Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells.

PubMed

Lu, Gang; Sun, Haipeng; She, Pengxiang; Youn, Ji-Youn; Warburton, Sarah; Ping, Peipei; Vondriska, Thomas M; Cai, Hua; Lynch, Christopher J; Wang, Yibin

2009-06-01

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55γ as a Stress-Sensitive Inhibitor of c-SRC

PubMed Central

Eichhorn, Pieter J. A; Creyghton, Menno P; Wilhelmsen, Kevin; van Dam, Hans; Bernards, René

2007-01-01

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55γ and PR55δ as inhibitors of c-Jun NH2-terminal kinase (JNK) activation by UV irradiation. We show that PR55γ binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55γ and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55γ. PMID:18069897

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

PubMed Central

Schmitz, Michael H. A.; Held, Michael; Janssens, Veerle; Hutchins, James R. A.; Hudecz, Otto; Ivanova, Elitsa; Goris, Jozef; Trinkle-Mulcahy, Laura; Lamond, Angus I.; Poser, Ina; Hyman, Anthony A.; Mechtler, Karl; Peters, Jan-Michael; Gerlich, Daniel W.

2013-01-01

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. PMID:20711181

Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity.

PubMed

Vijayaraghavan, S; Stephens, D T; Trautman, K; Smith, G D; Khatra, B; da Cruz e Silva, E F; Greengard, P

1996-03-01

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.

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

PubMed Central

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

2016-01-01

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells. PMID:27780869

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

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

DOE PAGES

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; ...

2015-09-04

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. Furthemore, these analyses, which were confirmed usingmore » 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. Our analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases.« less

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

PubMed

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

2016-12-02

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1.

PubMed

Tellier, Géraldine; Lenne, Astrid; Cailliau-Maggio, Katia; Cabezas-Cruz, Alejandro; Valdés, James J; Martoriati, Alain; Aliouat, El M; Gosset, Pierre; Delaire, Baptiste; Fréville, Aline; Pierrot, Christine; Khalife, Jamal

2016-01-01

Protein phosphatase 1 (PP1c) is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2β of Plasmodium falciparum (PfeIF2β) is an interactor of PfPP1c. Sequence analysis of PfeIF2β revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 ((29)FGEKKK(34), (103)KVAW(106)). As expected, we showed that PfeIF2β binds PfeIF2γ and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2β-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2β is able to induce Germinal Vesicle Break Down (GVBD) when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2β seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2β in the nucleus. Hence, the role played by PfeIF2β in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1.

Protein Phosphatase 1-α Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle.

PubMed

Sharma, Pragya; Arias, Edward B; Cartee, Gregory D

2016-09-01

Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) and Ser(588) by Akt is essential for insulin's full effect on glucose transport. However, protein phosphorylation is determined by the balance of actions by kinases and phosphatases, and the specific phosphatase(s) controlling AS160 dephosphorylation is (are) unknown. Accordingly, we assessed roles of highly expressed skeletal muscle serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used an AS160 dephosphorylation assay. Lysates from insulin-stimulated skeletal muscle were treated with pharmacological phosphatase inhibitors and assessed for AS160 Ser(588) and Thr(642) dephosphorylation. AS160 dephosphorylation on both phosphorylation sites was unaltered by PP2B or PP2C inhibitors. Okadaic acid (low dose inhibits PP2A; high dose inhibits PP1) delayed AS160 Ser(588) (both doses) and Thr(642) (high dose only) dephosphorylation concomitant with greater Akt phosphorylation (both doses). AS160 was coimmunoprecipitated with PP1-α but not with PP1-β, PP1-γ1, or PP2A. Recombinant inhibitor-2 protein (a selective PP1 inhibitor) delayed AS160 dephosphorylation on both phosphorylation sites without altering Akt phosphorylation. Furthermore, knockdown of PP1-α but not PP1-β or PP1-γ1 by small interfering RNA caused greater AS160 Ser(588) and Thr(642) phosphorylation concomitant with unaltered Akt phosphorylation. Together, these results identified PP1-α as a regulator of AS160 Thr(642) and Ser(588) dephosphorylation in skeletal muscle. © 2016 by the American Diabetes Association.

Structural Basis of PP2A Inhibition by Small t Antigen

PubMed Central

Cho, Uhn Soo; Morrone, Seamus; Sablina, Anna A; Arroyo, Jason D; Hahn, William C; Xu, Wenqing

2007-01-01

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST. PMID:17608567

Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d*

PubMed Central

Joseph, Biny K.; Liu, Hsing-Yin; Francisco, Jamie; Pandya, Devanshi; Donigan, Melissa; Gallo-Ebert, Christina; Giordano, Caroline; Bata, Adam; Nickels, Joseph T.

2015-01-01

AMP kinase is a heterotrimeric serine/threonine protein kinase that regulates a number of metabolic processes, including lipid biosynthesis and metabolism. AMP kinase activity is regulated by phosphorylation, and the kinases involved have been uncovered. The particular phosphatases counteracting these kinases remain elusive. Here we discovered that the protein phosphatase 2A heterotrimer, PP2APpp2r2d, regulates the phosphorylation state of AMP kinase by dephosphorylating Thr-172, a residue that activates kinase activity when phosphorylated. Co-immunoprecipitation and co-localization studies indicated that PP2APpp2r2d directly interacted with AMP kinase. PP2APpp2r2d dephosphorylated Thr-172 in rat aortic and human vascular smooth muscle cells. A positive correlation existed between decreased phosphorylation, decreased acetyl-CoA carboxylase Acc1 phosphorylation, and sterol response element-binding protein 1c-dependent gene expression. PP2APpp2r2d protein expression was up-regulated in the aortas of mice fed a high fat diet, and the increased expression correlated with increased blood lipid levels. Finally, we found that the aortas of mice fed a high fat diet had decreased AMP kinase Thr-172 phosphorylation, and contained an Ampk-PP2APpp2r2d complex. Thus, PP2APpp2r2d may antagonize the aortic AMP kinase activity necessary for maintaining normal aortic lipid metabolism. Inhibiting PP2APpp2r2d or activating AMP kinase represents a potential pharmacological treatment for many lipid-related diseases. PMID:25694423

Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells.

PubMed

Kim, Myeong-Ok; Choe, Min Ho; Yoon, Yi Na; Ahn, Jiyeon; Yoo, Minjin; Jung, Kwan-Young; An, Sungkwan; Hwang, Sang-Gu; Oh, Jeong Su; Kim, Jae-Sung

2017-11-15

Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged asan anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide asa CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide asa PP2A-activating drug in the clinical treatment of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

Developmental and light regulation of tumor suppressor protein PP2A in the retina

PubMed Central

Rajala, Ammaji; Wang, Yuhong; Abcouwer, Steven F.; Gardner, Thomas W.; Rajala, Raju V.S.

2018-01-01

Protein phosphatases are a group of universal enzymes that are responsible for the dephosphorylation of various proteins and enzymes in cells. Cellular signal transduction events are largely governed by the phosphorylation of key proteins. The length of cellular response depends on the activation of protein phosphatase that dephosphorylates the phosphate groups to halt a biological response, and fine-tune the defined cellular outcome. Dysregulation of these phosphatase(s) results in various disease phenotypes. The retina is a post-mitotic tissue, and oncogenic tyrosine and serine/ threonine kinase activities are important for retinal neuron survival. Aberrant activation of protein phosphatase(s) may have a negative effect on retinal neurons. In the current study, we characterized tumor suppressor protein phosphatase 2 (PP2A), a major serine/ threonine kinase with a broad substrate specificity. Our data suggest that PP2A is developmentally regulated in the retina, localized predominantly in the inner retina, and expressed in photoreceptor inner segments. Our findings indicate that PKCα and mTOR may serve as PP2A substrates. We found that light regulates PP2A activity. Our studies also suggest that rhodopsin regulates PP2A and its substrate(s) dephosphorylation. PP2A substrate phosphorylation is increased in mice lacking the A-subunit of PP2A. However, there is no accompanying effect on retina structure and function. Together, our findings suggest that controlling the activity of PP2A in the retina may be neuroprotective. PMID:29416710

Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR Is Essential for PROTEIN PHOSPHATASE 2A Holoenzyme Assembly and Plays Important Roles in Hormone Signaling, Salt Stress Response, and Plant Development1[W][OPEN

PubMed Central

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

2014-01-01

PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation. PMID:25281708

The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

PubMed

Kück, Ulrich; Beier, Anna M; Teichert, Ines

2016-05-01

The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-06-29

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

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

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradshaw, Niels; Levdikov, Vladimir M.; Zimanyi, Christina M.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activitymore » in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.« less

Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit*

PubMed Central

Migueleti, Deivid L. S.; Smetana, Juliana H. C.; Nunes, Hugo F.; Kobarg, Jörg; Zanchin, Nilson I. T.

2012-01-01

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation. PMID:22167190

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

PubMed

Kónya, Zoltán; Bécsi, Bálint; Kiss, Andrea; Tamás, István; Lontay, Beáta; Szilágyi, László; Kövér, Katalin E; Erdődi, Ferenc

2018-05-01

Aralkyl and aryl selenoglycosides as well as glycosyl selenocarboxylate derivatives were assayed on the activity of protein phosphatase-1 (PP1) and -2A (PP2A) catalytic subunits (PP1c and PP2Ac) in search of compounds for PP1c and PP2Ac effectors. The majority of tested selenoglycosides activated both PP1c and PP2Ac by ∼2-4-fold in a phosphatase assay with phosphorylated myosin light chain substrate when the hydroxyl groups of the glycosyl moiety were acetylated, but they were without any effects in the non-acetylated forms. A peptide from the myosin phosphatase target subunit-1 (MYPT1 23-38 ) that included an RVxF PP1c-binding motif attenuated activation of PP1c by 2-Trifluoromethylbenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (TFM-BASG) and 4-Bromobenzyl 2,3,4,6-tetra-O-acetyl-1-seleno-β-d-glucopyranoside (Br-BASG). MYPT1 23-38 stimulated PP2Ac and contributed to PP2Ac activation exerted by either Br-BASG or TFM-BASG. Br-BASG and TFM-BASG suppressed partially binding of PP1c to MYPT1 in surface plasmon resonance based binding experiments. Molecular docking predicted that the hydrophobic binding surfaces in PP1c for interaction with either the RVxF residues of PP1c-interactors or selenoglycosides are partially overlapped. Br-BASG and TFM-BASG caused a moderate increase in the phosphatase activity of HeLa cells in 1 h, and suppressed cell viability in 24 h incubations. In conclusion, our present study identified selenoglycosides as novel activators of PP1 and PP2A as well as provided insights into the structural background of their interactions establishing a molecular model for future design of more efficient phosphatase activator molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

PP2A regulates autophagy in two alternative ways in Drosophila.

PubMed

Bánréti, Ágnes; Lukácsovich, Tamás; Csikós, György; Erdélyi, Miklós; Sass, Miklós

2012-04-01

Protein phosphatase 2A (PP2A) holoenzyme is a heterotrimeric complex, consisting of A, B and C subunits. The catalytic subunit PP2A-C (microtubule star/mts) binds to the C-terminal part of the scaffold protein PP2A-A (PP2A-29B). In Drosophila, there are three different forms of B subunits (widerborst/wdb, twins/tws and PP2A-B'), which determine the subcellular localization and substrate specificity of the holoenzyme. Previous studies demonstrated that PP2A is involved in the control of TOR-dependent autophagy both in yeast and mammals. Furthermore, in Drosophila, wdb genetically interacts with the PtdIns3K/PTEN/Akt signaling cascade, which is a main upstream regulatory system of dTOR. Here we demonstrate that in Drosophila, two different PP2A complexes (containing B' or wdb subunit) play essential roles in the regulation of starvation-induced autophagy. The PP2A-A/wdb/C complex acts upstream of dTOR, whereas the PP2A-A/B'/C complex functions as a target of dTOR and may regulate the elongation of autophagosomes and their subsequent fusion with lysosomes. We also identified three Drosophila Atg orthologs (Atg14, Atg17 and Atg101), which represent potential targets of the PP2A-A/B'/C complex during autophagy.

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

PubMed

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

2016-11-04

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

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases

PubMed Central

Bradshaw, Niels; Levdikov, Vladimir M; Zimanyi, Christina M; Gaudet, Rachelle; Wilkinson, Anthony J; Losick, Richard

2017-01-01

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. DOI: http://dx.doi.org/10.7554/eLife.26111.001 PMID:28527238

MRAS: A Close but Understudied Member of the RAS Family.

PubMed

Young, Lucy C; Rodriguez-Viciana, Pablo

2018-01-08

MRAS is the closest relative to the classical RAS oncoproteins and shares most regulatory and effector interactions. However, it also has unique functions, including its ability to function as a phosphatase regulatory subunit when in complex with SHOC2 and protein phosphatase 1 (PP1). This phosphatase complex regulates a crucial step in the activation cycle of RAF kinases and provides a key coordinate input required for efficient ERK pathway activation and transformation by RAS. MRAS mutations rarely occur in cancer but deregulated expression may play a role in tumorigenesis in some settings. Activating mutations in MRAS (as well as SHOC2 and PP1) do occur in the RASopathy Noonan syndrome, underscoring a key role for MRAS within the RAS-ERK pathway. MRAS also has unique roles in cell migration and differentiation and has properties consistent with a key role in the regulation of cell polarity. Further investigations should shed light on what remains a relatively understudied RAS family member. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

PubMed

Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

2017-06-14

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

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

PubMed Central

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

2016-01-01

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

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

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

PubMed

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

The Ndc80 complex targets Bod1 to human mitotic kinetochores

PubMed Central

2017-01-01

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores. PMID:29142109

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation.

PubMed

Amsailale, Rachid; Beyaert, Maxime; Smal, Caroline; Janssens, Veerle; Van Den Neste, Eric; Bontemps, Françoise

2014-03-03

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn(2+)-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Phosphatidic acid (PA) binds PP2AA1 to regulate PP2A activity and PIN1 polar localization.

PubMed

Gao, Hong-Bo; Chu, Yu-Jia; Xue, Hong-Wei

2013-09-01

Phospholipase D (PLD) exerts broad biological functions in eukaryotes through regulating downstream effectors by its product, phosphatidic acid (PA). Protein kinases and phosphatases, such as mammalian target of rapamycin (mTOR), Protein Phosphatase 1 (PP1) and Protein Phosphatase 2C (PP2C), are PA-binding proteins that execute crucial regulatory functions in both animals and plants. PA participates in many signaling pathways by modulating the enzymatic activity and/or subcellular localization of bound proteins. In this study, we demonstrated that PLD-derived PA interacts with the scaffolding A1 subunit of Protein Phosphatase 2A (PP2A) and regulates PP2A-mediated PIN1 dephosphorylation in Arabidopsis. Genetic and pharmacological studies showed that both PA and PP2A participate in the regulation of auxin distribution. In addition, both the phosphorylation status and polar localization of PIN1 protein were affected by PLD inhibitors. Exogenous PA triggered the membrane accumulation of PP2AA1 and enhanced the PP2A activity at membrane, while PLD inhibition resulted in the reduced endosomal localization and perinuclear aggregation of PP2AA1. These results demonstrate the important role of PLD-derived PA in normal PP2A-mediated PIN dephosphorylation and reveal a novel mechanism, in which PA recruits PP2AA1 to the membrane system and regulates PP2A function on membrane-targeted proteins. As PA and PP2A are conserved among eukaryotes, other organisms might use similar mechanisms to mediate multiple biological processes.

Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

PubMed Central

Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

2017-01-01

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

Phosphorylation of FMRP and alterations of FMRP complex underlie enhanced mLTD in adult rats triggered by early life seizures.

