collection Activities; Submissions for OMB Review; Comment Request; H-2A...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-30

    ... Request; H-2A Foreign Labor Certification Program; Labor Certification Letter for H-2A Agricultural Foreign Workers, H-2B Foreign Labor Certification Program; and Application for Prevailing Wage...) of 1995 (44 U.S.C. 3501 et seq.). The ICR titles are H-2A Foreign Labor Certification Program,...

  11. Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism.

    PubMed

    Parrish, Jason C; Nichols, David E

    2006-11-01

    To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors. PMID:17010161

  12. Protein Phosphatase 2A Holoenzyme Is Targeted to Peroxisomes by Piggybacking and Positively Affects Peroxisomal β-Oxidation1[OPEN

    PubMed Central

    Kataya, Amr R.A.; Heidari, Behzad; Hagen, Lars; Kommedal, Roald; Slupphaug, Geir; Lillo, Cathrine

    2015-01-01

    The eukaryotic, highly conserved serine (Ser)/threonine-specific protein phosphatase 2A (PP2A) functions as a heterotrimeric complex composed of a catalytic (C), scaffolding (A), and regulatory (B) subunit. In Arabidopsis (Arabidopsis thaliana), five, three, and 17 genes encode different C, A, and B subunits, respectively. We previously found that a B subunit, B′θ, localized to peroxisomes due to its C-terminal targeting signal Ser-Ser-leucine. This work shows that PP2A C2, C5, andA2 subunits interact and colocalize with B′θ in peroxisomes. C and A subunits lack peroxisomal targeting signals, and their peroxisomal import depends on B′θ and appears to occur by piggybacking transport. B′θ knockout mutants were impaired in peroxisomal β-oxidation as shown by developmental arrest of seedlings germinated without sucrose, accumulation of eicosenoic acid, and resistance to protoauxins indole-butyric acid and 2,4-dichlorophenoxybutyric acid. All of these observations strongly substantiate that a full PP2A complex is present in peroxisomes and positively affects β-oxidation of fatty acids and protoauxins. PMID:25489022

  13. Pluripotency Activity of Nanog Requires Biochemical Stabilization by Variant Histone Protein H2A.Z.

    PubMed

    Wang, Jiaxu; Qiao, Mengran; He, Qianqian; Shi, Ronghua; Loh, Sharon Jia Hui; Stanton, Lawrence W; Wu, Mian

    2015-07-01

    The variant histone protein H2A.Z plays a critical role in early development. Likewise, Nanog, a master regulator of embryonic stem cells (ESCs), is essential for proper development in early embryogenesis. In this study, we establish that these two factors work together to maintain pluripotency. It is shown that H2A.Z influences the protein level of Nanog through the ubiquitin-proteasome pathway. Knockdown of H2A.Z causes differentiation of mouse ESCs and disrupts the reprogramming of somatic cells, which can be partially rescued by overexpression of Nanog. We conclude that the H2A.Z-Nanog partnership is involved in ESC pluripotency and reprogramming of somatic cells. Stem Cells 2015;33:2126-2134. PMID:25809870

  14. Adenosine is required for sustained inflammasome activation via the A2A receptor and the HIF-1α pathway

    NASA Astrophysics Data System (ADS)

    Ouyang, Xinshou; Ghani, Ayaz; Malik, Ahsan; Wilder, Tuere; Colegio, Oscar Rene; Flavell, Richard Anthony; Cronstein, Bruce Neil; Mehal, Wajahat Zafar

    2013-12-01

    Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1β, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.

  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. Synthesis and Structure–Activity Relationships of N-Benzyl Phenethylamines as 5-HT2A/2C Agonists

    PubMed Central

    2014-01-01

    N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor. PMID:24397362

  17. 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. PMID:24055686

  18. macroH2A1 Histone Variants Are Depleted on Active Genes but Concentrated on the Inactive X Chromosome†

    PubMed Central

    Changolkar, Lakshmi N.; Pehrson, John R.

    2006-01-01

    Using a novel thiol affinity chromatography approach to purify macroH2A1-containing chromatin fragments, we examined the distribution of macroH2A1 histone variants in mouse liver chromatin. We found that macroH2A1 was depleted on the transcribed regions of active genes. This depletion was observed on all of the 20 active genes that we probed, with only one site showing a small amount of enrichment. In contrast, macroH2A1 was concentrated on the inactive X chromosome, consistent with our previous immunofluorescence studies. This preferential localization was seen on genes that are active in liver, genes that are inactive in liver, and intergenic regions but was absent from four regions that escape X inactivation. These results support the hypothesis that macroH2As function as transcriptional repressors. Also consistent with this hypothesis is our finding that the heterochromatin protein HP1β copurifies with the macroH2A1-containing chromatin fragments. This study presents the first detailed examination of the distribution of macroH2A1 variants on specific sequences. Our results indicate that macroH2As have complex distribution patterns that are influenced by both local factors and long-range mechanisms. PMID:16738309

  19. Design and synthesis of some substituted thiazolo[3,2-a]pyrimidine derivatives of potential biological activities.

    PubMed

    Abdel Moty, Samia G; Hussein, Mostafa A; Abdel Aziz, Salah A; Abou-Salim, Mahrous A

    2016-03-01

    In continuation to our previous work, thiazolopyrimidines 2a-x were synthesized through intramolecular cyclization of 2-phenacylthio-dihydropyrimidine hydrobromides 1a-x using polyphosphoric acid. On the other hand, thiazolo[3,2-a]pyrimidine-3-one 3 was coupled with aryldiazonium salts or condensed with isatin to afford compounds 4a-c or 5, respectively. Chemical structure of the target compounds was substantiated by IR, FT-IR, (1)H-, (13)C and DEPT-(13)C NMR, MS as well as microanalyses. Moreover, the lipophilicity of the target compounds is expressed as Clog P. The antimicrobial screening of the test compounds 2a-x, 4a-c and 5 revealed moderate activity in comparison to reference drugs. Compounds 2a-c, 2e, 2o and 2v showed a gradual increase in their anti-inflammatory activity reaching its maximum at 5 h compared to indomethacin. Furthermore, the analgesic activity of compounds 2a-c, 2e, 2o and 2v revealed a maximum activity after 5 h of injection compared to aspirin and the LD50 of compounds 2e and 2v was determined. PMID:27013904

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

    PubMed

    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. 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

  2. Novel imidazo[1,2-a]naphthyridinic systems (part 1): synthesis, antiproliferative and DNA-intercalating activities.

    PubMed

    Andaloussi, Mounir; Moreau, Emmanuel; Masurier, Nicolas; Lacroix, Jacques; Gaudreault, René C; Chezal, Jean-Michel; El Laghdach, Anas; Canitrot, Damien; Debiton, Eric; Teulade, Jean-Claude; Chavignon, Olivier

    2008-11-01

    Novel imidazo[1,2-a]naphthyridinic systems 6a-15a and 6b-15b were obtained from Friedländer's reaction in imidazo[1,2-a]pyridine series. Most of the compounds were evaluated for their antitumor activity in the NCIs in vitro human tumor cell line screening panel. Among them, pentacyclic derivatives 13b and 14a exhibited in vitro activity comparable to anticancer agent such as amsacrine. Their mechanism of cytotoxicity action was unrelated to poisoning or inhibiting abilities against topo1. On the contrary, we highlighted a direct intercalation of the drugs into DNA by electrophoresis on agarose gel. PMID:18403058

  3. Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes vaporariorum.

    PubMed

    Walia, Yashika; Dhir, Sunny; Zaidi, Aijaz Asghar; Hallan, Vipin

    2015-01-01

    Nucleic acid transfer between plants is a phenomenon which is likely to occur in many ways in nature. We report here the active transmission of Apple scar skin viroid (ASSVd) naked ssRNA species by the whitefly Trialeurodes vaporariorum (Tv). Not only the viroid RNA, its DNA form was also identified from the insect. The viroid transfer efficiency was enhanced with the help of Cucumis sativus Phloem protein 2 (CsPP2), a plant protein known to translocate viroid RNAs. This PP2/ASSVd complex is stably present in the viroid infected cucumber plants, as was identified with the help of immunological reaction. As viroid-like secondary structures are found in some plant RNAs, and PP2 is known to bind and translocate several RNAs, the results have huge implications in transfer of these RNA species between plants visited by the whitefly. PMID:26327493

  4. Purification and identification of a novel heteropolysaccharide RBPS2a with anti-complementary activity from defatted rice bran.