PubMed

Bernard, Paul B; Castano, Anna M; O'Leary, Heather; Simpson, Kameron; Browning, Michael D; Benke, Tim A

2013-11-01

Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS). We now show that mGluR-mediated LTD (mLTD) is specifically enhanced following ELS, similar to FMRP knock-outs. Total FMRP expression is unchanged but S6K is hyperphosphorylated, consistent with S6K overactivation. We postulated that either disruption of the FMRP-S6K-PP2A complex and/or removal of this complex from synapses could explain our findings. Using subcellular fractionation, we were surprised to find that concentrations of FMRP and PP2A were undisturbed in the synaptosomal compartment but reduced in parallel in the cytosolic compartment. Following ELS FMRP phosphorylation was reduced in the cytosolic compartment and increased in the synaptic compartment, in parallel with the compartmentalization of S6K activation. Furthermore, FMRP and PP2A remain bound following ELS. In contrast, the interaction of S6K with FMRP is reduced by ELS. Blockade of PP2A results in enhanced mLTD; this is occluded by ELS. This suggests a critical role for the location and function of the FMRP-S6K-PP2A signaling complex in limiting the amount of mLTD. Specifically, non-synaptic targeting and the function of the complex may influence the "set-point" for regulating mLTD. Consistent with this, striatal-enriched protein tyrosine phosphatase (STEP), an FMRP "target" which regulates mLTD expression, is specifically increased in the synaptosomal compartment following ELS. Further, we provide behavioral data to suggest that FMRP complex dysfunction may underlie altered socialization, a symptom associated and observed in other rodent models of autism, including FXS. © 2013.

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

PubMed Central

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

2015-01-01

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

The M Phase Kinase Greatwall (Gwl) Promotes Inactivation of PP2A/B55δ, a Phosphatase Directed Against CDK Phosphosites

PubMed Central

Castilho, Priscila V.; Williams, Byron C.; Mochida, Satoru; Zhao, Yong

2009-01-01

We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, “antimitotic” phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55δ regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55δ inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55δ remains active even when MPF levels are high. The removal of PP2A/B55δ corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations. PMID:19793917

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. © The Author 2015. Published by Oxford University Press.

Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

PubMed

Thai, Vu; Dephoure, Noah; Weiss, Amit; Ferguson, Jacqueline; Leitao, Ricardo; Gygi, Steven P; Kellogg, Douglas R

2017-03-24

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2A Cdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2A Cdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2A Cdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2A Cdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2A Cdc55 by multiple overlapping mechanisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

USDA-ARS?s Scientific Manuscript database

The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

The master Greatwall kinase, a critical regulator of mitosis and meiosis.

PubMed

Vigneron, Suzanne; Robert, Perle; Hached, Khaled; Sundermann, Lena; Charrasse, Sophie; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

2016-01-01

Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.

Memantine Attenuates Alzheimer’s Disease-Like Pathology and Cognitive Impairment

PubMed Central

Wang, Xiaochuan; Blanchard, Julie; Iqbal, Khalid

2015-01-01

Deficiency of protein phosphatase-2A is a key event in Alzheimer’s disease. An endogenous inhibitor of protein phosphatase-2A, inhibitor-1, I1 PP2A, which inhibits the phosphatase activity by interacting with its catalytic subunit protein phosphatase-2Ac, is known to be upregulated in Alzheimer’s disease brain. In the present study, we overexpressed I1 PP2A by intracerebroventricular injection with adeno-associated virus vector-1-I1 PP2A in Wistar rats. The I1 PP2A rats showed a decrease in brain protein phosphatase-2A activity, abnormal hyperphosphorylation of tau, neurodegeneration, an increase in the level of activated glycogen synthase kinase-3beta, enhanced expression of intraneuronal amyloid-beta and spatial reference memory deficit; littermates treated identically but with vector only, i.e., adeno-associated virus vector-1-enhanced GFP, served as a control. Treatment with memantine, a noncompetitive NMDA receptor antagonist which is an approved drug for treatment of Alzheimer’s disease, rescued protein phosphatase-2A activity by decreasing its demethylation at Leu309 selectively and attenuated Alzheimer’s disease-like pathology and cognitive impairment in adeno-associated virus vector-1-I1 PP2A rats. These findings provide new clues into the possible mechanism of the beneficial therapeutic effect of memantine in Alzheimer’s disease patients. PMID:26697860

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

PubMed

Baro, Barbara; Játiva, Soraya; Calabria, Inés; Vinaixa, Judith; Bech-Serra, Joan-Josep; de LaTorre, Carolina; Rodrigues, João; Hernáez, María Luisa; Gil, Concha; Barceló-Batllori, Silvia; Larsen, Martin R; Queralt, Ethel

2018-05-01

Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus

PubMed Central

Scorsato, Valéria; Lima, Tatiani B.; Righetto, Germanna L.; Zanchin, Nilson I. T.; Brandão-Neto, José; Sandy, James; Pereira, Humberto D’Muniz; Ferrari, Állan J. R.; Gozzo, Fabio C.; Smetana, Juliana H. C.; Aparicio, Ricardo

2016-01-01

TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition. PMID:27489114

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus

NASA Astrophysics Data System (ADS)

Scorsato, Valéria; Lima, Tatiani B.; Righetto, Germanna L.; Zanchin, Nilson I. T.; Brandão-Neto, José; Sandy, James; Pereira, Humberto D.'Muniz; Ferrari, Állan J. R.; Gozzo, Fabio C.; Smetana, Juliana H. C.; Aparicio, Ricardo

2016-08-01

TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition.

Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

USDA-ARS?s Scientific Manuscript database

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 o...

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

PubMed

Chattopadhyay, Debasish; Swingle, Mark R; Salter, Edward A; Wood, Eric; D'Arcy, Brandon; Zivanov, Catherine; Abney, Kevin; Musiyenko, Alla; Rusin, Scott F; Kettenbach, Arminja; Yet, Larry; Schroeder, Chad E; Golden, Jennifer E; Dunham, Wade H; Gingras, Anne-Claude; Banerjee, Surajit; Forbes, David; Wierzbicki, Andrzej; Honkanen, Richard E

2016-06-01

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

PubMed Central

Kimura, Toru; Han, WonSun; Pagel, Philipp; Nairn, Angus C.; Caplan, Michael J.

2011-01-01

Background The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na+,K+-ATPase. The catalytic subunit of the Na+,K+-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na+,K+-ATPase interacting protein. PP-2A C-subunit interacted with the Na+,K+-ATPase, but not with the homologous sequences of the H+,K+-ATPase. We confirmed that the Na+,K+-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na+,K+-ATPase trafficking through direct association. PP2A inhibits association between the Na+,K+-ATPase and arrestin, and diminishes the effect of arrestin on Na+,K+-ATPase trafficking. GRK phosphorylates the Na+,K+-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase. PMID:22242112

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

PubMed

Yeh, Po-An; Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

PubMed Central

Chang, Ching-Jin

2017-01-01

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis.

PubMed

Lee, Sun Joo; Rodriguez-Bravo, Veronica; Kim, Hyunjung; Datta, Sutirtha; Foley, Emily A

2017-05-15

PP2A comprising B56 regulatory subunit isoforms (PP2A B56 ) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2A B56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/C Cdc20 ), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/C Cdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/C Cdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/C Cdc20 assembly does not require binding between PP2A B56 and BubR1, and thus this contribution of PP2A B56 towards mitotic exit is distinct from its functions at kinetochores. PP2A B56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2A B56 , modulates APC/C Cdc20 assembly. These results elucidate the contributions of PP2A B56 towards completion of mitosis. © 2017. Published by The Company of Biologists Ltd.

Nuclear-cytoplasmic shuttling protein PP2AB56 contributes to mTORC1-dependent dephosphorylation of FOXK1.

PubMed

Nakatsumi, Hirokazu; Oka, Takeru; Higa, Tsunaki; Shirane, Michiko; Nakayama, Keiichi I

2018-05-29

Mammalian target of rapamycin complex 1 (mTORC1) kinase is a master regulator of the cellular response to nutrition-related signals such as insulin and amino acids. mTORC1 is activated on the lysosomal membrane and induces phosphorylation of a variety of downstream molecules. We previously showed that activated mTORC1 induces protein phosphatase 2A (PP2A)-mediated dephosphorylation of the transcription factor forkhead box K1 (FOXK1). The mechanism underlying the signal transduction from the cytoplasmic mTORC1 to the nuclear FOXK1 has remained unclear, however, we now show that a nuclear-cytoplasmic transport system is necessary for the mTORC1-FOXK1 signal transduction. This reaction is mediated by a shuttling protein B56, which is a regulatory subunit of PP2A and plays an essential role in the mTORC1-dependent dephosphorylation of FOXK1. These results suggest that PP2A B56 phosphatase contributes to the signaling for mTORC1-dependent transcriptional regulation. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Epigallocatechin-3-gallate and penta-O-galloyl-β-D-glucose inhibit protein phosphatase-1.

PubMed

Kiss, Andrea; Bécsi, Bálint; Kolozsvári, Bernadett; Komáromi, István; Kövér, Katalin E; Erdődi, Ferenc

2013-01-01

Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine/threonine residues in cells. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant δ-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC(50) values of 0.47-1.35 μm and 0.26-0.4 μm, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC(50) values of 15 and 6.6 μm, respectively. The structure-inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl/oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence. © 2012 The Authors Journal compilation © 2012 FEBS.

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 displaces a restricted subset of PP2A B subunits, which is insufficient to cause tumor cell formation in vitro. MCV sT instead transforms tumor cells through another region called the large T stabilization domain. The PP2A targeting and transforming activities lie on opposite faces of the MCV sT molecule and can be genetically separated from each other. PMID:25631078

DARPP-32 Is a Robust Integrator of Dopamine and Glutamate Signals

PubMed Central

Fernandez, Éric; Schiappa, Renaud; Girault, Jean-Antoine; Novère, Nicolas Le

2006-01-01

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca2+ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca2+ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca2+ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons. PMID:17194217

Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2Cγ governs Cajal body localization and stability of the SMN complex

PubMed Central

Petri, Sebastian; Grimmler, Matthias; Over, Sabine; Fischer, Utz; Gruss, Oliver J.

2007-01-01

The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cγ interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex. PMID:17984321

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

PubMed

Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

2014-02-15

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

NASA Astrophysics Data System (ADS)

Qi, Fei; Guo, Huarong; Wang, Jian

2008-02-01

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

PP2B and PP1α cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

PubMed Central

Chen, Ruichuan; Liu, Min; Li, Huan; Xue, Yuhua; Ramey, Wanichaya N.; He, Nanhai; Ai, Nanping; Luo, Haohong; Zhu, Ying; Zhou, Nan; Zhou, Qiang

2008-01-01

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)–calmodulin–protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1α (PP1α) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1α releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1α alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription. PMID:18483222

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation

PubMed Central

Neufeld, Thomas P.

2017-01-01

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration. PMID:28100687

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.

Transcriptional responses to cantharidin a protein phosphatase inhibitor in Arabidopsis thaliana reveal the involvement of multiple signal transduction pathways

USDA-ARS?s Scientific Manuscript database

Cantharidin is a natural compound isolated from the blister beetle (Epicauta spp.). It is a very potent inhibitor of serine/threonine protein phosphatases PPP, especially PP2A and PP4. Protein phosphatases and kinases maintain a sensitive balance between phosphorylated and dephosphorylated forms of ...

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis.

PubMed

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-02-09

PR65 is the two-layered (alpha-alpha solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix-helix separation along one layer ("fracturing") leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme's substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A's response to spindle tension) or arise spontaneously (e.g., in PP2A's interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer's-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

PubMed Central

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-01-01

PR65 is the two-layered (α-α solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix–helix separation along one layer (“fracturing”) leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme’s substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A’s response to spindle tension) or arise spontaneously (e.g., in PP2A’s interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer’s-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance. PMID:20133745

Control of seed dormancy and germination by DOG1-AHG1 PP2C phosphatase complex via binding to heme.

PubMed

Nishimura, Noriyuki; Tsuchiya, Wataru; Moresco, James J; Hayashi, Yuki; Satoh, Kouji; Kaiwa, Nahomi; Irisa, Tomoko; Kinoshita, Toshinori; Schroeder, Julian I; Yates, John R; Hirayama, Takashi; Yamazaki, Toshimasa

2018-06-06

Abscisic acid (ABA) regulates abiotic stress and developmental responses including regulation of seed dormancy to prevent seeds from germinating under unfavorable environmental conditions. ABA HYPERSENSITIVE GERMINATION1 (AHG1) encoding a type 2C protein phosphatase (PP2C) is a central negative regulator of ABA response in germination; however, the molecular function and regulation of AHG1 remain elusive. Here we report that AHG1 interacts with DELAY OF GERMINATION1 (DOG1), which is a pivotal positive regulator in seed dormancy. DOG1 acts upstream of AHG1 and impairs the PP2C activity of AHG1 in vitro. Furthermore, DOG1 has the ability to bind heme. Binding of DOG1 to AHG1 and heme are independent processes, but both are essential for DOG1 function in vivo. Our study demonstrates that AHG1 and DOG1 constitute an important regulatory system for seed dormancy and germination by integrating multiple environmental signals, in parallel with the PYL/RCAR ABA receptor-mediated regulatory system.

Global loss of Leucine Carboxyl Methyltransferase-1 causes severe defects in fetal liver hematopoiesis.

PubMed

Lee, Jocelyn A; Wang, Zhengqi; Sambo, Danielle; Bunting, Kevin D; Pallas, David C

2018-05-07

Leucine Carboxyl Methyltransferase-1 (LCMT-1) 3 methylates the carboxy-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In the current study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down ~30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and death by embryonic day 16.5 (E16.5). Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit + Lin - Sca1 + (KLS) cells. The percent cycling cells and mitotic indexes of wild-type and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, non-competitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multi-lineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint

PubMed Central

Petersen, Paris; Chou, Danny M.; You, Zhongsheng; Hunter, Tony; Walter, Johannes C.; Walter, Gernot

2006-01-01

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts. PMID:16479016

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

PubMed Central

Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

2013-01-01

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway

PubMed Central

Mui, Melissa Z.; Zhou, Yiwang; Blanchette, Paola; Chughtai, Naila; Knight, Jennifer F.; Gruosso, Tina; Papadakis, Andreas I.; Huang, Sidong; Park, Morag; Gingras, Anne-Claude

2015-01-01

ABSTRACT When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2AB55 phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2AB55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2AB55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCE The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets, we performed, for the first time, a large-scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway, which enhances cell viability in many cancers, and results of preliminary studies suggested a link between inhibition of Hippo signaling and E4orf4 toxicity. PMID:26085163

Oxidative impairment of hippocampal long-term potentiation involves activation of protein phosphatase 2A and is prevented by ketone bodies.

PubMed

Maalouf, Marwan; Rho, Jong M

2008-11-15

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

Protein Phosphatase 2A Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung

PubMed Central

Wallace, Alison M.; Hardigan, Andrew; Geraghty, Patrick; Salim, Shaneeza; Gaffney, Adam; Thankachen, Jincy; Arellanos, Leo; D'Armiento, Jeanine M.; Foronjy, Robert F.

2012-01-01

Protein phosphatase 2A (PP2A) is the primary serine-threonine phosphatase of eukaryotic cells, and changes in its activity have been linked to neoplastic and neurodegenerative diseases. However, the role of PP2A in noncancerous lung diseases such as chronic obstructive pulmonary disease (COPD) has not been previously examined. This study determined that PP2A activity was significantly increased in the lungs of advanced emphysema subjects compared with age-matched controls. Furthermore, we found that cigarette smoke exposure increases PP2A activity in mouse lung in vivo and in primary human small airway epithelial (SAE) cells in vitro. In mice, intratracheal transfection of PP2A protein prior to cigarette smoke exposure prevented acute smoke–induced lung inflammation. Conversely, inhibiting PP2A activity during smoke exposure exacerbated inflammatory responses in the lung. To further determine how PP2A modulates the responses to cigarette smoke in the lung, enzyme levels were manipulated in SAE cells using protein transfection and short hairpin RNA (shRNA) techniques. Increasing PP2A activity in SAE cells via PP2A protein transfection downregulated cytokine expression and prevented the induction of proteases following cigarette smoke extract (CSE) treatment. Conversely, decreasing enzymatic activity by stably transfecting SAE cells with shRNA for the A subunit of PP2A exacerbated these smoke-mediated responses. This study establishes that PP2A induction by cigarette smoke modulates immune and proteolytic responses to cigarette smoke exposure. Together, these findings suggest that manipulation of PP2A activity may be a plausible means to treat COPD and other inflammatory diseases. PMID:22223484

Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis.

PubMed

Domingo-Sananes, Maria Rosa; Kapuy, Orsolya; Hunt, Tim; Novak, Bela

2011-12-27

Activation of the cyclin-dependent kinase (Cdk1) cyclin B (CycB) complex (Cdk1:CycB) in mitosis brings about a remarkable extent of protein phosphorylation. Cdk1:CycB activation is switch-like, controlled by two auto-amplification loops--Cdk1:CycB activates its activating phosphatase, Cdc25, and inhibits its inhibiting kinase, Wee1. Recent experimental evidence suggests that parallel to Cdk1:CycB activation during mitosis, there is inhibition of its counteracting phosphatase activity. We argue that the downregulation of the phosphatase is not just a simple latch that suppresses futile cycles of phosphorylation/dephosphorylation during mitosis. Instead, we propose that phosphatase regulation creates coherent feed-forward loops and adds extra amplification loops to the Cdk1:CycB regulatory network, thus forming an integral part of the mitotic switch. These network motifs further strengthen the bistable characteristic of the mitotic switch, which is based on the antagonistic interaction of two groups of proteins: M-phase promoting factors (Cdk1:CycB, Cdc25, Greatwall and Endosulfine/Arpp19) and interphase promoting factors (Wee1, PP2A-B55 and a Greatwall counteracting phosphatase, probably PP1). The bistable character of the switch implies the existence of a CycB threshold for entry into mitosis. The end of G2 phase is determined by the point where CycB level crosses the CycB threshold for Cdk1 activation.

Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs

PubMed Central

Farelli, Jeremiah D.; Galvin, Brendan D.; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E.; Causey, Thomas B.; Ali-Reynolds, Alana; Saltzberg, Daniel J.; Carlow, Clotilde K. S.; Dunaway-Mariano, Debra; Allen, Karen N.

2014-01-01

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible. PMID:24992307

A novel mitochondrial matrix serine/threonine protein phosphatase regulates the mitochondria permeability transition pore and is essential for cellular survival and development

PubMed Central

Lu, Gang; Ren, Shuxun; Korge, Paavo; Choi, Jayoung; Dong, Yuan; Weiss, James; Koehler, Carla; Chen, Jau-nian; Wang, Yibin

2007-01-01

Mitochondria play a central role in the regulation of programmed cell death signaling. Here, we report the finding of a mitochondrial matrix-targeted protein phosphatase 2C family member (PP2Cm) that regulates mitochondrial membrane permeability transition pore (MPTP) opening and is essential for cell survival, embryonic development, and cardiac function. PP2Cm is highly conserved among vertebrates, with the highest expression levels detected in the heart and brain. Small hairpin RNA (shRNA)-mediated knockdown of PP2Cm resulted in cell death associated with loss of mitochondrial membrane potential in cultured cardiac mycoytes and an induction of hepatocyte apoptosis in vivo. PP2Cm-deficient mitochondria showed elevated susceptibility to calcium-induced MPTP opening, whereas mitochondrial oxidative phosphorylation activities were not affected. Finally, inactivation of PP2Cm in developing zebrafish embryos caused abnormal cardiac and neural development as well as heart failure associated with induced apoptosis. These data suggest that PP2Cm is a novel mitochondrial protein phosphatase that has a critical function in cell death and survival, and may play a role in regulating the MPTP opening. PMID:17374715

A New Protein Phosphatase 2C (FsPP2C1) Induced by Abscisic Acid Is Specifically Expressed in Dormant Beechnut Seeds1

PubMed Central

Lorenzo, Oscar; Rodríguez, Dolores; Nicolás, Gregorio; Rodríguez, Pedro L.; Nicolás, Carlos

2001-01-01

An abscisic acid (ABA)-induced cDNA fragment encoding a putative protein phosphatase 2C (PP2C) was obtained by means of differential reverse transcriptase-polymerase chain reaction approach. The full-length clone was isolated from a cDNA library constructed using mRNA from ABA-treated beechnut (Fagus sylvatica) seeds. This clone presents all the features of plant type PP2C and exhibits homology to members of this family such as AthPP2CA (61%), ABI1 (48%), or ABI2 (47%), therefore it was named FsPP2C1. The expression of FsPP2C1 is detected in dormant seeds and increases after ABA treatment, when seeds are maintained dormant, but it decreases and tends to disappear when dormancy is being released by stratification or under gibberellic acid treatment. Moreover, drought stress seems to have no effect on FsPP2C1 transcript accumulation. The FsPP2C1 transcript expression is tissue specific and was found to accumulate in ABA-treated seeds rather than in other ABA-treated vegetative tissues examined. These results suggest that the corresponding protein could be related to ABA-induced seed dormancy. By expressing FsPP2C1 in Escherichia coli as a histidine tag fusion protein, we have obtained direct biochemical evidence supporting Mg2+-dependent phosphatase activity of this protein. PMID:11299374

Arctigenin inhibits triple-negative breast cancers by targeting CIP2A to reactivate protein phosphatase 2A.

PubMed

Huang, Qiuyue; Qin, Shanshan; Yuan, Xiaoning; Zhang, Liang; Ji, Juanli; Liu, Xuewen; Ma, Wenjing; Zhang, Yunfei; Liu, Pengfei; Sun, Zhiting; Zhang, Jingxuan; Liu, Ying

2017-07-01

We have shown that a novel STAT3 inhibitor arctigenin (Atn) induces significant cytotoxicity in triple-negative breast cancer (TNBC) cells. This study further delineated molecular mechanisms where by Atn triggered cytotoxicity in TNBC cells. We found Atn can also inhibit metastasis in TNBC cells through cancerous inhibitor of protein phosphatase 2A (CIP2A) pathway. CIP2A is an endogenous inhibitor of protein phosphatase 2A (PP2A), which can increase the migration and invasion of various cancer cells. PP2A is a tumor suppressor, which is functionally defective in various cancers. Atn-induced metastasis inhibition was associated with reactivation of PP2A, downregulation of CIP2A and Akt phosphorylation. Silencing CIP2A enhanced Atn-induced metastasis inhibition and apoptosis in TNBCs. Furthermore, ectopic expression of CIP2A or inhibition of PP2A in TNBC cells abolished the effects of Atn. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A, at least in part, promotes the anti-metastasis effect induced by Atn. Our findings disclose the novel therapeutic mechanism of this targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.

Fundamental role of the fostriecin unsaturated lactone and implications for selective protein phosphatase inhibition.

PubMed

Buck, Suzanne B; Hardouin, Christophe; Ichikawa, Satoshi; Soenen, Danielle R; Gauss, C-M; Hwang, Inkyu; Swingle, Mark R; Bonness, Kathy M; Honkanen, Richard E; Boger, Dale L

2003-12-24

Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP beta12-beta13 active site loop accounting for PP2A/4 potency and selectivity.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xin, E-mail: fangfei6073@126.com; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn; Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, andmore » abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.« less

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme.

PubMed

Cho, Uhn Soo; Xu, Wenqing

2007-01-04

Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase, the deregulation of which is associated with multiple human cancers, Alzheimer's disease and increased susceptibility to pathogen infections. How PP2A is structurally organized and functionally regulated remains unclear. Here we report the crystal structure of an AB'C heterotrimeric PP2A holoenzyme. The structure reveals that the HEAT repeats of the scaffold A subunit form a horseshoe-shaped fold, holding the catalytic C and regulatory B' subunits together on the same side. The regulatory B' subunit forms pseudo-HEAT repeats and interacts with the C subunit near the active site, thereby defining substrate specificity. The methylated carboxy-terminal tail of the C subunit interacts with a highly negatively charged region at the interface between A and B' subunits, suggesting that the C-terminal carboxyl methylation of the C subunit promotes B' subunit recruitment by neutralizing charge repulsion. Together, our structural results establish a crucial foundation for understanding PP2A assembly, substrate recruitment and regulation.

Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia.

PubMed Central

Kissmehl, R; Treptau, T; Hofer, H W; Plattner, H

1996-01-01

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells. PMID:8694788

Protein Phosphatase-1 Inhibitor-2 Is a Novel Memory Suppressor.

PubMed

Yang, Hongtian; Hou, Hailong; Pahng, Amanda; Gu, Hua; Nairn, Angus C; Tang, Ya-Ping; Colombo, Paul J; Xia, Houhui

2015-11-11

Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation. We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor-2 as a critical contributor to suppression of memory formation by PP1 may provide a novel therapeutic target for memory-related diseases. Copyright © 2015 the authors 0270-6474/15/3515082-06$15.00/0.

RRP1B Targets PP1 to Mammalian Cell Nucleoli and Is Associated with Pre-60S Ribosomal Subunits

PubMed Central

Chamousset, Delphine; De Wever, Veerle; Moorhead, Greg B.; Chen, Yan; Boisvert, Francois-Michel; Lamond, Angus I.

2010-01-01

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions. PMID:20926688

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15*

PubMed Central

Oberg, Elizabeth A.; Nifoussi, Shanna K.; Gingras, Anne-Claude; Strack, Stefan

2012-01-01

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events. PMID:23135275

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

2016-06-02

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.

Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).

PubMed

Leira, F; Vieites, J M; Vieytes, M R; Botana, L M

2000-12-01

Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate assay: 4-methylumbelliferyl phosphate and fluorescein diphosphate (FDP). In this report, we present the results obtained using a new fluorogenic substrate of PP-2A, the compound dimethylacridinone phosphate (DDAO). A linear relationship between PP-2A concentration and DDAO-induced fluorescence was observed. Okadaic acid (0.0157-9.43 nM)-dependent inhibition of phosphatase activity showed similar results using FDP and DDAO. Recovery percentages obtained with FDP and DDAO in spiked mussel samples (both raw and canned) were very similar and reproducible. Comparative analysis of DSP-contaminated mussel samples by HPLC and FDP/DDAO-PP-2A showed a good correlation among all methods, thus demonstrating that DDAO can be used as a fluorogenic substrate to quantify okadaic acid and related toxins in bivalve molluscs with optimum reliability.

Meet the terminator: The phosphatase PP2A puts brakes on IRF-3 activation.

PubMed

Chattopadhyay, Saurabh; Sen, Ganes C

2014-04-24

Cellular interferon response to microbial infection is transient. In a recent paper in Immunity, Long et al. (2014) identify protein phosphatase 2A (PP2A) as a deactivator of phospho-interferon regulatory factor 3, the key transcription factor for interferon synthesis, thus providing one basis for the observed transiency. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2011-01-01

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis. PMID:22163007

Impaired adiponectin signaling contributes to disturbed catabolism of branched-chain amino acids in diabetic mice.

PubMed

Lian, Kun; Du, Chaosheng; Liu, Yi; Zhu, Di; Yan, Wenjun; Zhang, Haifeng; Hong, Zhibo; Liu, Peilin; Zhang, Lijian; Pei, Haifeng; Zhang, Jinglong; Gao, Chao; Xin, Chao; Cheng, Hexiang; Xiong, Lize; Tao, Ling

2015-01-01

The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN(-/-)) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN(-/-) and diabetic animals. Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

PubMed

Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

2018-02-01

Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

PubMed

Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane; Dietzel, Erik; Stalmann, Gertrud; López-Méndez, Blanca; Davey, Norman E; Nilsson, Jakob; Becker, Stephan

2018-01-04

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

NASA Technical Reports Server (NTRS)

Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

2004-01-01

Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

A wheat (Triticum aestivum) protein phosphatase 2A catalytic subunit gene provides enhanced drought tolerance in tobacco.

PubMed

Xu, Chongyi; Jing, Ruilian; Mao, Xinguo; Jia, Xiaoyun; Chang, Xiaoping

2007-03-01

Multiple copies of genes encoding the catalytic subunit (c) of protein phosphatase 2A (PP2A) are commonly found in plants. For some of these genes, expression is up-regulated under water stress. The aim of this study was to investigate expression and characterization of TaPP2Ac-1 from Triticum aestivum, and to evaluate the effects of TaPP2Ac-1 on Nicotiana benthamiana in response to water stress. TaPP2Ac-1 cDNA was isolated from wheat by in silico identification and RT-PCR amplification. Transcript levels of TaPP2Ac-1 were examined in wheat responding to water deficit. Copy numbers of TaPP2Ac-1 in wheat genomes and subcellular localization in onion epidermal cells were studied. Enzyme properties of the recombinant TaPP2Ac-1 protein were determined. In addition, studies were carried out in tobacco plants with pCAPE2-TaPP2Ac-1 under water-deficit conditions. TaPP2Ac-1 cDNA was cloned from wheat. Transcript levels of TaPP2Ac-1 in wheat seedlings were up-regulated under drought condition. One copy for this TaPP2Ac-1 was present in each of the three wheat genomes. TaPP2Ac-1 fused with GFP was located in the nucleus and cytoplasm of onion epidermis cells. The recombinant TaPP2Ac-1 gene was over-expressed in Escherichia coli and encoded a functional serine/threonine phosphatase. Transgenic tobacco plants over-expressing TaPP2Ac-1 exhibited stronger drought tolerance than non-transgenic tobacco plants. Tobacco plants with pCAPE2-TaPP2Ac-1 appeared to be resistant to water deficit, as shown by their higher capacity to maintain leaf relative water content, leaf cell-membrane stability index, water-retention ability and water use efficiency under water stress. The results suggest that the physiological role of TaPP2Ac-1 is related to drought stress response, possibly through its involvement in drought-responding signal transduction pathways.

Ferulic Acid Attenuates the Injury-Induced Decrease of Protein Phosphatase 2A Subunit B in Ischemic Brain Injury

PubMed Central

Koh, Phil-Ok

2013-01-01

Background Ferulic acid provides a neuroprotective effect during cerebral ischemia through its anti-oxidant function. Protein phosphatase 2A (PP2A) is a serine and threonine phosphatase that contributes broadly to normal brain function. This study investigated whether ferulic acid regulates PP2A subunit B in a middle cerebral artery occlusion (MCAO) animal model and glutamate toxicity-induced neuronal cell death. Methodology/Principal Findings MCAO was surgically induced to yield permanent cerebral ischemic injury in rats. The rats were treated with either vehicle or ferulic acid (100 mg/kg, i.v.) immediately after MCAO, and cerebral cortex tissues were collected 24 h after MCAO. A proteomics approach, RT-PCR, and Western blot analyses performed to identification of PP2A subunit B expression levels. Ferulic acid significantly reduced the MCAO-induced infarct volume of the cerebral cortex. A proteomics approach elucidated the reduction of PP2A subunit B in MCAO-induced animals, and ferulic acid treatment prevented the injury-induced reduction in PP2A subunit B levels. RT-PCR and Western blot analyses also showed that ferulic acid treatment attenuates the injury-induced decrease in PP2A subunit B levels. Moreover, the number of PP2A subunit B-positive cells was reduced in MCAO-induced animals, and ferulic acid prevented these decreases. In cultured neuronal cells, ferulic acid treatment protected cells against glutamate toxicity and prevented the glutamate-induced decrease in PP2A subunit B. Conclusions/Significance These results suggest that the maintenance of PP2A subunit B by ferulic acid in ischemic brain injury plays an important role for the neuroprotective function of ferulic acid. PMID:23349830

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)*

PubMed Central

Aranda-Orgillés, Beatriz; Rutschow, Désirée; Zeller, Raphael; Karagiannidis, Antonios I.; Köhler, Andrea; Chen, Changwei; Wilson, Timothy; Krause, Sven; Roepcke, Stefan; Lilley, David; Schneider, Rainer; Schweiger, Susann

2011-01-01

We have shown previously that the ubiquitin ligase MID1, mutations of which cause the midline malformation Opitz BBB/G syndrome (OS), serves as scaffold for a microtubule-associated protein complex that regulates protein phosphatase 2A (PP2A) activity in a ubiquitin-dependent manner. Here, we show that the MID1 protein complex associates with mRNAs via a purine-rich sequence motif called MIDAS (MID1 association sequence) and thereby increases stability and translational efficiency of these mRNAs. Strikingly, inclusion of multiple copies of the MIDAS motif into mammalian mRNAs increases production of the encoded proteins up to 20-fold. Mutated MID1, as found in OS patients, loses its influence on MIDAS-containing mRNAs, suggesting that the malformations in OS patients could be caused by failures in the regulation of cytoskeleton-bound protein translation. This is supported by the observation that the majority of mRNAs that carry MIDAS motifs is involved in developmental processes and/or energy homeostasis. Further analysis of one of the proteins encoded by a MIDAS-containing mRNA, namely PDPK-1 (3-phosphoinositide dependent protein kinase-1), which is an important regulator of mammalian target of rapamycin/PP2A signaling, showed that PDPK-1 protein synthesis is significantly reduced in cells from an OS patient compared with an age-matched control and can be rescued by functional MID1. Together, our data uncover a novel messenger ribonucleoprotein complex that regulates microtubule-associated protein translation. They suggest a novel mechanism underlying OS and point at an enormous potential of the MIDAS motif to increase the efficiency of biotechnological protein production in mammalian cells. PMID:21930711

Evolution of Abscisic Acid Synthesis and Signaling Mechanisms

PubMed Central

Hauser, Felix; Waadt, Rainer; Schroeder, Julian I.