    PubMed

    Wang, Li; Zhang, Hongbin; Zhang, Xiaoyan; Chen, Zhengxing

    2008-09-01

    A novel heteropolysaccharide RBPS2a with anti-complementary activity was obtained from defatted rice bran by hot water extraction, ethanol precipitation, and purified by gel chromatography after anion-exchange chromatography. This fraction exhibited more potent anti-complementary activity than other polysaccharide fractions. RBPS2a was eluted as a single symmetrical narrow peak on high-performance gel-permeation chromatography (HPGPC) and the average molecular weight was 90,000Da. We found RBPS2a contained 86.7% polysaccharide and 8.7% protein. The amino acid pattern showed that RBPS2a contained large amount of glutamic acid, arginine, aspartic acid, lysine, and alanine. The molar content of the above five amino acids constituted 59.31% of the total amino acids. Gas chromatography of absolute acid hydrolysate of RBPS2a suggested that it was composed of arabinose, xylose, glucose and galactose with a molar ratio of 4:2:1:4. The Fourier-transform infrared spectra (FT-IR) and (1)H, (13)C NMR spectroscopy analysis revealed that RBPS2a had a backbone consisting of β-(1→3)-linked d-galacopyranosyl residues substituted at O-2 with glycosyl residues composed of α-d-xylose-(1→4)-α-d-arabinose-(1→ and α-d-glucose-(1→4)-α-d-arabinose-(1→ linked residues. Furthermore, some of the fractions extracted and purified from defatted rice bran exhibited strong anti-complementary activity. Among these fractions, the purified polysaccharide RBPS2a had the highest activity. PMID:26050177

  5. Altered CD94/NKG2A and perforin expression reduce the cytotoxic activity in malignant pleural effusions.

    PubMed

    Pace, Elisabetta; Di Sano, Caterina; Ferraro, Maria; Tipa, Annalisa; Olivieri, Dario; Spatafora, Mario; Santagata, Roberta; Bellia, Vincenzo; Gjomarkaj, Mark

    2011-01-01

    CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism. This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A. The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n=19) and congestive heart failure (CHF) (n=11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n=10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay. Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression. In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space. PMID:20932742

  6. Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells.

    PubMed

    Akiba, Toshihiko; Abe, Yuichi; Kitada, Sakae; Kusaka, Yoshitomo; Ito, Akio; Ichimatsu, Tokio; Katayama, Hideki; Akao, Tetsuyuki; Higuchi, Kazuhiko; Mizuki, Eiichi; Ohba, Michio; Kanai, Ryuta; Harata, Kazuaki

    2004-12-01

    Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37 kDa inactive protein is proteolytically activated to a 30 kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 134.37, c = 121.24 A. Diffraction data from a native crystal were collected to 2.75 A resolution using a synchrotron-radiation source. PMID:15583389

  7. Serotonin 2A and 2B receptor-induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity

    PubMed Central

    MacFarlane, P.M.; Vinit, S.; Mitchell, G.S.

    2011-01-01

    Acute intermittent hypoxia (AIH) facilitates phrenic motor output by a mechanism that requires spinal serotonin (type 2) receptor activation, NADPH oxidase activity and formation of reactive oxygen species (ROS). Episodic spinal serotonin (5-HT) receptor activation alone, without changes in oxygenation, is sufficient to elicit NADPH oxidase-dependent phrenic motor facilitation (pMF). Here we investigated: 1) whether serotonin 2A and/or 2B (5-HT2a/b) receptors are expressed in identified phrenic motor neurons, and 2) which receptor subtype is capable of eliciting NADPH-oxidase-dependent pMF. In anesthetized, artificially ventilated adult rats, episodic C4 intrathecal injections (3 × 6µl injections, 5 min intervals) of a 5-HT2a (DOI) or 5-HT2b (BW723C86) receptor agonist elicited progressive and sustained increases in integrated phrenic nerve burst amplitude (i.e. pMF), an effect lasting at least 90 minutes post-injection for both receptor subtypes. 5-HT2a and 5-HT2b receptor agonist-induced pMF were both blocked by selective antagonists (ketanserin and SB206553, respectively), but not by antagonists to the other receptor subtype. Single injections of either agonist failed to elicit pMF, demonstrating a need for episodic receptor activation. Phrenic motor neurons retrogradely labeled with cholera toxin B fragment expressed both 5-HT2a and 5-HT2b receptors. Pre-treatment with NADPH oxidase inhibitors (apocynin and DPI) blocked 5-HT2b, but not 5-HT2a-induced pMF. Thus, multiple spinal type 2 serotonin receptors elicit pMF, but they act via distinct mechanisms that differ in their requirement for NADPH oxidase activity. PMID:21223996

  8. Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6.

    PubMed

    Hu, Hao; Yu, Ting; Arpiainen, Satu; Lang, Matti A; Hakkola, Jukka; Abu-Bakar, A'edah

    2015-11-15

    Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5'-Luc constructs--down to -160bp from the TSS--showed p53 responsiveness in p53 overexpressed C3A cells. However, a further deletion from -160 to -74bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene--a well-known p53 activator--increased the expression of the p53 responsive positive control and the CYP2A6-5'-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5'-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6. PMID:26343999

  9. Activation of α2A-Containing Nicotinic Acetylcholine Receptors Mediates Nicotine-Induced Motor Output in Embryonic Zebrafish

    PubMed Central

    Menelaou, Evdokia; Udvadia, Ava J.; Tanguay, Robert L.; Svoboda, Kurt R.

    2014-01-01

    It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as evidence from others suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon-Beard (RB) neurons and olfactory sensory neurons in young embryos. In order to examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine-induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A-containing nAChR are dispensable for normal RB development. However, in the context of nicotine-induced motor output, α2A-containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself. PMID:24738729

  10. 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. PMID:23276608

  11. 12 CFR 584.2a - Exempt savings and loan holding companies and grandfathered activities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... pursuant to section 13(c) or 13(k) of the Federal Deposit Insurance Act, or section 406(f) or 408(m) of the... pursuant to an acquisition under section 13(c) or 13(k) of the Federal Deposit Insurance Act, or section... thrift lender pursuant to 12 U.S.C. 1467a(m) may not commence, or continue, any service or activity...

  12. 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.

  13. Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is mediated by protein phosphatase-2A.

    PubMed Central

    Gaussin, V; Hue, L; Stalmans, W; Bollen, M

    1996-01-01

    The activation of hepatic acetyl-CoA carboxylase by Na(+)-cotransported amino acids such as glutamine has been attributed mainly to the stimulation of its dephosphorylation by accumulating dicarboxylic acids, e.g. glutamate. We report here on a hepatic species of protein phosphatase-2A that activates acetyl-CoA carboxylase in the presence of physiological concentrations of glutamate or Mg2+ and, under these conditions, accounts for virtually all the hepatic acetyl-CoA carboxylase phosphatase activity. Glutamate also stimulated the dephosphorylation of a synthetic pentadecapeptide encompassing the Ser-79 phosphorylation site of rat acetyl-CoA carboxylase, but did not affect the dephosphorylation of other substrates such as phosphorylase. Conversely, protamine, which stimulated the dephosphorylation of phosphorylase, inhibited the activation of acetyl-CoA carboxylase. A comparison with various species of muscle protein phosphatase-2A showed that the stimulatory effects of glutamate and Mg2+ on the acetyl-CoA carboxylase phosphatase activity are largely mediated by the regulatory A subunit. Glutamate and Mg2+ emerge from our study as novel regulators of protein phosphatase-2A when acting on acetyl-CoA carboxylase. PMID:8645208

  14. 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

  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. Synthesis and cytotoxic activity of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidine derivatives.

    PubMed

    Vilchis-Reyes, Miguel Angel; Zentella, Alejandro; Martínez-Urbina, Miguel Angel; Guzmán, Angel; Vargas, Omar; Ramírez Apan, María Teresa; Ventura Gallegos, José Luis; Díaz, Eduardo

    2010-01-01

    A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-2-amine (compound 1) by trifluoromethyl groups and the isosteric substitution of the 2-methylimidazo[1,2-a]pyridin-3-yl scaffold by quinolin-4-yl or quinolin-3-yl moieties. The replacement of hydrogen by fluorine does not affect notably the cytotoxic activity and CDK inhibitor activity in this series. Quinolin-4-yl-substituted compound, 8, presents cytotoxic activity and is most effective and selective against CDK1/CycA than against CDK2/CycB. Compound 11, which has a quinolin-3-yl moiety is CDK inhibitor but presents null cytotoxic activity. Quinolin-4-yl-substituted compounds constitute a new lead of cytotoxic and CDK inhibitor compounds from which more compelling and selective inhibitors can be designed. PMID:19879023

  17. 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.