2011-01-01

The plant hormone abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. Core ABA signaling components consist of a recently identified group of ABA receptor proteins of the PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family that act as negative regulators of members of the PROTEIN PHOSPHATASE 2C (PP2C) family. Inhibition of PP2C activity enables activation of SNF1-RELATED KINASE 2 (SnRK2) protein kinases, which target downstream components, including transcription factors, ion channels and NADPH oxidases. These and other components form a complex ABA signaling network. Here, an in depth analysis of the evolution of components in this ABA signaling network shows that (i) PYR/RCAR ABA receptor and ABF-type transcription factor families arose during land colonization of plants and are not found in algae and other species, (ii) ABA biosynthesis enzymes have evolved to plant- and fungal-specific forms, leading to different ABA synthesis pathways, (iii) existing stress signaling components, including PP2C phosphatases and SnRK kinases, were adapted for novel roles in this plant-specific network to respond to water limitation. In addition, evolutionarily conserved secondary structures in the PYR/RCAR ABA receptor family are visualized. PMID:21549957

Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

NASA Technical Reports Server (NTRS)

Swingle, M. R.; Honkanen, R.; Ciszak, E.

2004-01-01

Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression

PubMed Central

Xu, Peng; Raetz, Elizabeth A.; Kitagawa, Mayumi; Virshup, David M.; Lee, Sang Hyun

2013-01-01

Summary BUBR1 is a mitotic phosphoprotein essential for the maintenance of chromosome stability by promoting chromosome congression and proper kinetochore–microtubule (K-fiber) attachment, but the underlying mechanism(s) has remained elusive. Here we identify BUBR1 as a binding partner of the B56 family of Protein Phosphatase 2A regulatory subunits. The interaction between BUBR1 and the B56 family is required for chromosome congression, since point mutations in BUBR1 that block B56 binding abolish chromosome congression. The BUBR1:B56-PP2A complex opposes Aurora B kinase activity, since loss of the complex can be reverted by inhibiting Aurora B. Importantly, we show that the failure of BUBR1 to recruit B56-PP2A also contributes to the chromosome congression defects found in cells derived from patients with the Mosaic Variegated Aneuploidy (MVA) syndrome. Together, we propose that B56-PP2A is a key mediator of BUBR1's role in chromosome congression and functions by antagonizing Aurora B activity at the kinetochore for establishing stable kinetochore–microtubule attachment at the metaphase plate. PMID:23789096

The PP4R1 sub-unit of protein phosphatase PP4 is essential for inhibition of NF-κB by merkel polyomavirus small tumour antigen.

PubMed

Abdul-Sada, Hussein; Müller, Marietta; Mehta, Rajni; Toth, Rachel; Arthur, J Simon C; Whitehouse, Adrian; Macdonald, Andrew

2017-04-11

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with a high metastatic potential. The majority of MCC cases are caused by the Merkel cell polyomavirus (MCPyV), through expression of the virus-encoded tumour antigens. Whilst mechanisms attributing tumour antigen expression to transformation are being uncovered, little is known of the mechanisms by which MCPyV persists in the host. We previously identified the MCPyV small T antigen (tAg) as a novel inhibitor of nuclear factor kappa B (NF-kB) signalling and a modulator of the host anti-viral response. Here we demonstrate that regulation of NF-kB activation involves a previously undocumented interaction between tAg and regulatory sub-unit 1 of protein phosphatase 4 (PP4R1). Formation of a complex with PP4R1 and PP4c is required to bridge MCPyV tAg to the NEMO adaptor protein, allowing deactivation of the NF-kB pathway. Mutations in MCPyV tAg that fail to interact with components of this complex, or siRNA depletion of PP4R1, prevents tAg-mediated inhibition of NF-kB and pro-inflammatory cytokine production. Comparison of tAg binding partners from other human polyomavirus demonstrates that interactions with NEMO and PP4R1 are unique to MCPyV. Collectively, these data identify PP4R1 as a novel target for virus subversion of the host anti-viral response.

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boylan, Joan M.; Salomon, Arthur R.; Department of Chemistry, Brown University, Providence, RI

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase inmore » apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.« less

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein

PubMed Central

Ferrar, Tony; Chamousset, Delphine; De Wever, Veerle; Nimick, Mhairi; Andersen, Jens; Trinkle-Mulcahy, Laura; Moorhead, Greg B. G.

2012-01-01

Summary The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway. PMID:23213405

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

2015-05-18

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.

A novel regulatory defect in the branched-chain α-keto acid dehydrogenase complex due to a mutation in the PPM1K gene causes a mild variant phenotype of maple syrup urine disease.

PubMed

Oyarzabal, Alfonso; Martínez-Pardo, Mercedes; Merinero, Begoña; Navarrete, Rosa; Desviat, Lourdes R; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2013-02-01

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others. © 2012 WILEY PERIODICALS, INC.

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

PubMed Central

Xu, Yanhui; Chen, Yu; Zhang, Ping; Jeffrey, Philip D.; Shi, Yigong

2009-01-01

Summary 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 Bα. We show that Bα specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The Bα subunit comprises a seven-bladed β propeller, with an acidic, substrate-binding groove located in the center of the propeller. The β propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding β hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau. PMID:18922469

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

PubMed

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation.

PubMed

Cristóbal, Ion; Rincón, Raúl; Manso, Rebeca; Madoz-Gúrpide, Juan; Caramés, Cristina; del Puerto-Nevado, Laura; Rojo, Federico; García-Foncillas, Jesús

2014-09-01

The tumor suppressor protein phosphatase 2A (PP2A) is frequently inactivated in human cancer and phosphorylation of its catalytic subunit (p-PP2A-C) at tyrosine-307 (Y307) has been described to inhibit this phosphatase. However, its molecular and clinical relevance in colorectal cancer (CRC) remains unclear. p-PP2A-C Y307 was determined by immunoblotting in 7 CRC cell lines and 35 CRC patients. CRC cells were treated with the PP2A activator forskolin alone or combined with the PP2A inhibitor okadaic acid, 5-fluorouracil and oxaliplatin. We examined cell growth, colonosphere formation, caspase activity and AKT and ERK activation. PP2A-C was found hyperphosphorylated in CRC cell lines. Forskolin dephosphorylated and activated PP2A, impairing proliferation and colonosphere formation, and inducing activation of caspase 3/7 and changes in AKT and ERK phosphorylation. Moreover, forskolin showed additive effects with 5-fluorouracil and oxaliplatin treatments. Analysis of p-PP2A-C Y307 in primary tumors confirmed the presence of this alteration in a subgroup of CRC patients. Our data show that PP2A-C hyperphosphorylation is a frequent event that contributes to PP2A inhibition in CRC. Antitumoral effects of forskolin-mediated PP2A activation suggest that the analysis of p-PP2A-C Y307 status could be used to identify a subgroup of patients who would benefit from treatments based on PP2A activators. Copyright © 2014 Elsevier B.V. All rights reserved.

Myosin phosphatase and RhoA-activated kinase modulate neurotransmitter release by regulating SNAP-25 of SNARE complex

PubMed Central

Sipos, Adrienn; Darula, Zsuzsanna; Bécsi, Bálint; Nagy, Dénes; Iván, Judit; Erdődi, Ferenc

2017-01-01

Reversible phosphorylation of neuronal proteins plays an important role in the regulation of neurotransmitter release. Myosin phosphatase holoenzyme (MP) consists of a protein phosphatase-1 (PP1) catalytic subunit (PP1c) and a regulatory subunit, termed myosin phosphatase targeting subunit (MYPT1). The primary function of MP is to regulate the phosphorylation level of contractile proteins; however, recent studies have shown that MP is localized to neurons, and is also involved in the mediation of neuronal processes. Our goal was to investigate the effect of RhoA-activated kinase (ROK) and MP on the phosphorylation of one potential neuronal substrate, the synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is a member of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, along with synaptobrevin and syntaxin, and the primary role of SNAP25 is to mediate vesicle fusion. We showed that MYPT1 interacts with SNAP-25, as revealed by immunoprecipitation and surface plasmon resonance based binding studies. Mass spectrometry analysis and in vitro phosphorylation/dephosphorylation assays demonstrated that ROK phosphorylates, while MP dephosphorylates, SNAP-25 at Thr138. Silencing MYPT1 in B50 neuroblastoma cells increased phosphorylation of SNAP-25 at Thr138. Inhibition of PP1 with tautomycetin increased, whereas inhibition of ROK by H1152, decreased the phosphorylation of SNAP-25 at Thr138 in B50 cells, in cortical synaptosomes, and in brain slices. In response to the transduction of the MP inhibitor, kinase-enhanced PP1 inhibitor (KEPI), into synaptosomes, an increase in phosphorylation of SNAP-25 and a decrease in the extent of neurotransmitter release were detected. The interaction between SNAP-25 and syntaxin increased with decreasing phosphorylation of SNAP-25 at Thr138, upon inhibition of ROK. Our data suggest that ROK/MP play a crucial role in vesicle trafficking, fusion, and neurotransmitter release by oppositely regulating the phosphorylation of SNAP-25 at Thr138. PMID:28486561

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

PubMed Central

Sangodkar, Jaya; Perl, Abbey; Tohme, Rita; Kiselar, Janna; Kastrinsky, David B.; Izadmehr, Sudeh; Mazhar, Sahar; Wiredja, Danica D.; O’Connor, Caitlin M.; Hoon, Divya; Dhawan, Neil S.; Schlatzer, Daniela; Yao, Shen; Leonard, Daniel; Borczuk, Alain C.; Gokulrangan, Giridharan; Wang, Lifu; Svenson, Elena; Farrington, Caroline C.; Yuan, Eric; Avelar, Rita A.; Stachnik, Agnes; Smith, Blake; Gidwani, Vickram; Giannini, Heather M.; McQuaid, Daniel; McClinch, Kimberly; Wang, Zhizhi; Levine, Alice C.; Sears, Rosalie C.; Chen, Edward Y.; Duan, Qiaonan; Datt, Manish; Ma’ayan, Avi; DiFeo, Analisa; Sharma, Neelesh; Galsky, Matthew D.; Brautigan, David L.; Ioannou, Yiannis A.; Xu, Wenqing; Chance, Mark R.; Ohlmeyer, Michael

2017-01-01

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins. PMID:28504649

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets.

PubMed

Uhrig, R Glen; Moorhead, Greg B

2011-12-01

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development. © 2011 Landes Bioscience

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.

Downregulation of protein phosphatase 2A carboxyl methylation and methyltransferase may contribute to Alzheimer disease pathogenesis.

PubMed

Sontag, Estelle; Hladik, Christa; Montgomery, Lisa; Luangpirom, Ampa; Mudrak, Ingrid; Ogris, Egon; White, Charles L

2004-10-01

ABalphaC, a major protein phosphatase 2A (PP2A) heterotrimeric enzyme, binds to and regulates the microtubule cytoskeleton and tau. We have shown that ABalphaC protein expression levels are selectively reduced in Alzheimer disease (AD). Notably, the carboxyl methylation of PP2A catalytic subunit (PP2A(C)) is critically required for ABalphaC holoenzyme assembly, and catalyzed by a specific methyltransferase (PPMT). Here, we provide the first analysis of human PPMT and methylated PP2A(C) in brain regions from AD, non-AD demented, and aged control autopsy cases. Immunoblotting analyses revealed that PPMT protein expression and PP2A(C) methylation levels were quantitatively decreased in AD-affected brain regions. Immunohistochemical studies showed that PPMT was abundant in neurons throughout the cortex in normal control and non-AD demented cases. However, in AD, there was a regional loss of PPMT immunoreactivity that closely paralleled the severity of tau pathology, but not amyloid plaque burden. We propose that the deregulation of PPMT and PP2A methylation/demethylation cycles contributes to AD pathogenesis, by inducing changes in PP2A heteromultimeric composition and substrate specificity. In turn, PP2A dysfunction compromises the mechanisms that control tau, neuronal plasticity, and survival.

A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

PubMed Central

Passalacqua, Karla D.; Satola, Sarah W.; Crispell, Emily K.

2012-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype. PMID:22850507

Highly sensitive detection and discrimination of LR and YR microcystins based on protein phosphatases and an artificial neural network.

PubMed

Covaci, O I; Sassolas, A; Alonso, G A; Muñoz, R; Radu, G L; Bucur, B; Marty, J-L

2012-08-01

The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants--LR, YR, and RR--were studied. The corresponding K (I) for each enzyme-MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR. The sensitivity of the enzymes increased in the following order: mutant PP2A < mutant PP1 < natural PP2A. The best limit of detection obtained was 21.2 pM MC-LR using the most sensible enzyme. Methanol, ethanol, and acetonitrile up to 2 % (v/v) may be used in inhibition measurements. An artificial neural network (ANN) was used to discriminate two MC variants--LR and YR--using the differences in inhibition percentages measured with mutant PP1 and natural PP2A. The ANN is able to analyze mixtures with concentrations ranging from 8 to 98 pM MC-LR and 31 to 373 pM MC-YR.

Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation.

PubMed

Takeuchi, Tomoharu; Kobayashi, Takayasu; Tamura, Shinri; Yokosawa, Hideyoshi

2006-08-07

ISG15, an interferon-upregulated ubiquitin-like protein, is covalently conjugated to various cellular proteins (ISGylation). In this study, we found that protein phosphatase 2Cbeta (PP2Cbeta), which functions in the nuclear factor kappaB (NF-kappaB) pathway via dephosphorylation of TGF-beta-activated kinase, was ISGylated, and analysis by NF-kappaB luciferase reporter assay revealed that PP2Cbeta activity was suppressed by co-expression of ISG15, UBE1L, and UbcH8. We determined the ISGylation sites of PP2Cbeta and constructed its ISGylation-resistant mutant. In contrast to the wild type, this mutant suppressed the NF-kappaB pathway even in the presence of ISG15, UBE1L, and UbcH8. Thus, we propose that ISGylation negatively regulates PP2Cbeta activity.

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

PubMed

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

2004-05-01

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

Structure of the protein phosphatase 2A holoenzyme.

PubMed

Xu, Yanhui; Xing, Yongna; Chen, Yu; Chao, Yang; Lin, Zheng; Fan, Eugene; Yu, Jong W; Strack, Stefan; Jeffrey, Philip D; Shi, Yigong

2006-12-15

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.

Rearrangement of the Protein Phosphatase 1 Interactome During Heart Failure Progression.