  18. Endotoxin Tolerance Inhibits Lyn and c-Src Phosphorylation and Association with Toll-Like Receptor 4 but Increases Expression and Activity of Protein Phosphatases.

    PubMed

    Xiong, Yanbao; Murphy, Michael; Manavalan, Tissa T; Pattabiraman, Goutham; Qiu, Fu; Chang, Hui-Hsin; Ho, I-Cheng; Medvedev, Andrei E

    2016-01-01

    Endotoxin tolerance protects the host by limiting excessive 'cytokine storm' during sepsis, but compromises the ability to counteract infections in septic shock survivors. It reprograms Toll-like receptor (TLR) 4 responses by attenuating the expression of proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators, but the mechanisms of reprogramming remain unclear. In this study, we demonstrate that the induction of endotoxin tolerance in human monocytes, THP-1 and MonoMac-6 cells inhibited lipopolysaccharide (LPS)-mediated phosphorylation of Lyn, c-Src and their recruitment to TLR4, but increased total protein phosphatase (PP) activity and the expression of protein tyrosine phosphatase (PTP) 1B, PP2A, PTP nonreceptor type (PTPN) 22 and mitogen-activated protein kinase phosphatase (MKP)-1. Chemical PP inhibitors, okadaic acid, dephostatin and cantharidic acid markedly decreased or completely abolished LPS tolerance, indicating the importance of phosphatases in endotoxin tolerization. Overexpression of PTPN22 decreased LPS-mediated nuclear factor (NF)-x03BA;B activation, p38 phosphorylation and CXCL8 gene expression, while PTPN22 ablation upregulated LPS-induced p65 NF-x03BA;B and p38 phosphorylation and the expression of TNF-α and pro-IL-1β mRNA, indicating PTPN22 as an inhibitor of TLR4 signaling. Thus, LPS tolerance interferes with TLR4 signaling by inhibiting Lyn and c-Src phosphorylation and their recruitment to TLR4, while increasing the phosphatase activity and expression of PP2A, PTPN22, PTP1B and MKP1. PMID:26457672

  19. 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. PMID:24252101

  20. Folic Acid Inhibits Amyloid β-Peptide Production through Modulating DNA Methyltransferase Activity in N2a-APP Cells

    PubMed Central

    Li, Wen; Jiang, Mingyue; Zhao, Shijing; Liu, Huan; Zhang, Xumei; Wilson, John X.; Huang, Guowei

    2015-01-01

    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. PMID:26492244

  1. 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

    2015-01-01

    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. PMID:26492244

  2. Role of "oncogenic nexus" of CIP2A in breast oncogenesis: how does it work?

    PubMed

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

    2015-01-01

    The CIP2A gene is an oncogene associated with solid and hematologic malignancies [1]. CIP2A protein is an oncoprotein and a potential cancer therapy target [2]. Literature shows that CIP2A inhibits the tumor suppressor protein PP2A [3] which downregulates phophorylation of AKT, a hallmark of cancers [4] and stabilizes the proto-oncogene, c-MYC in tumor cells [5], the comprehensive action of CIP2A and its functional interaction(s) with other oncoproteins and tumor suppressors is not clearly established. Recently we tried to put forward a contextual mode-of-action of CIP2A protein in a review which proposed that CIP2A influences oncogenesis via an "oncogenic nexus" [1]. In this review we critically evaluated the potential relevance of the mode-of-action of the "oncogenic nexus" of CIP2A in breast carcinogenesis and appraised the role of this nexus in different PAM50 luminal A, PAM50 luminal B, PAM50 HER2-enriched and PAM50 basal BC. This review has a novel approach. Here we have not only compiled and discussed the latest developments in this field but also presented data obtained from c-BioPortal and STRING10 in order to substantiate our view regarding the mode-of-action of the "oncogenic nexus" of CIP2A. We functionally correlated alterations of genes pertaining to the "oncogenic nexus" of CIP2A with protein-protein interactions between the different components of the nexus including (1) subunits of PP2A, (2) multiple transcription factors including MYC oncogene and (3) components of the PI3K-mTOR and the MAPK-ERK oncogenic pathways. Using these proteins as "input" to STRING10 we studied the association, Action view, at the highest Confidence level. OncoPrints (c-BioPortal) showed alterations (%) of regulatory subunits genes of PP2A (PPP2R1A and PPP2R1B) along with alterations of CIP2A in breast invasive carcinoma (TCGA, Nature 2012 & TCGA, Provisional). Similar genetic alterations of PP2A were also observed in samples of breast tumors at our Avera Research

  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. PMID:25462167

  4. Threshold Levels of Gfi1 Maintain E2A Activity for B Cell Commitment via Repression of Id1

    PubMed Central

    Fraszczak, Jennifer; Helness, Anne; Chen, Riyan; Vadnais, Charles; Robert, François; Khandanpour, Cyrus; Möröy, Tarik

    2016-01-01

    A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage. PMID:27467586

  5. Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

    PubMed

    Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

    2016-04-01

    The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. PMID:26851736

  6. NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A*S⃞

    PubMed Central

    Frasch, S. Courtney; Berry, Karin Zemski; Fernandez-Boyanapalli, Ruby; Jin, Hyun-Sun; Leslie, Christina; Henson, Peter M.; Murphy, Robert C.; Bratton, Donna L.

    2008-01-01

    Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. PMID:18824544

  7. 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. PMID:26360629

  8. The AD1 and AD2 Transactivation Domains of E2A Are Essential for the Antiapoptotic Activity of the Chimeric Oncoprotein E2A-HLF

    PubMed Central

    Inukai, Takeshi; Inaba, Toshiya; Ikushima, Satoshi; Look, A. Thomas

    1998-01-01

    The chimeric oncoprotein E2A-HLF, generated by the t(17;19) chromosomal translocation in pro-B-cell acute lymphoblastic leukemia, incorporates the transactivation domains of E2A and the basic leucine zipper (bZIP) DNA-binding and protein dimerization domain of HLF (hepatic leukemic factor). The ability of E2A-HLF to prolong the survival of interleukin-3 (IL-3)-dependent murine pro-B cells after IL-3 withdrawal suggests that it disrupts signaling pathways normally responsible for cell suicide, allowing the cells to accumulate as transformed lymphoblasts. To determine the structural motifs that contribute to this antiapoptotic effect, we constructed a panel of E2A-HLF mutants and programmed their expression in IL-3-dependent murine pro-B cells (FL5.12 line), using a zinc-inducible vector. Neither the E12 nor the E47 product of the E2A gene nor the wild-type HLF protein was able to protect the cells from apoptosis induced by IL-3 deprivation. Surprisingly, different combinations of disabling mutations within the HLF bZIP domain had little effect on the antiapoptotic property of the chimeric protein, so long as the amino-terminal portion of E2A remained intact. In the context of a bZIP domain defective in DNA binding, mutants retaining either of the two transactivation domains of E2A were able to extend cell survival after growth factor deprivation. Thus, the block of apoptosis imposed by E2A-HLF in pro-B lymphocytes depends critically on the transactivating regions of E2A. Since neither DNA binding nor protein dimerization through the bZIP domain of HLF is required for this effect, we propose mechanisms whereby protein-protein interactions with the amino-terminal region of E2A allow the chimera to act as a transcriptional cofactor to alter the expression of genes regulating the apoptotic machinery in pro-B cells. PMID:9742120

  9. Guaiazulene biochemical activity and cytotoxic and genotoxic effects on rat neuron and N2a neuroblastom cells

    PubMed Central

    Togar, Basak; Turkez, Hasan; Hacimuftuoglu, Ahmet; Tatar, Abdulgani; Geyikoglu, Fatime

    2015-01-01

    Aim: Neuroblastoma (NB)cells are often used in cancer researches such as glioblastoma cells since they have the potential of high mitotic activity, nuclear pleomorphism, and tumor necrosis. Guaiazulene (GYZ 1,4-dimethyl-7-isopropylazulene)is present in several essential oils of medicinal and aromatic plants. Many studies have reported the cytotoxic effect of GYZ; however, there are no studies that compare such effects between cancer cell lines and normal human cells after treatment with GYZ. Materials and Methods: In this study, we aimed to describe in vitro antiproliferative and/or cytotoxic properties (by 3-[4,5 dimetylthiazol -2-yl]-2,5 diphenlytetrazolium bromide [MTT] test), oxidative effects (by total antioxidant capacity [TAC] and total oxidative stress [TOS] analysis)and genotoxic damage potentials (by single cell gel electrophoresis)of GYZ. Result: The results indicated that GYZ have anti-proliferative activity suppressing the proliferation of neuron and N2a-NB cells at high doses. In addition, GYZ treatments at higher doses led to decreases of TAC levels and increases of TOS levels in neuron and N2a-NB cells. On the other hand, the mean values of the total scores of cells showing DNA damage were not found different from the control values. Conclusion: From this study, it is observed that GYZ has in vitro cytotoxic activity against neuron and N2a-NB cells. PMID:26401381

  10. Lipocalin 2, a Regulator of Retinoid Homeostasis and Retinoid-mediated Thermogenic Activation in Adipose Tissue.

    PubMed

    Guo, Hong; Foncea, Rocio; O'Byrne, Sheila M; Jiang, Hongfeng; Zhang, Yuanyuan; Deis, Jessica A; Blaner, William S; Bernlohr, David A; Chen, Xiaoli

    2016-05-20

    We have recently characterized the role of lipocalin 2 (Lcn2) as a new adipose-derived cytokine in the regulation of adaptive thermogenesis via a non-adrenergic pathway. Herein, we explored a potential non-adrenergic mechanism by which Lcn2 regulates thermogenesis and lipid metabolism. We found that Lcn2 is a retinoic acid target gene, and retinoic acid concurrently stimulated UCP1 and Lcn2 expression in adipocytes. Lcn2 KO mice exhibited a blunted effect of all-trans-retinoic acid (ATRA) on body weight and fat mass, lipid metabolism, and retinoic acid signaling pathway activation in adipose tissue under the high fat diet-induced obese condition. We further demonstrated that Lcn2 is required for the full action of ATRA on the induction of UCP1 and PGC-1α expression in brown adipocytes and the restoration of cold intolerance in Lcn2 KO mice. Interestingly, we discovered that Lcn2 KO mice have decreased levels of retinoic acid and retinol in adipose tissue. The protein levels of STRA6 responsible for retinol uptake were significantly decreased in adipose tissue. The retinol transporter RBP4 was increased in adipose tissue but decreased in the circulation, suggesting the impairment of RBP4 secretion in Lcn2 KO adipose tissue. Moreover, Lcn2 deficiency abolished the ATRA effect on RBP4 expression in adipocytes. All the data suggest that the decreased retinoid level and action are associated with impaired retinol transport and storage in adipose tissue in Lcn2 KO mice. We conclude that Lcn2 plays a critical role in regulating metabolic homeostasis of retinoids and retinoid-mediated thermogenesis in adipose tissue. PMID:27008859

  11. Curriculum Change Part 2a. Programme Development in Colleges. Part 2a: The Work Activities of Teachers on TEC Certificate Programmes.

    ERIC Educational Resources Information Center

    Dodd, S.; And Others

    A study examined the day-to-day work activities of teachers of Technician Education Council (TEC) certificate programs in Great Britain. During the study, a detailed questionnaire dealing with the tasks performed by TEC teachers, the amount of time spent on TEC teachers on individual activities, and the impact of the TEC programs on teacher…

  12. Design, synthesis and anxiolytic-like activity of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides.

    PubMed

    Mokrov, G V; Deeva, O A; Gudasheva, T A; Yarkov, S A; Yarkova, M A; Seredenin, S B

    2015-07-01

    A series of 1-arylpyrrolo[1,2-a]pyrazine-3-carboxamides were designed and synthesized as 18kDa translocator protein (TSPO) ligands. Anxiolytic-like activity of compounds was evaluated in the open field test and elevated plus maze test. Compounds 1a and 1b demonstrated high anxiolytic-like effect in the dose range of 0.1-1.0mg/kg comparable with that of diazepam. The involvement of TSPO receptor in the mechanism of anxiolytic-like activity of new compounds was proved by antagonism of the most active compound 1a with TSPO selective inhibitor PK11195. In vitro binding studies demonstrated high TSPO affinities for compounds 1a and 1b. PMID:25937237

  13. 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. PMID:27039035

  14. Inhibition of human Cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

    PubMed Central

    Kandagatla, Suneel K.; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M.

    2014-01-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ±0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ±1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5–12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. PMID:24924949

  15. PDE3A Regulates Basal Myocardial Contractility through Interacting with SERCA2a-Signaling Complexes in Mouse Heart

    PubMed Central

    Beca, Sanja; Ahmad, Faiyaz; Shen, Weixing; Liu, Jie; Makary, Samy; Polidovitch, Nazari; Sun, Junhui; Hockman, Steven; Chung, Youn Wook; Movesian, Matthew; Murphy, Elizabeth; Manganiello, Vincent; Backx, Peter H.

    2013-01-01

    Rationale cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP–mediated signaling. The role of different PDE isozymes, particularly PDE3A versus PDE3B, in the regulation of heart function remains unclear. Objective To determine the relative contribution of PDE3A versus PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Methods and Results Compared to wild-type (WT) littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A−/−, but not PDE3B−/−, mice. Furthermore, PDE3 inhibition had no effect on PDE3A−/− hearts but increased contractility in WT (as expected) and PDE3B−/− hearts to levels indistinguishable from PDE3A−/−. The enhanced contractility in PDE3A−/− hearts was associated with cAMP-dependent elevations in Ca2+ transient amplitudes and increased SR Ca2+ content, without changes in L-type Ca2+ currents (ICa,L) of cardiomyocytes, as well as with increased SR Ca2+-ATPase (SERCA2a) activity, SR Ca2+ uptake rates, and phospholamban (PLN) phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ~8-fold in SR fractions from PDE3A−/− hearts. Co-immunoprecipitation experiments further revealed that PDE3A associates with both SERCA2a and PLN in a complex which also contains AKAP-18, PKA-RII and PP2A. Conclusion Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca2+ content by regulating cAMP in microdomains containing macromolecular complexes of SERCA2a-PLN-PDE3A. PMID:23168336

  16. Inhibition of Protein Phosphatase 2A: Focus on the Glutamatergic System.

    PubMed

    Zimmer, Eduardo R; Leuzy, Antoine; Souza, Diogo O; Portela, Luis V

    2016-08-01

    In a recent review published in Molecular Neurobiology, Kamat and colleagues (Mol Neurobiol. 2014 Dec;50(3):852-65) highlighted the cellular and molecular mechanisms involved in Okadaic acid (OKA)-induced neurotoxicity. In this review, the authors underline a wide range of pathological signaling pathways involved in OKA-induced neurotoxicity; however, the role of glutamate was only briefly described. We believe that the hyperactivation of the glutamatergic system is a key pathophysiological player in OKA-induced neurotoxicity and deserves serious attention. In this commentary, we propose an integrative model linking glutamate and PP2A and put forward some unanswered questions. PMID:26141124

  17. Inhibitors of 7-Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells.

    PubMed

    Kim, Hye-Young H; Korade, Zeljka; Tallman, Keri A; Liu, Wei; Weaver, C David; Mirnics, Karoly; Porter, Ned A

    2016-05-16

    A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS. PMID:27097157

  18. 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.

  19. A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model.

    PubMed

    Roy, S; Patil, D; Ghadigaonkar, S; Roy, R; Mukherjee, S; Chowdhary, A; Deshmukh, R

    2015-01-01

    Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 10(4.25) LD50/0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD50 titre, challenge dose of 50 LD 50 was administered along with rhuIFN α-2a with pre-exposure (primed) and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These findings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the efficacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease. PMID:25560017

  20. Design and synthesis of some substituted thiazolo[3,2-a]pyrimidine derivatives of potential biological activities

    PubMed Central

    Abdel Moty, Samia G.; Hussein, Mostafa A.; Abdel Aziz, Salah A.; Abou-Salim, Mahrous A.