PubMed

Chiang, David Y; Alsina, Katherina M; Corradini, Eleonora; Fitzpatrick, Martin; Ni, Li; Lahiri, Satadru K; Reynolds, Julia; Pan, Xiaolu; Scott, Larry; Heck, Albert J R; Wehrens, Xander H

2018-04-18

Background -Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. Methods -Mice were subjected to transverse aortic constriction and grouped based on ejection fraction (EF) into sham, hypertrophy, moderate HF (EF 30-40%), and severe HF (EF<30%). Cardiac lysates were subjected to affinity-purification using anti-PP1c antibodies followed by high-resolution mass spectrometry. Ppp1r7 was knocked down in mouse cardiomyocytes and HeLa cells using adeno-associated virus serotype 9 (AAV9) and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes. Results -Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome dataset ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression including two known (Ppp1r7, Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. Conclusions -PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The nine key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

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

PubMed

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

2016-02-26

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

Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes.

PubMed

Toranzo, G Sánchez; Bonilla, F; Bühler, M C Gramajo; Bühler, M I

2011-05-01

The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK-MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO₃ did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK-MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc-PKA, which affects the activity of the Cdc25 phosphatase.

Lysophosphatidic acid modulates the association of PTP1B with N-cadherin/catenin complex in SKOV3 ovarian cancer cells.

PubMed

Huang, Ruby Yun-Ju; Wen, Chen-Chen; Liao, Chih-Kai; Wang, Shu-Huei; Chou, Liang-Yin; Wu, Jiahn-Chun

2012-09-01

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA-induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N-cadherin-β-catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of β-catenin and alter the balance of β-catenin-bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the β-catenin-α-catenin, but not the β-catenin-N-cadherin complex. This disintegration of β-catenin from α-catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to β-catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA. © The Author(s) Journal compilation © 2012 International Federation for Cell Biology.

Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs

PubMed Central

Waadt, Rainer; Schroeder, Julian I.

2016-01-01

The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by “monomeric” PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA. PMID:27192441

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

The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis.

PubMed

Yu, Hong-Guo; Koshland, Douglas

2007-03-26

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.

Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

PubMed

Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

2018-04-25

Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

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

PubMed

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

2014-09-29

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

Serine/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma

PubMed Central

Shen, Y; Wang, Y; Sheng, K; Fei, X; Guo, Q; Larner, J; Kong, X; Qiu, Y; Mi, J

2011-01-01

Increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma multiforme (GBM). This study aims to determine if serine/threonine phosphatase (protein phosphatase 6 (PP6)) is a molecular target for GBM radiosensitization treatment. The GBM orthotopic xenograft mice model was used in this study. Our data demonstrated that the protein level of PP6 catalytic subunit (PP6c) was upregulated in the GBM tissue from about 50% patients compared with the surrounding tissue or control tissue. Both the in vitro survival fraction of GBM cells and the patient survival time were highly correlated or inversely correlated with PP6c expression (R2=0.755 and −0.707, respectively). We also found that siRNA knockdown of PP6c reduced DNA-dependent protein kinase (DNA-PK) activity in three different GBM cell lines, increasing their sensitivity to radiation. In the orthotopic mice model, the overexpression of PP6c in GBM U87 cells attenuated the effect of radiation treatment, and reduced the survival time of mice compared with the control mice, while the PP6c knocking-down improved the effect of radiation treatment, and increased the survival time of mice. These findings demonstrate that PP6 regulates the sensitivity of GBM cells to radiation, and suggest small molecules disrupting or inhibiting PP6 association with DNA-PK is a potential radiosensitizer for GBM. PMID:22158480

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

PubMed Central

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

2003-01-01

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

A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

PubMed

Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

2002-10-25

Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

Pharmacological Activation of Protein Phosphatase 2 A (PP2A): A Novel Strategy to Fight Against Human Malignancies?

PubMed

Carratù, Maria Rosaria; Signorile, Anna; De Rasmo, Domenico; Reale, Antonia; Vacca, Angelo

2016-01-01

The serine-threonine protein phosphatase 2A (PP2A) regulates multiple cell signaling cascades and its inactivation by viral oncoproteins, mutation of specific structural subunits or upregulation of the cellular endogenous inhibitors may contribute to malignant transformation by regulating specific phosphorylation events. Pharmacological modulation of PP2A activity is becoming an attractive strategy for cancer treatment. Some compounds targeting PP2A are able to induce PP2A reactivation and subsequent cell death in several types of cancer. We undertook a search of bibliographic databases for peer-reviewed articles focusing on the main item of the review. We selected articles published in indexed journals. The quality of retrieved papers was appraised using the standard bibliometric indicators. One hundred and fourteen papers were included in the review. Twenty-seven papers gave an overview of structure and physiological role of PP2A. Twenty-five papers outlined the role of PP2A in tumor suppression. Forty papers analyzed the mechanism involved in PP2A reactivation by synthetic compounds, and twenty-two papers outlined the capability of natural compounds of restoring PP2A activity and how this could be beneficial. Findings analyzed in this review underline the central role of PP2A as a regulator of cell growth and survival, hence its function as tumor suppressor. The discovery that some compounds, either synthetic or natural, are capable of reactivating PP2A opens up new perspectives for future strategies to fully exploit therapeutic potential in human cancer. Thus, this review could also be of particular interest to pharmaceutical or biotechnology companies for drug design and targeted delivery.

Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

PubMed

Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

2017-11-01

Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential Effects of Tautomycetin and Its Derivatives on Protein Phosphatase Inhibition, Immunosuppressive Function and Antitumor Activity

PubMed Central

Niu, Mingshan; Sun, Yan; Liu, Bo

2012-01-01

In the present work, we studied the structure-activity relationship (SAR) of tautomycetin (TMC) and its derivatives. Further, we demonstrated the correlation between the immunosuppressive fuction, anticancer activity and protein phosphatase type 1 (PP1) inhibition of TMC and its derivatives. We have prepared some TMC derivatives via combinatorial biosynthesis, isolation from fermentation broth or chemical degradation of TMC. We found that the immunosuppressive activity was correlated with anticancer activity for TMC and its analog compounds, indicating that TMC may home at the same targets for its immunosuppressive and anticancer activities. Interestingly, TMC-F1, TMC-D1 and TMC-D2 all retained significant, albeit reduced PP1 inhibitory activity compared to TMC. However, only TMC-D2 showed immunosuppressive and anticancer activities in studies carried out in cell lines. Moreover, TMC-Chain did not show any significant inhibitory activity towards PP1 but showed strong growth inhibitory effect. This observation implicates that the maleic anhydride moiety of TMC is critical for its phosphatase inhibitory activity whereas the C1-C18 moiety of TMC is essential for the inhibition of tumor cell proliferation. Furthermore, we measured in vivo phosphatase activities of PP1 in MCF-7 cell extracts treated with TMC and its related compounds, and the results indicate that the cytotoxicity of TMC doesn't correlate with its in vivo PP1 inhibition activity. Taken together, our study suggests that the immunosuppressive and anticancer activities of TMC are not due to the inhibition of PP1. Our results provide a novel insight for the elucidation of the underlying molecular mechanisms of TMC's important biological functions. PMID:22563261

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

PubMed Central

Daempfling, Lea

2013-01-01

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals. PMID:23716589

Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Ziyin; Tu Xiaoming; Wang, Ching C.

2006-11-01

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrestedmore » in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.« less

Regulation of cardiac excitation and contraction by p21 activated kinase-1.

PubMed

Ke, Yunbo; Lei, Ming; Solaro, R John

2008-01-01

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.

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

PubMed

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

2006-11-30

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

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

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A.

PubMed

Han, Min; Pendem, Suresh; Teh, Suet Ling; Sukumaran, Dinesh K; Wu, Feng; Wilson, John X

2010-01-01

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier. Copyright 2009 Elsevier Inc. All rights reserved.

Leishmania mexicana: promastigotes and amastigotes secrete protein phosphatases and this correlates with the production of inflammatory cytokines in macrophages.

PubMed

Escalona-Montaño, A R; Ortiz-Lozano, D M; Rojas-Bernabé, A; Wilkins-Rodriguez, A A; Torres-Guerrero, H; Mondragón-Flores, R; Mondragón-Gonzalez, R; Becker, I; Gutiérrez-Kobeh, L; Aguirre-Garcia, M M

2016-09-01

Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages.

Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction.

PubMed

Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S; Somlyo, Avril V; Eto, Masumi

2009-08-07

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.

Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered.

PubMed

Rieder, Franz J J; Kastner, Marie-Theres; Hartl, Markus; Puchinger, Martin G; Schneider, Martina; Majdic, Otto; Britt, William J; Djinović-Carugo, Kristina; Steininger, Christoph

2017-03-01

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis1[W][OA

PubMed Central

Trotta, Andrea; Wrzaczek, Michael; Scharte, Judith; Tikkanen, Mikko; Konert, Grzegorz; Rahikainen, Moona; Holmström, Maija; Hiltunen, Hanna-Maija; Rips, Stephan; Sipari, Nina; Mulo, Paula; Weis, Engelbert; von Schaewen, Antje; Aro, Eva-Mari; Kangasjärvi, Saijaliisa

2011-01-01

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B′γ (B′γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b′γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b′γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b′γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b′γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b′γ leaves. We suggest that the specific B′γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance. PMID:21571669

Overexpression of a Protein Phosphatase 2C from Beech Seeds in Arabidopsis Shows Phenotypes Related to Abscisic Acid Responses and Gibberellin Biosynthesis1

PubMed Central

Reyes, David; Rodríguez, Dolores; González-García, Mary Paz; Lorenzo, Oscar; Nicolás, Gregorio; García-Martínez, José Luis; Nicolás, Carlos

2006-01-01

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis. PMID:16815952

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

PubMed

Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

2015-10-13

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

PubMed Central

Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

2017-01-01

Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution.

PubMed

Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C

2017-01-27

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca 2+ -regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Glutamate Counteracts Dopamine/PKA Signaling via Dephosphorylation of DARPP-32 Ser-97 and Alteration of Its Cytonuclear Distribution*

PubMed Central

Nishi, Akinori; Matamales, Miriam; Musante, Veronica; Valjent, Emmanuel; Kuroiwa, Mahomi; Kitahara, Yosuke; Rebholz, Heike; Greengard, Paul; Girault, Jean-Antoine; Nairn, Angus C.

2017-01-01

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1 receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels. PMID:27998980

A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melcher, Karsten; Ng, Ley-Moy; Zhou, X Edward

2010-01-12

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved β-loopsmore » that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling.« less

Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1) from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

PubMed Central

Kumar, Rajinder; Adams, Brian; Oldenburg, Anja; Musiyenko, Alla; Barik, Sailen

2002-01-01

Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA) led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes. PMID:12057017

Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

NASA Astrophysics Data System (ADS)

Gao, Lan; Li, Hao-Ming

2017-08-01

Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

1E7-03, a low MW compound targeting host protein phosphatase-1, inhibits HIV-1 transcription

PubMed Central

Ammosova, Tatyana; Platonov, Maxim; Ivanov, Andrei; Kont, Yasemin Saygideğer; Kumari, Namita; Kehn-Hall, Kylene; Jerebtsova, Marina; Kulkarni, Amol A; Üren, Aykut; Kovalskyy, Dmytro; Nekhai, Sergei

2014-01-01

Background and Purpose HIV-1 transcription is activated by the Tat protein which recruits the cyclin-dependent kinase CDK9/cyclin T1 to TAR RNA. Tat binds to protein phosphatase-1 (PP1) through the Q35VCF38 sequence and translocates PP1 to the nucleus. PP1 dephosphorylates CDK9 and activates HIV-1 transcription. We have synthesized a low MW compound 1H4, that targets PP1 and prevents HIV-1 Tat interaction with PP1 and inhibits HIV-1 gene transcription. Here, we report our further work with the 1H4-derived compounds and analysis of their mechanism of action. Experimental Approach Using the 1H4-PP1 complex as a model, we iteratively designed and synthesized follow-up libraries that were analysed for the inhibition of HIV-1 transcription and toxicity. We also confirmed the mechanism of action of the PP1-targeting molecules by determining the affinity of binding of these molecules to PP1, by analysing their effects on PP1 activity, disruption of PP1 binding to Tat and shuttling of PP1 to the nucleus. Key Results We identified a tetrahydroquinoline derivative, compound 7, which disrupted the interaction of Tat with PP1. We further optimized compound 7 and obtained compound 7c, renamed 1E7-03, which inhibited HIV-1 with low IC50 (fivefold lower than the previously reported compound, 1H4), showed no cytotoxicity and displayed a plasma half-life greater than 8 h in mice. 1E7-03 bound to PP1 in vitro and prevented shuttling of PP1 into the nucleus. Conclusions and Implications Our study shows that low MW compounds that functionally mimic the PP1-binding RVxF peptide can inhibit HIV-1 transcription by deregulating PP1. PMID:25073485

Molecular Evolution of Phosphoprotein Phosphatases in Drosophila

PubMed Central

Miskei, Márton; Ádám, Csaba; Kovács, László; Karányi, Zsolt; Dombrádi, Viktor

2011-01-01

Phosphoprotein phosphatases (PPP), these ancient and important regulatory enzymes are present in all eukaryotic organisms. Based on the genome sequences of 12 Drosophila species we traced the evolution of the PPP catalytic subunits and noted a substantial expansion of the gene family. We concluded that the 18–22 PPP genes of Drosophilidae were generated from a core set of 8 indispensable phosphatases that are present in most of the insects. Retropositons followed by tandem gene duplications extended the phosphatase repertoire, and sporadic gene losses contributed to the species specific variations in the PPP complement. During the course of these studies we identified 5, up till now uncharacterized phosphatase retrogenes: PpY+, PpD5+, PpD6+, Pp4+, and Pp6+ which are found only in some ancient Drosophila. We demonstrated that all of these new PPP genes exhibit a distinct male specific expression. In addition to the changes in gene numbers, the intron-exon structure and the chromosomal localization of several PPP genes was also altered during evolution. The G−C content of the coding regions decreased when a gene moved into the heterochromatic region of chromosome Y. Thus the PPP enzymes exemplify the various types of dynamic rearrangements that accompany the molecular evolution of a gene family in Drosophilidae. PMID:21789237

Rapid detection of protein phosphatase activity using Zn(II)-coordinated gold nanosensors based on His-tagged phosphopeptides.

PubMed

Lee, Jin Oh; Kim, Eun-Ji; Lim, Butaek; Kim, Tae-Wuk; Kim, Young-Pil

2015-01-20

We report a rapid colorimetric assay to detect protein phosphatase (PP) activity based on the controlled assembly and disassembly of gold nanoparticles (AuNPs) via Zn(II)-specific coordination in the presence of His6-tagged phosphopeptides. Among divalent metal ions including Ni(II), Cu(II), Co(II), Mg(II), Mn(II), and Zn(II), only Zn(II) triggered a strong association between phosphopeptides with hexahistidine at a single end and nitrilotriacetic acid (NTA)-modified AuNPs (21.3 nm in core diameter), leading to the self-assembly of AuNPs and consequently changes in color of the AuNP solution. In contrast, unphosphorylated peptides and His6-deficient phosphopeptides did not change the color of the AuNP solution. As a result, protein phosphatase 1 (PP1) activity and its inhibition were easily quantified with high sensitivity by determining the extinction ratio (E520/E700) of colloidal AuNPs. Most importantly, this method was capable of detecting protein phosphatase 2A (PP2A) activity in immunoprecipitated plant extracts. Because PPs play pivotal roles in mediating diverse signal transduction pathways as primary effectors of protein dephosphorylation, we anticipate that our method will be applied as a rapid format method to analyze the activities of various PPs and their inhibition.

Suppressing Type 2C Protein Phosphatases Alters Fruit Ripening and the Stress Response in Tomato.