    2013-01-01

    In continuation to our previous work, thiazolopyrimidines 2a–x were synthesized through intramolecular cyclization of 2-phenacylthio-dihydropyrimidine hydrobromides 1a–x using polyphosphoric acid. On the other hand, thiazolo[3,2-a]pyrimidine-3-one 3 was coupled with aryldiazonium salts or condensed with isatin to afford compounds 4a–c or 5, respectively. Chemical structure of the target compounds was substantiated by IR, FT-IR, 1H-, 13C and DEPT-13C NMR, MS as well as microanalyses. Moreover, the lipophilicity of the target compounds is expressed as Clog P. The antimicrobial screening of the test compounds 2a–x, 4a–c and 5 revealed moderate activity in comparison to reference drugs. Compounds 2a–c, 2e, 2o and 2v showed a gradual increase in their anti-inflammatory activity reaching its maximum at 5 h compared to indomethacin. Furthermore, the analgesic activity of compounds 2a–c, 2e, 2o and 2v revealed a maximum activity after 5 h of injection compared to aspirin and the LD50 of compounds 2e and 2v was determined. PMID:27013904

  1. IKKα negatively regulates ASC-dependent inflammasome activation.

    PubMed

    Martin, Bradley N; Wang, Chenhui; Willette-Brown, Jami; Herjan, Tomasz; Gulen, Muhammet F; Zhou, Hao; Bulek, Katarzyna; Franchi, Luigi; Sato, Takashi; Alnemri, Emad S; Narla, Goutham; Zhong, Xiao-Ping; Thomas, James; Klinman, Dennis; Fitzgerald, Katherine A; Karin, Michael; Nuñez, Gabriel; Dubyak, George; Hu, Yinling; Li, Xiaoxia

    2014-01-01

    The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IκB kinase α (IKKα) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes. PMID:25266676

  2. 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

  3. 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.

  4. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGESBeta

    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, 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

  5. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids.

    PubMed

    Naranjo, Andrea N; McNeely, Patrick M; Katsaras, John; Robinson, Anne Skaja

    2016-08-01

    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. 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. PMID:27241126

  6. Role of “oncogenic nexus” of CIP2A in breast oncogenesis: how does it work?

    PubMed Central

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

    2015-01-01

    The CIP2A gene is an oncogene associated with solid and hematologic malignancies [1]. CIP2A protein is an oncoprotein and a potential cancer therapy target [2]. Literature shows that CIP2A inhibits the tumor suppressor protein PP2A [3] which downregulates phophorylation of AKT, a hallmark of cancers [4] and stabilizes the proto-oncogene, c-MYC in tumor cells [5], the comprehensive action of CIP2A and its functional interaction(s) with other oncoproteins and tumor suppressors is not clearly established. Recently we tried to put forward a contextual mode-of-action of CIP2A protein in a review which proposed that CIP2A influences oncogenesis via an “oncogenic nexus” [1]. In this review we critically evaluated the potential relevance of the mode-of-action of the “oncogenic nexus” of CIP2A in breast carcinogenesis and appraised the role of this nexus in different PAM50 luminal A, PAM50 luminal B, PAM50 HER2-enriched and PAM50 basal BC. This review has a novel approach. Here we have not only compiled and discussed the latest developments in this field but also presented data obtained from c-BioPortal and STRING10 in order to substantiate our view regarding the mode-of-action of the “oncogenic nexus” of CIP2A. We functionally correlated alterations of genes pertaining to the “oncogenic nexus” of CIP2A with protein-protein interactions between the different components of the nexus including (1) subunits of PP2A, (2) multiple transcription factors including MYC oncogene and (3) components of the PI3K-mTOR and the MAPK-ERK oncogenic pathways. Using these proteins as “input” to STRING10 we studied the association, Action view, at the highest Confidence level. OncoPrints (c-BioPortal) showed alterations (%) of regulatory subunits genes of PP2A (PPP2R1A and PPP2R1B) along with alterations of CIP2A in breast invasive carcinoma (TCGA, Nature 2012 & TCGA, Provisional). Similar genetic alterations of PP2A were also observed in samples of breast tumors at our

  7. IKKα negatively regulates ASC-dependent inflammasome activation

    PubMed Central

    Martin, Bradley N.; Wang, Chenhui; Willette-Brown, Jami; Herjan, Tomasz; Gulen, Muhammet F.; Zhou, Hao; Bulek, Katarzyna; Franchi, Luigi; Sato, Takashi; Narla, Goutham; Zhong, Xiao-Ping; Thomas, James; Klinman, Dennis; Fitzgerald, Katherine A.; Karin, Michael; Nuñez, Gabriel; Dubyak, George; Hu, Yinling; Li, Xiaoxia

    2014-01-01

    The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes. PMID:25266676

  8. HSP105 recruits protein phosphatase 2A to dephosphorylate β-catenin.

    PubMed

    Yu, Nancy; Kakunda, Michael; Pham, Victoria; Lill, Jennie R; Du, Pan; Wongchenko, Matthew; Yan, Yibing; Firestein, Ron; Huang, XiaoDong

    2015-04-01

    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

  9. 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

  10. Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A

    SciTech Connect

    Van Hoof, C.; Cayla, X.; Merlevede, W.; Goris, J.

    1995-07-20

    The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5{prime} flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-{kappa}B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5{prime} of a luciferase reporter gene revealed that the 5{prime} flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis. 28 refs., 8 figs., 1 tab.

  11. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels.

    PubMed

    Liu, Yumei; Zou, Haifeng; Zhao, Ping; Sun, Bo; Wang, Jinghua; Kong, Qingfei; Mu, Lili; Zhao, Sihan; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Zhao, Jiaying; Yin, Pengqi; Liu, Lei; Zhao, Xiuli; Li, Hulun

    2016-08-25

    Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4(+) T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca(2+)]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which may offer a therapeutic option for this disease. PMID:27217214

  12. Synthesis, antiproliferative activity and DNA binding properties of novel 5-aminobenzimidazo[1,2-a]quinoline-6-carbonitriles.

    PubMed

    Perin, Nataša; Nhili, Raja; Ester, Katja; Laine, William; Karminski-Zamola, Grace; Kralj, Marijeta; David-Cordonnier, Marie-Hélène; Hranjec, Marijana

    2014-06-10

    The synthesis of 5-amino substituted benzimidazo[1,2-a]quinolines prepared by microwave assisted amination from halogeno substituted precursor was described. The majority of compounds were active at micromolar concentrations against colon, lung and breast carcinoma cell lines in vitro. The N,N-dimethylaminopropyl 9 and piperazinyl substituted derivative 19 showed the most pronounced activity towards all of the three tested tumor cell lines, which could be correlated to the presence of another N heteroatom and its potential interactions with biological targets. The DNA binding studies, consisting of UV/Visible absorbency, melting temperature studies, and fluorescence and circular dichroism titrations, revealed that compounds 9, 19 and 20 bind to DNA as strong intercalators. The cellular distribution analysis, based on compounds' intrinsic fluorescence, showed that compound 20 does not enter the cell, while compounds 9 and 19 do, which is in agreement with their cytotoxic effects. Compound 9 efficiently targets the nucleus whereas 19, which also showed DNA intercalating properties in vitro, was mostly localised in the cytoplasm suggesting that the antitumor mechanism of action is DNA-independent. PMID:24780599

  13. 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

  14. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    PubMed

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation. PMID:26611209

  15. The Histone Variant MacroH2A1.2 is Necessary for the Activation of Muscle Enhancers and Recruitment of the Transcription Factor Pbx1

    PubMed Central

    Dell’Orso, Stefania; Wang, A. Hongjun; Shih, Han-Yu; Saso, Kayoko; Berghella, Libera; Gutierrez-Cruz, Gustavo; Ladurner, Andreas G.; O’Shea, John J.; Sartorelli, Vittorio; Zare, Hossein

    2016-01-01

    SUMMARY Histone variants complement and integrate histone post-translational modifications in regulating transcription. The histone variant macroH2A1 (mH2A1) is almost three times the size of its canonical H2A counterpart due to the presence of a ~25kDa evolutionarily conserved non-histone macro domain. Strikingly, mH2A1 can mediate both gene repression and activation. However, the molecular determinants conferring these alternative functions remain elusive. Here, we report that mH2A1.2 is required for the activation of the myogenic gene regulatory network and muscle cell differentiation. H3K27 acetylation at prospective enhancers is exquisitely sensitive to mH2A1.2, indicating a role of mH2A1.2 in imparting enhancer activation. Both H3K27 acetylation and recruitment of the transcription factor Pbx1 at prospective enhancers are regulated by mH2A1.2. Overall, our findings indicate a role of mH2A1.2 in marking regulatory regions for activation. PMID:26832413

  16. Axonal elongation and dendritic branching is enhanced by adenosine A2A receptors activation in cerebral cortical neurons.

    PubMed

    Ribeiro, Filipa F; Neves-Tomé, Raquel; Assaife-Lopes, Natália; Santos, Telma E; Silva, Rui F M; Brites, Dora; Ribeiro, Joaquim A; Sousa, Mónica M; Sebastião, Ana M

    2016-06-01

    Axon growth and dendrite development are key processes for the establishment of a functional neuronal network. Adenosine, which is released by neurons and glia, is a known modulator of synaptic transmission but its influence over neuronal growth has been much less investigated. We now explored the action of adenosine A2A receptors (A2AR) upon neurite outgrowth, discriminating actions over the axon or dendrites, and the mechanisms involved. Morphometric analysis of primary cultures of cortical neurons from E18 Sprague-Dawley rats demonstrated that an A2AR agonist, CGS 21680, enhances axonal elongation and dendritic branching, being the former prevented by inhibitors of phosphoinositide 3-kinase, mitogen-activated protein kinase and phospholipase C, but not of protein kinase A. By testing the influence of a scavenger of BDNF (brain-derived neurotrophic factor) over the action of the A2AR agonist and the action of a selective A2AR antagonist over the action of BDNF, we could conclude that while the action of A2ARs upon dendritic branching is dependent on the presence of endogenous BDNF, the influence of A2ARs upon axonal elongation is independent of endogenous BDNF. In consonance with the action over axonal elongation, A2AR activation promoted a decrease in microtubule stability and an increase in microtubule growth speed in axonal growth cones. In conclusion, we disclose a facilitatory action of A2ARs upon axonal elongation and microtubule dynamics, providing new insights for A2ARs regulation of neuronal differentiation and axonal regeneration. PMID:26068054

  17. Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficile infection in mice following vancomycin treatment

    PubMed Central

    2012-01-01

    Background Activation of the A2A adenosine receptor (A2AAR) decreases production of inflammatory cytokines, prevents C. difficile toxin A-induced enteritis and, in combination with antibiotics, increases survival from sepsis in mice. We investigated whether A2AAR activation improves and A2AAR deletion worsens outcomes in a murine model of C. difficile (strain VPI10463) infection (CDI). Methods C57BL/6 mice were pretreated with an antibiotic cocktail prior to infection and then treated with vancomycin with or without an A2AAR agonist. A2AAR-/- and littermate wild-type (WT) mice were similarly infected, and IFNγ and TNFα were measured at peak of and recovery from infection. Results Infected, untreated mice rapidly lost weight, developed diarrhea, and had mortality rates of 50-60%. Infected mice treated with vancomycin had less weight loss and diarrhea during antibiotic treatment but mortality increased to near 100% after discontinuation of antibiotics. Infected mice treated with both vancomycin and an A2AAR agonist, either ATL370 or ATL1222, had minimal weight loss and better long-term survival than mice treated with vancomycin alone. A2AAR KO mice were more susceptible than WT mice to death from CDI. Increases in cecal IFNγ and blood TNFα were pronounced in the absence of A2AARs. Conclusion In a murine model of CDI, vancomycin treatment resulted in reduced weight loss and diarrhea during acute infection, but high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. The administration of vancomycin plus an A2AAR agonist reduced inflammation and improved survival rates, suggesting a possible benefit of A2AAR agonists in the management of CDI to prevent recurrent disease. PMID:23217055

  18. DYRK1A-mediated phosphorylation of GluN2A at Ser1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

    PubMed Central

    Grau, Cristina; Arató, Krisztina; Fernández-Fernández, José M.; Valderrama, Aitana; Sindreu, Carlos; Fillat, Cristina; Ferrer, Isidre; de la Luna, Susana; Altafaj, Xavier

    2014-01-01

    N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons. PMID:25368549

  19. 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

  20. Age-related changes in AMPK activation: Role for AMPK phosphatases and inhibitory phosphorylation by upstream signaling pathways.

    PubMed

    Salminen, Antero; Kaarniranta, Kai; Kauppinen, Anu

    2016-07-01

    AMP-activated protein kinase (AMPK) is a fundamental regulator of energy metabolism, stress resistance, and cellular proteostasis. AMPK signaling controls an integrated signaling network which is involved in the regulation of healthspan and lifespan e.g. via FoxO, mTOR/ULK1, CRCT-1/CREB, and SIRT1 signaling pathways. Several studies have demonstrated that the activation capacity of AMPK signaling declines with aging, which impairs the maintenance of efficient cellular homeostasis and enhances the aging process. However, it seems that the aging process affects AMPK activation in a context-dependent manner since occasionally, it can also augment AMPK activation, possibly attributable to the type of insult and tissue homeostasis. Three protein phosphatases, PP1, PP2A, and PP2C, inhibit AMPK activation by dephosphorylating the Thr172 residue of AMPKα, required for AMPK activation. In addition, several upstream signaling pathways can phosphorylate Ser/Thr residues in the β/γ interaction domain of the AMPKα subunit that subsequently blocks the activation of AMPK. These inhibitory pathways include the insulin/AKT, cyclic AMP/PKA, and RAS/MEK/ERK pathways. We will examine the evidence whether the efficiency of AMPK responsiveness declines during the aging process. Next, we will review the mechanisms involved in curtailing the activation of AMPK. Finally, we will elucidate the potential age-related changes in the inhibitory regulation of AMPK signaling that might be a part of the aging process. PMID:27060201

  1. The 2 A resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium.

    PubMed

    Pflugrath, J W; Quiocho, F A

    1988-03-01

    The crystal structure of the liganded form of the sulfate-binding protein, an initial receptor for active transport of sulfate in Salmonella typhimurium, has been solved and refined at 2.0 A resolution (1 A = 0.1 nm). The final model, which consists of 2422 non-hydrogen atoms, one sulfate substrate and 143 water molecules, yields a crystallographic R-factor of 14.0% for 16,959 reflections between 8 and 2 A. The structure deviates from ideal bond lengths and angle distances by 0.015 A and 0.037 A, respectively. The protein is ellipsoid with overall dimensions of 35 A x 35 A x 65 A and consists of two similar globular domains. The two domains are linked by three distinct peptide segments, which though widely separated in the amino acid sequence, are in close proximity in the tertiary structure. As these connecting segments are located near the periphery of the molecule, they further serve as the base or a "boundary" of the deep cleft formed between the two domains. Despite the unusual interdomain connectivity, both domains have similar supersecondary structure consisting of a central five-stranded beta-pleated sheet sandwiched by alpha-helices on either side. The arrangement of the two domains gives rise to the ellipsoidal shape and to the cleft between the two domains wherein the sulfate substrate is found and completely engulfed. A discovery of considerable importance is that the sulfate substrate is tightly held in place primarily by seven hydrogen bonds, five of which are donated by main-chain peptide NH groups, another by a serine hydroxyl and the last by the indole NH moiety of a tryptophan side-chain; there are no positively charged residues, nor cations, nor water molecules within van der Waals' distance to the sulfate dianion. All the main-chain peptide units associated with the sulfate are in turn linked (via the peptide CO group) to arrays of hydrogen bonds. Three of these arrays are composed of alternating peptide units and hydrogen bonds within the

  2. 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. PMID:27154620

  3. Synthesis and evaluation of the anticoccidial activity of trifluoropyrido[1,2-a]pyrimidin-2-one derivatives.

    PubMed

    Silpa, Laurence; Niepceron, Alisson; Laurent, Fabrice; Brossier, Fabien; Pénichon, Mélanie; Enguehard-Gueiffier, Cécile; Abarbri, Mohamed; Silvestre, Anne; Petrignet, Julien

    2016-01-01

    Screening of our chemical library to discover new molecules exhibiting in vitro activity against the invasion of host cells by Eimeria tenella revealed a lead compound with an IC50 of 15μM. Structure-activity relationship studies were conducted with 34 newly synthesized compounds to identify more active molecules and enhance in vitro activity against the parasite. Four compounds were more effective in inhibiting MDBK cell invasion in vitro than the lead compound. PMID:26597537

  4. Melatonin reversal of DOI-induced hypophagia in rats; possible mechanism by suppressing 5-HT(2A) receptor-mediated activation of HPA axis.