PubMed

Zhang, Yushu; Li, Qian; Jiang, Li; Kai, Wenbin; Liang, Bin; Wang, Juan; Du, Yangwei; Zhai, Xiawan; Wang, Jieling; Zhang, Yingqi; Sun, Yufei; Zhang, Lusheng; Leng, Ping

2018-01-01

Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Deprotonation states of the two active site water molecules regulate the binding of protein phosphatase 5 with its substrate: A molecular dynamics study.

PubMed

Wang, Lingyun; Yan, Feng

2017-10-01

Protein phosphatase 5 (PP5), mainly localized in human brain, can dephosphorylate tau protein whose high level of phosphorylation is related to Alzheimer's disease. Similar to other protein phosphatases, PP5 has a conserved motif in the catalytic domain that contains two binding sites for manganese (Mn 2+ ) ions. Structural data indicate that two active site water molecules, one bridging the two Mn 2+ ions and the other terminally coordinated with one of the Mn 2+ ions (Mn1), are involved in catalysis. Recently, a density functional theory study revealed that the two water molecules can be both deprotonated to keep a neutral active site for catalysis. The theoretical study gives us an insight into the catalytic mechanism of PP5, but the knowledge of how the deprotonation states of the two water molecules affect the binding of PP5 with its substrate is still lacking. To approach this problem, molecular dynamics simulations were performed to model the four possible deprotonation states. Through structural, dynamical and energetic analyses, the results demonstrate that the deprotonation states of the two water molecules affect the structure of the active site including the distance between the two Mn 2+ ions and their coordination, impact the interaction energy of residues R275, R400 and H304 which directly interact with the substrate phosphoserine, and mediate the dynamics of helix αJ which is involved in regulation of the enzyme's activity. Furthermore, the deprotonation state that is preferable for PP5 binding of its substrate has been identified. These findings could provide new design strategy for PP5 inhibitor. © 2017 The Protein Society.

The protein phosphatase 2A catalytic subunit StPP2Ac2b acts as a positive regulator of tuberization induction in Solanum tuberosum L.

PubMed

Muñiz García, María Noelia; Muro, María Catalina; Mazzocchi, Luciana Carla; País, Silvia Marina; Stritzler, Margarita; Schlesinger, Mariana; Capiati, Daniela Andrea

2017-02-01

This study provides the first genetic evidence for the role of PP2A in tuberization, demonstrating that the catalytic subunit StPP2Ac2b positively modulates tuber induction, and that its function is related to the regulation of gibberellic acid metabolism. The results contribute to a better understanding of the molecular mechanism controlling tuberization induction, which remains largely unknown. The serine/threonine protein phosphatases type 2A (PP2A) are implicated in several physiological processes in plants, playing important roles in hormone responses. In cultivated potato (Solanum tuberosum), six PP2A catalytic subunits (StPP2Ac) were identified. The PP2Ac of the subfamily I (StPP2Ac1, 2a and 2b) were suggested to be involved in the tuberization signaling in leaves, where the environmental and hormonal signals are perceived and integrated. The aim of this study was to investigate the role of PP2A in the tuberization induction in stolons. We selected one of the catalytic subunits of the subfamily I, StPP2Ac2b, to develop transgenic plants overexpressing this gene (StPP2Ac2b-OE). Stolons from StPP2Ac2b-OE plants show higher tuber induction rates in vitro, as compared to wild type stolons, with no differences in the number of tubers obtained at the end of the process. This effect is accompanied by higher expression levels of the gibberellic acid (GA) catabolic enzyme StGA2ox1. GA up-regulates StPP2Ac2b expression in stolons, possibly as part of the feedback system by which the hormone regulates its own level. Sucrose, a tuber-promoting factor in vitro, increases StPP2Ac2b expression. We conclude that StPP2Ac2b acts in stolons as a positive regulator tuber induction, integrating different tuberization-related signals mainly though the modulation of GA metabolism.

Protein Phosphatase 2A Signaling in Human Prostate Cancer

DTIC Science & Technology

2011-06-01

centrifugation, 25 mL of supernatant was transferred to an assay plate; 100 mL of Malachite Green phosphate detection solution was added and incu...prostate cancer cells was resolved and immunoblotted for PP2AC and b-actin (internal control). PP2A activity was determined by malachite green–based

The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network.

PubMed

Kendall, Ryan T; Strungs, Erik G; Rachidi, Saleh M; Lee, Mi-Hye; El-Shewy, Hesham M; Luttrell, Deirdre K; Janech, Michael G; Luttrell, Louis M

2011-06-03

The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

Protein Phosphatase 2A Signaling in Human Prostate Cancer

DTIC Science & Technology

2014-08-01

transfectants. Later, these cells were characterized for the PP2Acα expression and activity by immunoblot and malachite green based assay, respectively. Data...After centrifugation, 25 mL of supernatant was transferred to an assay plate; 100 mL of Malachite Green phosphate detection solution was added and incu...cancer cells was resolved and immunoblotted for PP2AC and b-actin (internal control). PP2A activity was determined by malachite green–based

SCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana

PubMed Central

Ren, Ziyin; Zhi, Liya; Yao, Bin; Su, Chao; Liu, Liu; Li, Xia

2017-01-01

The phytohormone abscisic acid (ABA) is an essential part of the plant response to abiotic stressors such as drought. Upon the perception of ABA, pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins interact with co-receptor protein phosphatase type 2Cs to permit activation Snf1-related protein kinase2 (SnRK2) kinases, which switch on ABA signaling by phosphorylating various target proteins. Thus, SnRK2 kinases are central regulators of ABA signaling. However, the mechanisms that regulate SnRK2 degradation remain elusive. Here, we show that SnRK2.3 is degradated by 26S proteasome system and ABA promotes its degradation. We found that SnRK2.3 interacts with AtPP2-B11 directly. AtPP2-B11 is an F-box protein that is part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex that negatively regulates plant responses to ABA by specifically promoting the degradation of SnRK2.3. AtPP2-B11 was induced by ABA, and the knockdown of AtPP2-B11 expression markedly increased the ABA sensitivity of plants during seed germination and postgerminative development. Overexpression of AtPP2-B11 does not affect ABA sensitivity, but inhibits the ABA hypersensitive phenotypes of SnRK2.3 overexpression lines. These results reveal a novel mechanism through which AtPP2-B11 specifically degrades SnRK2.3 to attenuate ABA signaling and the abiotic stress response in Arabidopsis. PMID:28787436

Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products.

PubMed

Grossman, Elizabeth A; Ward, Carl C; Spradlin, Jessica N; Bateman, Leslie A; Huffman, Tucker R; Miyamoto, David K; Kleinman, Jordan I; Nomura, Daniel K

2017-11-16

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150

PubMed Central

Por, Elaine D.; Samelson, Bret K.; Belugin, Sergei; Akopian, Armen N.; Scott, John D.; Jeske, Nathaniel A.

2011-01-01

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150−/− mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150−/− mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components. PMID:20883208

PP2B/calcineurin-mediated desensitization of TRPV1 does not require AKAP150.

PubMed

Por, Elaine D; Samelson, Bret K; Belugin, Sergei; Akopian, Armen N; Scott, John D; Jeske, Nathaniel A

2010-12-15

Activation of protein kinases and phosphatases at the plasma membrane often initiates agonist-dependent signalling events. In sensory neurons, AKAP150 (A-kinase-anchoring protein 150) orientates PKA (protein kinase A), PKC (protein kinase C) and the Ca2+/calmodulin-dependent PP2B (protein phosphatase 2B, also known as calcineurin) towards membrane-associated substrates. Recent evidence indicates that AKAP150-anchored PKA and PKC phosphorylate and sensitize the TRPV1 (transient receptor potential subfamily V type 1 channel, also known as the capsaicin receptor). In the present study, we explore the hypothesis that an AKAP150-associated pool of PP2B catalyses the dephosphorylation and desensitization of TRPV1. Biochemical, electrophysiological and cell-based experiments indicate that PP2B associates with AKAP150 and TRPV1 in cultured TG (trigeminal ganglia) neurons. Gene silencing of AKAP150 reduces basal phosphorylation of TRPV1. However, functional studies in neurons isolated from AKAP150-/- mice indicate that the anchoring protein is not required for pharmacological desensitization of TRPV1. Behavioural analysis of AKAP150-/- mice further support this notion, demonstrating that agonist-stimulated desensitization of TRPV1 is sensitive to PP2B inhibition and does not rely on AKAP150. These findings allow us to conclude that pharmacological desensitization of TRPV1 by PP2B may involve additional regulatory components.

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.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans

PubMed Central

Lange, Karen I.; Heinrichs, Jeffrey; Cheung, Karen; Srayko, Martin

2013-01-01

Summary Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B′, B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo. PMID:23336080

Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans.

PubMed

Lange, Karen I; Heinrichs, Jeffrey; Cheung, Karen; Srayko, Martin

2013-01-15

Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B', B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

Influence of verapamil on tachycardia-induced alterations of PP1 and PP2A in rabbit atrium

PubMed Central

Laszlo, Roman; Winkler, Christoph; Wöhrl, Stefan; Laszlo, Sara; Eick, Christian; Schreieck, Jürgen; Bosch, Ralph F

2007-01-01

BACKGROUND Long-term atrial fibrillation changes in many regulatory and structural processes may result in stabilization of the arrhythmia. There is evidence that decreased amplitude of L-type Ca2+ current – probably a key mechanism of atrial remodelling –resulting from changes in expression of regulatory proteins is at least jointly responsible. OBJECTIVES To assess the expressions of protein phosphatases PP1 and PP2A, as well as the effect of verapamil pretreatment (VPT), in the early phases of atrial fibrillation in a rabbit model. METHODS Four groups, each consisting of six animals, were studied: ‘not paced, no drug’ group; ‘paced, no drug’ group (rapid atrial pacing [RAP] 600 beats/min for 24 h); ‘not paced, verapamil’ (NPV) group (duration of VPT was seven days, verapamil 7.5 mg/kg was administered every 12 h); and ‘paced, verapamil’ (PV) group (pacemaker stimulation after VPT). Protein expression was evaluated by Western blot analysis. RESULTS RAP resulted in an augmented (32%) PP1 expression (not paced, no drug group versus paced, no drug group). The increase in PP1 expression was prevented with VPT (NPV group versus PV group). Expression of PP2A was not influenced by RAP. However, VPT led to an increase of PP2A expression (16%) after RAP (NPV group versus PV group). CONCLUSIONS Fortified expression of protein phosphatases might be – besides transcriptional downregulation of channel subunits – another important cause of reduced L-type Ca2+ current after RAP. Blocking L-type Ca2+ channels with verapamil to prevent tachycardia-induced changes of PP1 expression might be expedient. PMID:18651001

A functional genomic analysis of Arabidopsis thaliana PP2C clade D

USDA-ARS?s Scientific Manuscript database

In the reference dicot plant Arabidopsis thaliana, the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into 10 multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among 3 chromosomes (PPD1, At3g12620; PPD2...

ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis[C][W

PubMed Central

Rodrigues, Américo; Adamo, Mattia; Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rabissi, Agnese; Lumbreras, Victoria; González-Guzmán, Miguel; Antoni, Regina; Rodriguez, Pedro L.; Baena-González, Elena

2013-01-01

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways. PMID:24179127

Sphingosine regulates the NLRP3-inflammasome and IL-1β release from macrophages

PubMed Central

Luheshi, Nadia M; Giles, James A; Lopez-Castejon, Gloria; Brough, David

2012-01-01

Abstract Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that regulates inflammatory responses to injury and infection. IL-1β secretion requires the protease caspase-1, which is activated following recruitment to inflammasomes. Endogenous danger-associated molecular patterns (DAMPs) released from necrotic cells activate caspase-1 through an NLRP3-inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3-inflammasome-dependent secretion of IL-1β from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph-induced IL-1β release. IL-1β release induced by other well-characterized NLRP3-inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)-dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation. PMID:22105559

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

PubMed Central

Harvey, Stacy L.; Enciso, Germán; Dephoure, Noah; Gygi, Steven P.; Gunawardena, Jeremy; Kellogg, Douglas R.

2011-01-01

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2ACdc55) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2ACdc55 sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2ACdc55 and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength. PMID:21849476

An ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants

PubMed Central

Cao, Minjie; Liu, Xue; Zhang, Yan; Xue, Xiaoqian; Zhou, X Edward; Melcher, Karsten; Gao, Pan; Wang, Fuxing; Zeng, Liang; Zhao, Yang; Zhao, Yang; Deng, Pan; Zhong, Dafang; Zhu, Jian-Kang; Xu, H Eric; Xu, Yong

2013-01-01

Abscisic acid (ABA) is the most important hormone for plants to resist drought and other abiotic stresses. ABA binds directly to the PYR/PYL family of ABA receptors, resulting in inhibition of type 2C phosphatases (PP2C) and activation of downstream ABA signaling. It is envisioned that intervention of ABA signaling by small molecules could help plants to overcome abiotic stresses such as drought, cold and soil salinity. However, chemical instability and rapid catabolism by plant enzymes limit the practical application of ABA itself. Here we report the identification of a small molecule ABA mimic (AM1) that acts as a potent activator of multiple members of the family of ABA receptors. In Arabidopsis, AM1 activates a gene network that is highly similar to that induced by ABA. Treatments with AM1 inhibit seed germination, prevent leaf water loss, and promote drought resistance. We solved the crystal structure of AM1 in complex with the PYL2 ABA receptor and the HAB1 PP2C, which revealed that AM1 mediates a gate-latch-lock interacting network, a structural feature that is conserved in the ABA-bound receptor/PP2C complex. Together, these results demonstrate that a single small molecule ABA mimic can activate multiple ABA receptors and protect plants from water loss and drought stress. Moreover, the AM1 complex crystal structure provides a structural basis for designing the next generation of ABA-mimicking small molecules. PMID:23835477

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

PubMed

Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

2016-11-01

Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

The Tea4-PP1 landmark promotes local growth by dual Cdc42 GEF recruitment and GAP exclusion.

PubMed

Kokkoris, Kyriakos; Gallo Castro, Daniela; Martin, Sophie G

2014-05-01

Cell polarization relies on small GTPases, such as Cdc42, which can break symmetry through self-organizing principles, and landmarks that define the axis of polarity. In fission yeast, microtubules deliver the Tea1-Tea4 complex to mark cell poles for growth, but how this complex activates Cdc42 is unknown. Here, we show that ectopic targeting of Tea4 to cell sides promotes the local activation of Cdc42 and cell growth. This activity requires that Tea4 binds the type I phosphatase (PP1) catalytic subunit Dis2 or Sds21, and ectopic targeting of either catalytic subunit is similarly instructive for growth. The Cdc42 guanine-nucleotide-exchange factor Gef1 and the GTPase-activating protein Rga4 are required for Tea4-PP1-dependent ectopic growth. Gef1 is recruited to ectopic Tea4 and Dis2 locations to promote Cdc42 activation. By contrast, Rga4 is locally excluded by Tea4, and its forced colocalization with Tea4 blocks ectopic growth, indicating that Rga4 must be present, but at sites distinct from Tea4. Thus, a Tea4-PP1 landmark promotes local Cdc42 activation and growth both through Cdc42 GEF recruitment and by creating a local trough in a Cdc42 GAP.

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.

PubMed

Voss, Martin; Paterson, James; Kelsall, Ian R; Martín-Granados, Cristina; Hastie, C James; Peggie, Mark W; Cohen, Patricia T W

2011-01-01

Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target. Copyright © 2010 Elsevier Inc. All rights reserved.