    PubMed

    Raghavendra, V; Kulkarni, S K

    2000-03-31

    Serotonin type 2A (5-HT(2A)) receptor-mediated neurotransmitter is known to activate hypothalamic-pituitary-adrenal (HPA) axis, regulate sleep-awake cycle, induce anorexia and hyperthermia. Interaction between melatonin and 5-HT(2A) receptors in the regulation of the sleep-awake cycle and head-twitch response in rat have been reported. Previous studies have shown that melatonin has suppressant effect on HPA axis activation, decreases core body temperature and induces hyperphagia in animals. However, melatonin interaction with 5-HT(2A) receptors in mediation of these actions is not yet reported. We have studied the acute effect of melatonin and its antagonist, luzindole on centrally administered (+/-)-1-(2, 5-dimethoxy-4-iodophenyl) 2-amino propane (DOI; a 5-HT(2A/2C) agonist)-induced activation of HPA axis, hypophagia and hyperthermia in 24-h food-deprived rats. Like ritanserin [(1 mg/kg, i.p.) 5-HT(2A/2C) antagonist], peripherally administered melatonin (1.5 and 3 mg/kg, i.p.) did not affect the food intake, rectal temperature or basal adrenal ascorbic acid level. However, pretreatment of rats with it significantly reversed DOI (10 microgram, intraventricular)-induced anorexia and activation of HPA axis. But the hyperthermia induced by DOI was not sensitive to reversal by melatonin. Mel(1) receptor subtype antagonist luzindole (5 microgram, intraventricular) did not modulate the DOI effect but antagonized the melatonin (3 mg/kg, i.p.) reversal of 5-HT(2A) agonist response. The present data suggest that melatonin reversal of DOI-induced hypophagia could be due to suppression of 5-HT(2A) mediated activation of HPA axis. PMID:10727629

  5. 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. PMID:26818358

  6. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

    PubMed

    Leatherbarrow, R J; Rademacher, T W; Dwek, R A; Woof, J M; Clark, A; Burton, D R; Richardson, N; Feinstein, A

    1985-04-01

    Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events. PMID:4033665

  7. High throughput screening and structure-activity relationship study of potential α2A-adrenoceptor agonists by LANCETM cAMP assay.

    PubMed

    Yang, Huan; He, Ling; Yan, Ming; He, Jian-Guo; Yu, Tao

    2013-06-28

    G protein-coupled receptors (GPCRs) are signaling molecules with a wide variety of skills. Members of this large family of membrane protein have been shown to regulate the activities of the different signaling pathways of the ligand specific manner. α2-adrenoceptors (α2-ARs) are one of the GPCRs and the stimulation of them could modulate many classical effects such as hypotension, bradycardia, etc. Recently, α2A-AR is more and more important for its role in the therapeutic applications in central nervous system (CNS) diseases.High throughput screening of α2A-AR agonists was established by LANCETM cAMP assay from a compound library of 80,000 small-molecule compounds to find out potential human α2A-adrenoceptor (α2A-AR) agonists that might have therapeutic effect in CNS diseases. From the preliminary and secondary screening, 37 compounds were identified as α2AAR agonists, and six compounds among them presented more pronounced α2A-AR stimulating activity than guanfacine, a selective α2A-AR agonist. The study provided referred data for the development of potent α2A-AR agonists and suggested that the existence of the parent structure (1, 2, 4-benzothiadiazine 1, 1-dioxide) bodes well for pharmaceutical development of α2A-AR agonists. PMID:23514320

  8. 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

  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. Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis.

    PubMed

    Flögel, Ulrich; Burghoff, Sandra; van Lent, Peter L E M; Temme, Sebastian; Galbarz, Lisa; Ding, Zhaoping; El-Tayeb, Ali; Huels, Sandra; Bönner, Florian; Borg, Nadine; Jacoby, Christoph; Müller, Christa E; van den Berg, Wim B; Schrader, Jürgen

    2012-08-01

    Adenosine A(2A) receptor (A(2A)R) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A(2A)R agonists (prodrugs) that require the presence of ecto-5'-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5'-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 ((19)F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A(2A)R. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A(2A)R expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A(2A)R agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation. PMID:22875828

  11. 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

  12. 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

    2015-01-01

    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 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+). PMID:26659126

  13. Okadaic acid-sensitive activation of Maxi Cl− channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells

    PubMed Central

    Diaz, Mario; Bahamonde, Maria I; Lock, Hagar; Muñoz, Francisco J; Hardy, Simon P; Posas, Francesc; Valverde, Miguel A

    2001-01-01

    The regulation of Maxi Cl− channels by 17β-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl− channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells.Whole-cell Maxi Cl− currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium.Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl− channels. The inhibitory effect of 17β-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine.Current activation was unaffected by the removal of intracellular Ca2+ and Mg2+, but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl− channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells.Although the role of these Maxi Cl− channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways. PMID:11579158

  14. Activity-induced synaptic delivery of the GluN2A-containing NMDA receptor is dependent on endoplasmic reticulum chaperone Bip and involved in fear memory.

    PubMed

    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

    2015-07-01

    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 Ca(2+)-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

  15. 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. PMID:26657313

  16. 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. PMID:23785166

  17. Advancement of Imidazo[1,2-a]pyridines with Improved Pharmacokinetics and nM Activity vs. Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    A set of 14 imidazo[1,2-a]pyridine-3-carboxamides was synthesized and screened against Mycobacterium tuberculosis H37Rv. The minimum inhibitory concentrations of 12 of these agents were ≤1 μM against replicating bacteria and 5 compounds (9, 12, 16, 17, and 18) had MIC values ≤0.006 μM. Compounds 13 and 18 were screened against a panel of MDR and XDR drug resistant clinical Mtb strains with the potency of 18 surpassing that of clinical candidate PA-824 by nearly 10-fold. The in vivo pharmacokinetics of compounds 13 and 18 were evaluated in male mice by oral (PO) and intravenous (IV) routes. These results indicate that readily synthesized imidazo[1,2-a]pyridine-3-carboxamides are an exciting new class of potent, selective anti-TB agents that merit additional development opportunities. PMID:23930153

  18. A novel fused 1,2,4-triazine aryl derivative as antioxidant and nonselective antagonist of adenosine A(2A) receptors in ethanol-activated liver stellate cells.

    PubMed

    Szuster-Ciesielska, Agnieszka; Sztanke, Krzysztof; Kandefer-Szerszeń, Martyna

    2012-01-01

    It has been detected that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis. In this paper we examined if our new triazine derivative (IMT) can inhibit ethanol-induced activation of HSCs measured as increased α-SMA, collagen synthesis and enhanced oxidative stress in rat liver stellate cells. We also investigated its influence on cytokines (TGF-β, TNF-α) synthesis, MMP-2 and TIMP-1 production and ethanol-induced intracellular signal transduction. Moreover, with using of known adenosine A(2A) receptor agonist (CGS 21680), and antagonist (SCH 58261) we examined if this triazine derivative acts on adenosine receptors. We detected a strong antagonistic action of new triazine derivative (IMT) on ethanol-induced rat liver stellate cells activation, observed as a significant decrease in α-SMA, collagen synthesis, reactive oxygen species production, TGF-β, TNF-α, MMP-2 and TIMP-1 production as well as JNK, p38MAPK, NFκB, IκB, Smad3 phosphorylation. Moreover, IMT strongly inhibited activation of stellate cells by known selective agonist of adenosine A(2A) receptor (CGS 21680). When known A(2A) receptor antagonist (SCH 58261) was used together with IMT this effect was not spectacular. Additionally, only slight enhancement of inhibition was observed when cells were pretreated both IMT with SCH 58261, hence we suppose that IMT acts as nonselective antagonist of A(2A) receptors, and, besides its antioxidant activity, also by this way inhibited ethanol-induced stellate cell activation. PMID:22063920

  19. A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals.

    PubMed

    Srikanth, Sonal; Kim, Kyun-Do; Gao, Yuanyuan; Woo, Jin Seok; Ghosh, Shubhamoy; Calmettes, Guillaume; Paz, Aviv; Abramson, Jeff; Jiang, Meisheng; Gwack, Yousang

    2016-03-22

    More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells. PMID:27016526

  20. TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response

    PubMed Central

    Rosales, Kimberly Romero; Reid, Michael A.; Yang, Ying; Tran, Thai Q.; Wang, Wen-I; Lowman, Xazmin; Pan, Min; Kong, Mei

    2015-01-01

    Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response. PMID:26717153

  1. PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2013-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  2. 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.