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

The metabolic advantage of tumor cells

PubMed Central

2011-01-01

1- Oncogenes express proteins of "Tyrosine kinase receptor pathways", a receptor family including insulin or IGF-Growth Hormone receptors. Other oncogenes alter the PP2A phosphatase brake over these kinases. 2- Experiments on pancreatectomized animals; treated with pure insulin or total pancreatic extracts, showed that choline in the extract, preserved them from hepatomas. Since choline is a methyle donor, and since methylation regulates PP2A, the choline protection may result from PP2A methylation, which then attenuates kinases. 3- Moreover, kinases activated by the boosted signaling pathway inactivate pyruvate kinase and pyruvate dehydrogenase. In addition, demethylated PP2A would no longer dephosphorylate these enzymes. A "bottleneck" between glycolysis and the oxidative-citrate cycle interrupts the glycolytic pyruvate supply now provided via proteolysis and alanine transamination. This pyruvate forms lactate (Warburg effect) and NAD+ for glycolysis. Lipolysis and fatty acids provide acetyl CoA; the citrate condensation increases, unusual oxaloacetate sources are available. ATP citrate lyase follows, supporting aberrant transaminations with glutaminolysis and tumor lipogenesis. Truncated urea cycles, increased polyamine synthesis, consume the methyl donor SAM favoring carcinogenesis. 4- The decrease of butyrate, a histone deacetylase inhibitor, elicits epigenic changes (PETEN, P53, IGFBP decrease; hexokinase, fetal-genes-M2, increase) 5- IGFBP stops binding the IGF - IGFR complex, it is perhaps no longer inherited by a single mitotic daughter cell; leading to two daughter cells with a mitotic capability. 6- An excess of IGF induces a decrease of the major histocompatibility complex MHC1, Natural killer lymphocytes should eliminate such cells that start the tumor, unless the fever prostaglandin PGE2 or inflammation, inhibit them... PMID:21649891

Regulation of Na+-K+-2Cl− cotransport by protein phosphorylation in ferret erythrocytes

PubMed Central

Flatman, Peter W; Creanor, James

1999-01-01

Na+-K+-2Cl− cotransport in ferret erythrocytes was measured as the bumetanide-sensitive uptake of 86Rb. The resting cotransport rate was high but could be increased threefold by treating erythrocytes with calyculin A, a potent inhibitor of serine/threonine phosphatases. Twenty nanomolar was sufficient to maximally and rapidly (within 4 min) stimulate transport. The effects of several kinase inhibitors were tested. High concentrations of K-252a, K-252b, calphostin C and hypericin caused less than 20 % inhibition. Staurosporine (IC50, 0.06 μm) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; IC50, 2.5 μm) were more potent but still only partially (40–50 %) inhibited transport, an effect mimicked by reducing ionized intracellular Mg2+ concentration to submicromolar levels. Genistein may inhibit all transport at a sufficiently high dose (IC50, 0.36 mM) perhaps by directly inhibiting the transporter. Staurosporine, PP1 and the removal of Mg2+ all prevented subsequent stimulation by calyculin A, and all inhibited calyculin-stimulated transport by 20–30 %. The effects of staurosporine, PP1 and Mg2+ removal were not additive. The phosphatase that dephosphorylates the cotransporter is probably Mg2+ (or possibly Ca2+ or Mn2+) sensitive and not the target for calyculin A. The data suggest that this phosphatase is inhibited by phosphorylation, and that it is the regulation of this process which is affected by calyculin A and the kinase inhibitors tested here. Phosphorylation of the phosphatase is probably regulated by members of the Src family of tyrosine kinases. PMID:10358111

Statin-activated nuclear receptor PXR promotes SGK2 dephosphorylation by scaffolding PP2C to induce hepatic gluconeogenesis.

PubMed

Gotoh, Saki; Negishi, Masahiko

2015-09-22

Statin therapy is known to increase blood glucose levels in humans. Statins utilize pregnane X receptor (PXR) and serum/glucocorticoid regulated kinase 2 (SGK2) to activate phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6Pase) genes, thereby increasing glucose production in human liver cells. Here, the novel statin/PXR/SGK2-mediated signaling pathway has now been characterized for hepatic gluconeogenesis. Statin-activated PXR scaffolds the protein phosphatase 2C (PP2C) and SGK2 to stimulate PP2C to dephosphorylate SGK2 at threonine 193. Non-phosphorylated SGK2 co-activates PXR-mediated trans-activation of promoters of gluconeogenic genes in human liver cells, thereby enhancing gluconeogenesis. This gluconeogenic statin-PXR-SGK2 signal is not present in mice, in which statin treatment suppresses hepatic gluconeogenesis. These findings provide the basis for statin-associated side effects such as an increased risk for Type 2 diabetes.

Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin-LR in human laryngeal epithelial cells (Hep-2).

PubMed

Wang, Beilei; Liu, Jinghui; Huang, Pu; Xu, Kailun; Wang, Hanying; Wang, Xiaofeng; Guo, Zonglou; Xu, Lihong

2017-03-01

The major toxic mechanism of Microcystin-LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin-LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep-2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton-associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep-2. A dose-dependent change in p-Tau and p-Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep-2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 890-903, 2017. © 2016 Wiley Periodicals, Inc.

Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

PubMed

Meyer-Roxlau, Stefanie; Lämmle, Simon; Opitz, Annett; Künzel, Stephan; Joos, Julius P; Neef, Stefan; Sekeres, Karolina; Sossalla, Samuel; Schöndube, Friedrich; Alexiou, Konstantin; Maier, Lars S; Dobrev, Dobromir; Guan, Kaomei; Weber, Silvio; El-Armouche, Ali

2017-07-01

Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology.

PubMed

Sontag, Estelle; Luangpirom, Ampa; Hladik, Christa; Mudrak, Ingrid; Ogris, Egon; Speciale, Samuel; White, Charles L

2004-04-01

The formation of amyloid-containing senile plaques and tau-rich neurofibrillary tangles are central events in Alzheimer disease (AD) pathogenesis. Significantly, ABalphaC, a major protein phosphatase 2A (PP2A) holoenzyme, specifically binds to and dephosphorylates tau. Deregulation of PP2A results in tau hyperphosphorylation in vivo. Here, we compared the expression levels and distribution of PP2A subunits in various brain regions from autopsy cases of AD and aged controls with or without histological evidence of age-related neurofibrillary degeneration. Immunoblotting analyses revealed that there was a significant reduction in the total amounts of ABalphaC in AD frontal and temporal cortices that matched the decrease in PP2A activity measured in the same brain homogenates. Immunohistochemical studies showed that neuronal ABalphaC expression levels were significantly and selectively decreased in AD-affected regions and in tangle-bearing neurons, but not in AD cerebellum and in non-AD dementias. Reduced neuronal ABalphaC immunoreactivity closely correlated with tangle load, but not plaque burden, suggesting that ABalphaC dysfunction contributes to AD tau pathology. Glial cells within senile plaques were also positive for ABalphaC. Increased glial PP2A immunoreactivity was observed in both AD and non-AD cases and may play a role in the brain's response to general inflammatory processes and amyloidogenesis.

Inhibition of glycogen synthase kinase-3beta downregulates total tau proteins in cultured neurons and its reversal by the blockade of protein phosphatase-2A.

PubMed

Martin, Ludovic; Magnaudeix, Amandine; Esclaire, Françoise; Yardin, Catherine; Terro, Faraj

2009-02-03

In tauopathies such as Alzheimer's disease (AD), the molecular mechanisms of tau protein aggregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration remain not understood. It was recently demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration. Therefore, reduction of tau levels might represent a mechanism of neuroprotection. Glycogen synthase kinase-3beta (GSK3beta) and protein phosphatase-2A (PP2A) are key enzymes involved in the regulation of tau phosphorylation, and have been suggested to be involved in the abnormal tau phosphorylation and aggregation in AD. Connections between PP2A and GSK3beta signaling have been reported. We have previously demonstrated that exposure of cultured cortical neurons to lithium decreased tau protein expression and provided neuroprotection against Abeta. Since lithium is not a specific inhibitor of GSK3beta (ID50=2.0 mM), whether or not the lithium-induced tau decrease involves GSK3beta remained to be determined. For that purpose, cultured cortical neurons were exposed to 6-bromo-indirubin-3'-oxime (6-BIO), a more selective and potent GSK3beta inhibitor (ID50=1.5 microM) or to lithium. Analysis of tau levels and phosphorylation by western-blot assays showed that lithium and 6-BIO dose-dependently decreased both tau protein levels and tau phosphorylation. Conversely, inhibition of cyclin-dependent kinase-5 (CDK5) by roscovitine decreased phosphorylated tau but failed to alter tau protein levels. These data indicate that GSK3beta might be selectively involved in the regulation of tau protein levels. Moreover, inhibition of PP2A by okadaic acid, but not that of PP2B (protein phosphatase-2B)/calcineurin by FK506, dose-dependently reversed lithium-induced tau decrease. These data indicate that GSK3beta regulates both tau phosphorylation and total tau levels through PP2A.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

PubMed

Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

1997-04-15

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barber, Claire; Netherton, Chris; Goatley, Lynnett

The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 ormore » the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α. - Highlights: •The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate translation initiation factor eIF2α (eIF2α). •The residues V{sup 16}, F{sup 18} of DP71L are required for binding to the α, β and γ isoforms of PP1 and for DP71L function. •The sequence LSAVL downstream from the PP1 binding site (residues 57–61) are also important for DP71L function. •DP71L mutants of the LSAVL sequence retain ability to co-precipitate with PP1 showing these sequences have a different role to PP1 binding.« less

Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in chordoma.

PubMed

Hao, Shuyu; Song, Hua; Zhang, Wei; Seldomridge, Ashlee; Jung, Jinkyu; Giles, Amber J; Hutchinson, Marsha-Kay; Cao, Xiaoyu; Colwell, Nicole; Lita, Adrian; Larion, Mioara; Maric, Dragan; Abu-Asab, Mones; Quezado, Martha; Kramp, Tamalee; Camphausen, Kevin; Zhuang, Zhengping; Gilbert, Mark R; Park, Deric M

2018-05-18

Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.

PubMed

Ilinykh, Philipp A; Tigabu, Bersabeh; Ivanov, Andrey; Ammosova, Tatiana; Obukhov, Yuri; Garron, Tania; Kumari, Namita; Kovalskyy, Dmytro; Platonov, Maxim O; Naumchik, Vasiliy S; Freiberg, Alexander N; Nekhai, Sergei; Bukreyev, Alexander

2014-08-15

The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Overexpression of the protein phosphatase 2A regulatory subunit a gene ZmPP2AA1 improves low phosphate tolerance by remodeling the root system architecture of maize

PubMed Central

Wang, Jiemin; Pei, Laming; Jin, Zhe; Zhang, Kewei; Zhang, Juren

2017-01-01

Phosphate (Pi) limitation is a constraint for plant growth and development in many natural and agricultural ecosystems. In this study, a gene encoding Zea mays L. protein phosphatase 2A regulatory subunit A, designated ZmPP2AA1, was induced in roots by low Pi availability. The function of the ZmPP2AA1 gene in maize was analyzed using overexpression and RNA interference. ZmPP2AA1 modulated root gravitropism, negatively regulated primary root (PR) growth, and stimulated the development of lateral roots (LRs). A detailed characterization of the root system architecture (RSA) in response to different Pi concentrations with or without indole-3-acetic acid and 1-N-naphthylphthalamic acid revealed that auxin was involved in the RSA response to low Pi availability. Overexpression of ZmPP2AA1 enhanced tolerance to Pi starvation in transgenic maize in hydroponic and soil pot experiments. An increased dry weight (DW), root-to-shoot ratio, and total P content and concentration, along with a delayed and reduced accumulation of anthocyanin in overexpressing transgenic maize plants coincided with their highly branched root system and increased Pi uptake capability under low Pi conditions. Inflorescence development of the ZmPP2AA1 overexpressing line was less affected by low Pi stress, resulting in higher grain yield per plant under Pi deprivation. These data reveal the biological function of ZmPP2AA1, provide insights into a linkage between auxin and low Pi responses, and drive new strategies for the efficient utilization of Pi by maize. PMID:28448624

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

2016-07-26

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.

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

PubMed Central

Zwaenepoel, Karen; Louis, Justin V; Goris, Jozef; Janssens, Veerle

2008-01-01

Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. PMID:18715506

Group A PP2Cs evolved in land plants as key regulators of intrinsic desiccation tolerance

PubMed Central

Komatsu, Kenji; Suzuki, Norihiro; Kuwamura, Mayuri; Nishikawa, Yuri; Nakatani, Mao; Ohtawa, Hitomi; Takezawa, Daisuke; Seki, Motoaki; Tanaka, Maho; Taji, Teruaki; Hayashi, Takahisa; Sakata, Yoichi

2013-01-01

Vegetative desiccation tolerance is common in bryophytes, although this character has been lost in most vascular plants. The moss Physcomitrella patens survives complete desiccation if treated with abscisic acid (ABA). Group A protein phosphatases type 2C (PP2C) are negative regulators of abscisic acid signalling. Here we show that the elimination of Group A PP2C is sufficient to ensure P. patens survival to full desiccation, without ABA treatment, although its growth is severely hindered. Microarray analysis shows that the Group A PP2C-regulated genes exclusively overlap with genes exhibiting a high level of ABA induction. Group A PP2C disruption weakly affects ABA-activated kinase activity, indicating Group A PP2C action downstream of these kinases in the moss. We propose that Group A PP2C emerged in land plants to repress desiccation tolerance mechanisms, possibly facilitating plants propagation on land, whereas ABA releases the intrinsic desiccation tolerance from Group A PP2C regulation. PMID:23900426

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

PubMed

Maciejowski, John; Drechsler, Hauke; Grundner-Culemann, Kathrin; Ballister, Edward R; Rodriguez-Rodriguez, Jose-Antonio; Rodriguez-Bravo, Veronica; Jones, Mathew J K; Foley, Emily; Lampson, Michael A; Daub, Henrik; McAinsh, Andrew D; Jallepalli, Prasad V

2017-04-24

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation. Copyright © 2017 Elsevier Inc. All rights reserved.

Interleukin-18 gene deletion protects against sepsis-induced cardiac dysfunction by inhibiting PP2A activity.

PubMed

Okuhara, Yoshitaka; Yokoe, Shunichi; Iwasaku, Toshihiro; Eguchi, Akiyo; Nishimura, Koichi; Li, Wen; Oboshi, Makiko; Naito, Yoshiro; Mano, Toshiaki; Asahi, Michio; Okamura, Haruki; Masuyama, Tohru; Hirotani, Shinichi

2017-09-15

Interleukin-18 (IL-18) neutralization protects against lipopolysaccharide (LPS)-induced injuries, including myocardial dysfunction. However, the mechanism is yet to be fully elucidated. The aim of the present study was to determine whether IL-18 gene deletion prevents sepsis-induced cardiac dysfunction and to elucidate the potential mechanisms underlying IL-18-mediated cardiotoxicity by LPS. Ten-week-old male wild-type (WT) and IL-18 knockout (IL-18 KO) mice were intraperitoneally administered LPS. Serial echocardiography showed better systolic pump function and less left ventricular (LV) dilatation in LPS-treated IL-18 KO mice compared with those in LPS-treated WT mice. LPS treatment significantly decreased the levels of phospholamban (PLN) and Akt phosphorylation in WT mice compared with those in saline-treated WT mice, while the LPS-induced decrease in the phosphorylation levels was attenuated in IL-18 KO mice compared with that in WT mice. IL-18 gene deletion also attenuated an LPS-induced increase of type 2 protein phosphatase 2A (PP2A) activity, a molecule that dephosphorylates PLN and Akt. There was no difference in type 1 protein phosphatase (PP1) activity. To address whether IL-18 affects PLN and Akt phosphorylation via PP2A activation in cardiomyocytes, rat neonatal cardiac myocytes were cultured and stimulated using 100ng/ml of recombinant rat IL-18. Exogenous IL-18 decreased the level of PLN and Akt phosphorylation in cardiomyocytes. PP2A activity but not PP1 activity was increased by IL-18 stimulation in cardiomyocytes. IL-18 plays a pivotal role in advancing sepsis-induced cardiac dysfunction, and the mechanisms underlying IL-18-mediated cardiotoxicity potentially involve the regulation of PLN and Akt phosphorylation through PP2A activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential regulation of the androgen receptor by protein phosphatase regulatory subunits

PubMed Central

Grey, James; Jones, Dominic; Wilson, Laura; Nakjang, Sirintra; Clayton, Jake; Temperley, Richard; Clark, Emma; Gaughan, Luke; Robson, Craig

2018-01-01

The Androgen Receptor (AR) is a key molecule in the development, maintenance and progression of prostate cancer (PC). However, the relationship between the AR and co-regulatory proteins that facilitate AR activity in castrate resistant settings remain understudied. Here we show that protein phosphatase 1 regulatory subunits, identified from a phosphatase RNAi screen, direct PP1 catalytic subunits to a varied yet significant response in AR function. As such, we have characterised the PP1β holoenzyme, myosin phosphatase (MLCP), as a novel ligand independent regulator of the AR. Sustained MLCP activity through down-regulation of the MLCP inhibitory subunit, PPP1R14C, results in impaired AR nuclear translocation, protein stability and transcriptional activity in distinct models of PC progression, culminating in restoration of a non-malignant prostate genotype. Phenotypically, a marked reduction in cell proliferation and migration, characterised by G1 cell cycle arrest is observed, confirming PP1 holoenzyme disruption as a novel treatment approach in PC. PMID:29423094

Mining the Immune Cell Proteome to Identify Ovarian Cancer-Specific Biomarkers

DTIC Science & Technology

2013-11-01

transcription 4 (STAT4) Ras-related C3 botulinum toxin substrate 3 (RAC3) Serine/ threonine -protein phosphatase 2A catalytic subunit beta isoform (PP2AB...Mitogen-activated protein kinase 14 (MK14) Wnt signaling pathway (6) Beta-arrestin-1 (ARRB1) Serine/ threonine -protein phosphatase 2A catalytic...carried the diagnosis of chronic hypertension, diabetes , anti-phospholipid lipid antibody syndrome, or systemic lupus erythematous. Subjects were also

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

A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

PubMed

Steidle, Elizabeth A; Chong, Lucy S; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C; Rolfes, Ronda J

2016-03-25

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5or IP7)in vitro. In vivo,siw14Δ yeast mutants possess increased IP7levels, whereas heterologousSIW14overexpression eliminates IP7from cells. IP7levels increased proportionately whensiw14Δ was combined withddp1Δ orvip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7isoform 5PP-IP5to IP6. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

PubMed

Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

2015-01-01

The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum.