  3. Investigations of Activated ACVR1/ALK2, a Bone Morphogenetic Protein Type I Receptor, That Causes Fibrodysplasia Ossificans Progressiva

    PubMed Central

    Kaplan, Frederick S.; Seemann, Petra; Haupt, Julia; Xu, Meiqi; Lounev, Vitali Y.; Mullins, Mary; Shore, Eileen M.

    2016-01-01

    Bone morphogenetic protein (BMP) type I receptors are serine-threonine kinase transmembrane signal transduction proteins that regulate a vast array of ligand-dependent cell-fate decisions with temporal and spatial fidelity during development and postnatal life. A recent discovery identified a recurrent activating heterozygous missense mutation in a BMP type I receptor [Activin receptor IA/activin-like kinase 2 (ACVR1; also known as ALK2)] in patients with the disabling genetic disorder fibrodysplasia ossificans progressiva (FOP). Individuals with FOP experience episodes of tissue metamorphosis that convert soft connective tissue such as skeletal muscle into a highly ramified and disabling second skeleton of heterotopic bone. The single nucleotide ACVR1/ALK2 mutation that causes FOP is one of the most specific disease-causing mutations in the human genome and to date the only known inherited activating mutation of a BMP receptor that causes a human disease. Thus, the study of FOP provides the basis for understanding the clinically relevant effects of activating mutations in the BMP signaling pathway. Here we briefly review methodologies that we have applied to studying activated BMP signaling in FOP. PMID:21036241

  4. AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

    PubMed Central

    Park, Hyojin; Lee, Sungwoon; Shrestha, Pravesh; Kim, Jihye; Park, Jeong Ae; Ko, Yeongrim; Ban, Young Ho; Park, Dae-Young; Ha, Sang-Jun; Koh, Gou Young; Hong, Victor Sukbong; Mochizuki, Naoki; Kim, Young-Myeong; Lee, Weontae

    2015-01-01

    The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor. PMID:26553931

  5. Dioxygen Binding in the Active Site of Histone Demethylase JMJD2A and the Role of the Protein Environment.

    PubMed

    Cortopassi, Wilian A; Simion, Robert; Honsby, Charles E; França, Tanos C C; Paton, Robert S

    2015-12-21

    JMJD2A catalyses the demethylation of di- and trimethylated lysine residues in histone tails and is a target for the development of new anticancer medicines. Mechanistic details of demethylation are yet to be elucidated and are important for the understanding of epigenetic processes. We have evaluated the initial step of histone demethylation by JMJD2A and demonstrate the dramatic effect of the protein environment upon oxygen binding using quantum mechanics/molecular mechanics (QM/MM) calculations. The changes in electronic structure have been studied for possible spin states and different conformations of O2 , using a combination of quantum and classical simulations. O2 binding to this histone demethylase is computed to occur preferentially as an end-on superoxo radical bound to a high-spin ferric centre, yielding an overall quintet ground state. The favourability of binding is strongly influenced by the surrounding protein: we have quantified this effect using an energy decomposition scheme into electrostatic and dispersion contributions. His182 and the methylated lysine assist while Glu184 and the oxoglutarate cofactor are deleterious for O2 binding. Charge separation in the superoxo-intermediate benefits from the electrostatic stabilization provided by the surrounding residues, stabilizing the binding process significantly. This work demonstrates the importance of the extended protein environment in oxygen binding, and the role of energy decomposition in understanding the physical origin of binding/recognition. PMID:26577067

  6. Modulation of cytochrome P450 2A5 activity by lipopolysaccharide: low-dose effects and non-monotonic dose-response relationship.

    PubMed

    De-Oliveira, Ana C A X; Poça, Kátia S; Totino, Paulo R R; Paumgartten, Francisco J R

    2015-01-01

    Mouse cytochrome P450 (CYP) 2A5 is induced by inflammatory conditions and infectious diseases that down-regulate the expression and activity of most other CYP isoforms. Enhanced oxidative stress and nuclear factor (erythroid 2-related factor) 2 (Nrf2) transcription factor activation have been hypothesised to mediate up-regulation of CYP2A5 expression in the murine liver. The unique and complex regulation of CYP2A5, however, is far from being thoroughly elucidated. Sepsis and high doses of bacterial lipopolysaccharide (LPS) elicit oxidative stress in the liver, but depression, not induction, of CYP2A5 has been observed in studies of mice treated with LPS. The foregoing facts prompted us to evaluate the response of CYP2A5 liver activity in female DBA-2 mice over a broad range of LPS doses (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 mg/kg). Cytokine levels (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon gamma, tumour necrosis factor alpha) and nitric oxide (NO) were measured in the blood serum. Activities of CYP1A (EROD) and CYP2B (BROD) in the liver were also determined for comparative purposes. LPS depressed CYP2A5 at low doses (0.025-2.0 mg/kg) but not at doses (>2 mg/kg) that increased pro-inflammatory cytokines and NO serum levels, and depressed CYP1A and CYP2B activities. Blockade of pro-inflammatory cytokines and the overproduction of NO induced by co-treatment with pentoxifylline and LPS and iNOS inhibition with aminoguanidine both extended down-regulation of CYP2A5 to the high dose range while not affecting LPS-induced depression of CYP1A and CYP2B. Overall, the results suggested that NO plays a role in the reversal of the low-dose LPS-induced depression of CYP2A5 observed when mice were challenged with higher doses of LPS. PMID:25635819

  7. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

    PubMed Central

    Bazovkina, Darya V.; Kondaurova, Elena M.; Naumenko, Vladimir S.; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  8. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells.

    PubMed

    Selby, Mark J; Engelhardt, John J; Quigley, Michael; Henning, Karla A; Chen, Timothy; Srinivasan, Mohan; Korman, Alan J

    2013-07-01

    Antitumor activity of CTLA-4 antibody blockade is thought to be mediated by interfering with the negative regulation of T-effector cell (Teff) function resulting from CTLA-4 engagement by B7-ligands. In addition, a role for CTLA-4 on regulatory T cells (Treg), wherein CTLA-4 loss or inhibition results in reduced Treg function, may also contribute to antitumor responses by anti-CTLA-4 treatment. We have examined the role of the immunoglobulin constant region on the antitumor activity of anti-CTLA-4 to analyze in greater detail the mechanism of action of anti-CTLA-4 antibodies. Anti-CTLA-4 antibody containing the murine immunoglobulin G (IgG)2a constant region exhibits enhanced antitumor activity in subcutaneous established MC38 and CT26 colon adenocarcinoma tumor models compared with anti-CTLA-4 containing the IgG2b constant region. Interestingly, anti-CTLA-4 antibodies containing mouse IgG1 or a mutated mouse IgG1-D265A, which eliminates binding to all Fcγ receptors (FcγR), do not show antitumor activity in these models. Assessment of Teff and Treg populations at the tumor and in the periphery showed that anti-CTLA-4-IgG2a mediated a rapid and dramatic reduction of Tregs at the tumor site, whereas treatment with each of the isotypes expanded Tregs in the periphery. Expansion of CD8(+) Teffs is observed with both the IgG2a and IgG2b anti-CTLA-4 isotypes, resulting in a superior Teff to Treg ratio for the IgG2a isotype. These data suggest that anti-CTLA-4 promotes antitumor activity by a selective reduction of intratumoral Tregs along with concomitant activation of Teffs. PMID:24777248

  9. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity.

    PubMed

    Bazovkina, Darya V; Kondaurova, Elena M; Naumenko, Vladimir S; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  10. Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

    PubMed

    Huh, G H; Nakayama, T; Meshi, T; Iwabuchi, M

    1997-03-01

    To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner. PMID:9106503

  11. X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis.

    PubMed

    Gomes, Marco T R; Teixeira, Raphael D; Lopes, Míriam T P; Nagem, Ronaldo A P; Salas, Carlos E

    2012-12-01

    CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Cryst