PubMed

Hollin, Thomas; De Witte, Caroline; Lenne, Astrid; Pierrot, Christine; Khalife, Jamal

2016-03-17

Protein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum (Pf). The activity of PP1 is regulated by the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite. To better understand the P. falciparum PP1 (PfPP1) regulatory network, we here report the use of three strategies to characterize the PfPP1 interactome: co-affinity purified proteins identified by mass spectrometry, yeast two-hybrid (Y2H) screening and in silico analysis of the P. falciparum predicted proteome. Co-affinity purification followed by MS analysis identified 6 PfPP1 interacting proteins (Pips) of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK] motif, also shown to be a PP1 binding motif and one with both binding motifs. The Y2H screens identified 134 proteins of which 30 present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the Pf predicted proteome using a consensus RVxF motif as template revealed the presence of 55 potential Pips. As further demonstration, 35 candidate proteins were validated as PfPP1 interacting proteins in an ELISA-based assay. To the best of our knowledge, this is the first study on PfPP1 interactome. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication of Pips in protein folding/proteolysis, transcription and pathogenicity networks is proposed. The present work provides a starting point for further studies on the structural basis of these interactions and their functions in P. falciparum.

NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

PubMed Central

Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

2010-01-01

Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

Centromeric binding and activity of Protein Phosphatase 4

PubMed Central

Lipinszki, Zoltan; Lefevre, Stephane; Savoian, Matthew S.; Singleton, Martin R.; Glover, David M.; Przewloka, Marcin R.

2015-01-01

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis. PMID:25562660

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis

PubMed Central

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura

2015-01-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. PMID:25986610

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

PubMed

Jonak, Katarzyna; Zagoriy, Ievgeniia; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-06-18

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast

PubMed Central

Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-01-01

ABSTRACT Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. PMID:28514186

Alp7/TACC recruits kinesin-8–PP1 to the Ndc80 kinetochore protein for timely mitotic progression and chromosome movement

PubMed Central

Tang, Ngang Heok; Toda, Takashi

2015-01-01

ABSTRACT Upon establishment of proper kinetochore–microtubule attachment, the spindle assembly checkpoint (SAC) must be silenced to allow onset of anaphase, which is when sister chromatids segregate equally to two daughter cells. However, how proper kinetochore–microtubule attachment leads to timely anaphase onset remains elusive. Furthermore, the molecular mechanisms of chromosome movement during anaphase A remain unclear. In this study, we show that the fission yeast Alp7/TACC protein recruits a protein complex consisting of the kinesin-8 (Klp5–Klp6) and protein phosphatase 1 (PP1) to the kinetochore upon kinetochore–microtubule attachment. Accumulation of this complex at the kinetochore, on the one hand, facilitates SAC inactivation through PP1, and, on the other hand, accelerates polewards chromosome movement driven by the Klp5–Klp6 motor. We identified an alp7 mutant that had specific defects in binding to the Klp5–Klp6–PP1 complex but with normal localisation to the microtubule and kinetochore. Consistent with our proposition, this mutant shows delayed anaphase onset and decelerated chromosome movement during anaphase A. We propose that the recruitment of kinesin-8–PP1 to the kinetochore through Alp7/TACC interaction plays a crucial role in regulation of timely mitotic progression and chromosome movement during anaphase A. PMID:25472718

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

PubMed

Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A; Affar, El Bachir; Milot, Eric

2014-12-01

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

The IKAROS Interaction with a Complex Including Chromatin Remodeling and Transcription Elongation Activities Is Required for Hematopoiesis

PubMed Central

Bottardi, Stefania; Mavoungou, Lionel; Pak, Helen; Daou, Salima; Bourgoin, Vincent; Lakehal, Yahia A.; Affar, El Bachir; Milot, Eric

2014-01-01

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation. PMID:25474253

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly

PubMed Central

Abdulkarim, Baroj; Igoillo-Esteve, Mariana; Daures, Mathilde; Romero, Sophie; Philippi, Anne; Senée, Valérie; Lopes, Miguel; Cunha, Daniel A.; Harding, Heather P.; Derbois, Céline; Bendelac, Nathalie; Hattersley, Andrew T.; Eizirik, Décio L.; Ron, David

2015-01-01

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic β-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in β-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to β-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities. PMID:26159176

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

PubMed

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

2014-05-01

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

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

PubMed

Parua, Pabitra K; Booth, Gregory T; Sansó, Miriam; Benjamin, Bradley; Tanny, Jason C; Lis, John T; Fisher, Robert P

2018-06-13

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .

Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases.

PubMed

Liu, Chunliang; Dunaway-Mariano, Debra; Mariano, Patrick S

2017-03-10

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (K i ) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The K i of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Role of serine threonine protein phosphatase type 5 (PP5) in the regulation of stress induced signaling networks and cancer

PubMed Central

Golden, Teresa; Swingle, Mark; Honkanen, Richard E.

2008-01-01

Although the aberrant actions of protein kinases have long been known to contribute to tumor promotion and carcinogenesis, roles for proteins phosphatases in the development of human cancer have only emerged in the last decade. In this review, we discuss the data obtained from studies examining the biological and pathological roles of a serine/threonine protein phosphate, PP5, which suggest that PP5 is a potentially important regulator of both hormone- and stress-induced networks that enable a cell to respond appropriately to genomic stress. PMID:18253812

The modulation of the phosphorylation status of NKCC1 in organ cultured bovine lenses: Implications for the regulation of fiber cell and overall lens volume.

PubMed

Vorontsova, Irene; Donaldson, Paul J; Kong, Zhiying; Wickremesinghe, Chiharu; Lam, Leo; Lim, Julie C

2017-12-01

In previous work, we have shown the Sodium/Potassium/2 Chloride Cotransporter (NKCC1) to be a key effector of lens fiber cell volume regulation. Since others have shown that the activity of NKCC1 is regulated via its phosphorylation status, the purpose of this study was to investigate whether NKCC1 phosphorylation can be modulated in organ cultured bovine lenses, and to see how this relates to changes in lens wet weight. Western blotting was first used to confirm the expression of NKCC1, phosphorylated NKCC1 (NKCC1-P) and the regulatory kinases WNK/SPAK and phosphatases PP1/PP2A in bovine lenses at the protein level. Changes to NKCC1-P status were then assessed by organ culturing bovine lenses in either isotonic, hypertonic or hypotonic solutions in the presence or absence of the NKCC inhibitor, bumetanide, or phosphatase inhibitors okadaic acid and calyculin A. After 1-22 h of culturing, lenses were weighed, assessed for transparency and the cortical protein fractions analyzed by western blot using antibodies to detect total NKCC1 and NKCC1-P. NKCC1, NKCC1-P, SPAK, PP1 and PP2A were all detected in the membrane fraction of bovine lenses. Under hypertonic conditions, NKCC1 is phosphorylated and activated to mediate a regulatory volume increase. Finally, NKCC1-P signal increased in the presence of phosphatase inhibitors indicating that PP1/PP2A can dephosphorylate NKCC1. These results show that the phosphorylation status and hence activity of NKCC1 is dynamically regulated and that in response to hypertonic stress, NKCC1 activity is increased to effect a regulatory volume increase that limits cell shrinkage. These findings support the view that the lens dynamically regulates ion fluxes to maintain steady state lens volume, and suggest that dysfunction of this regulation maybe an initiating factor in the localized fiber cell swelling that is a characteristic of diabetic lens cataract. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2011-01-01

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

Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stage C. elegans embryos

PubMed Central

Afshar, Katayoun; Werner, Michael E.; Tse, Yu Chung; Glotzer, Michael; Gönczy, Pierre

2010-01-01

Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development. PMID:20040490

In vitro extinction learning in Hermissenda: involvement of conditioned inhibition molecules

PubMed Central

Cavallo, Joel S.; Hamilton, Brittany N.; Farley, Joseph

2014-01-01

Extinction of a conditioned association is typically viewed as the establishment of new learning rather than the erasure of the original memory. However, recent research in the nudibranch, Hermissenda crassicornis (H.c.) demonstrated that extinction training (using repeated light-alone presentations) given 15 min, but not 23 h, after memory acquisition reversed both the cellular correlates of learning (enhanced Type B cell excitability) and the behavioral changes (reduced phototaxis) produced by associative conditioning (pairings of light, CS, and rotation, US). Here, we investigated the putative molecular signaling pathways that underlie this extinction in H.c. by using a novel in vitro protocol combined with pharmacological manipulations. After intact H.c. received either light-rotation pairings (Paired), random presentations of light and rotation (Random), or no stimulation (Untrained), B cells from isolated CNSs were recorded from during exposure to extinction training consisting of two series of 15 consecutive light-steps (LSs). When in vitro extinction was administered shortly (2 h, but not 24 h) after paired training, B cells from Paired animals showed progressive and robust declines in spike frequency by the 30th LS, while control cells (Random and Untrained) did not. We found that several molecules implicated in H.c. conditioned inhibitory (CI) learning, protein phosphatase 1 (PP1) and arachidonic acid (AA)/12-lipoxygenase (12-LOX) metabolites, also contributed to the spike frequency decreases produced by in vitro extinction. Protein phosphatase 2B (PP2B) also appeared to play a role. Calyculin A (PP1 inhibitor), cyclosporin A (PP2B inhibitor), and baicalein (a 12-LOX inhibitor) all blocked the spike frequency declines in Paired B cells produced by 30 LSs. Conversely, injection of catalytically-active PP1 (caPP1) or PP2B (caPP2B) into Untrained B cells partially mimicked the spike frequency declines observed in Paired cells, as did bath-applied AA, and occluded additional LS-produced reductions in spiking in Paired cells. PMID:25374517

Avirulence gene mapping in the Hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family.

PubMed

Zhao, Chaoyang; Shukle, Richard; Navarro-Escalante, Lucio; Chen, Mingshun; Richards, Stephen; Stuart, Jeffrey J

2016-01-01

The genetic tractability of the Hessian fly (HF, Mayetiola destructor) provides an opportunity to investigate the mechanisms insects use to induce plant gall formation. Here we demonstrate that capacity using the newly sequenced HF genome by identifying the gene (vH24) that elicits effector-triggered immunity in wheat (Triticum spp.) seedlings carrying HF resistance gene H24. vH24 was mapped within a 230-kb genomic fragment near the telomere of HF chromosome X1. That fragment contains only 21 putative genes. The best candidate vH24 gene in this region encodes a protein containing a secretion signal and a type-2 serine/threonine protein phosphatase (PP2C) domain. This gene has an H24-virulence associated insertion in its promoter that appears to silence transcription of the gene in H24-virulent larvae. Candidate vH24 is a member of a small family of genes that encode secretion signals and PP2C domains. It belongs to the fraction of genes in the HF genome previously predicted to encode effector proteins. Because PP2C proteins are not normally secreted, our results suggest that these are PP2C effectors that HF larvae inject into wheat cells to redirect, or interfere, with wheat signal transduction pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

The molecular basis for development of proinflammatory autoantibodies to progranulin.

PubMed

Thurner, Lorenz; Fadle, Natalie; Regitz, Evi; Kemele, Maria; Klemm, Philipp; Zaks, Marina; Stöger, Elisabeth; Bette, Birgit; Carbon, Gabi; Zimmer, Vincent; Assmann, Gunter; Murawski, Niels; Kubuschok, Boris; Held, Gerhard; Preuss, Klaus-Dieter; Pfreundschuh, Michael

2015-07-01

Recently we identified in a wide spectrum of autoimmune diseases frequently occurring proinflammatory autoantibodies directed against progranulin, a direct inhibitor of TNFR1 & 2 and of DR3. In the present study we investigated the mechanisms for the breakdown of self-tolerance against progranulin. Isoelectric focusing identified a second, differentially electrically charged progranulin isoform exclusively present in progranulin-antibody-positive patients. Alkaline phosphatase treatment revealed this additional progranulin isoform to be hyperphosphorylated. Subsequently Ser81, which is located within the epitope region of progranulin-antibodies, was identified as hyperphosphorylated serine residue by site directed mutagenesis of candidate phosphorylation sites. Hyperphosphorylated progranulin was detected exclusively in progranulin-antibody-positive patients during the courses of their diseases. The occurrence of hyperphosphorylated progranulin preceded seroconversions of progranulin-antibodies, indicating adaptive immune response. Utilizing panels of kinase and phosphatase inhibitors, PKCβ1 was identified as the relevant kinase and PP1 as the relevant phosphatase for phosphorylation and dephosphorylation of Ser81. In contrast to normal progranulin, hyperphosphorylated progranulin interacted exclusively with inactivated (pThr320) PP1, suggesting inactivated PP1 to cause the detectable occurrence of phosphorylated Ser81 PGRN. Investigation of possible functional alterations of PGRN due to Ser81 phosphorylation revealed, that hyperphosphorylation prevents the interaction and thus direct inhibition of TNFR1, TNFR2 and DR3, representing an additional direct proinflammatory effect. Finally phosphorylation of Ser81 PGRN alters the conversion pattern of PGRN. In conclusion, inactivated PP1 induces hyperphosphorylation of progranulin in a wide spectrum of autoimmune diseases. This hyperphosphorylation prevents direct inhibition of TNFR1, TNFR2 and DR3 by PGRN, alters the conversion of PGRN, and is strongly associated with the occurrence of neutralizing, proinflammatory PGRN-antibodies, indicating immunogenicity of this alternative secondary modification. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein*

PubMed Central

Hwang, Justin H.; Jiang, Tao; Kulkarni, Shreya; Faure, Nathalie; Schaffhausen, Brian S.

2013-01-01

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt. PMID:24052256

HONSU, a protein phosphatase 2C, regulates seed dormancy by inhibiting ABA signaling in Arabidopsis.

PubMed

Kim, Woohyun; Lee, Yeon; Park, Jeongmoo; Lee, Nayoung; Choi, Giltsu

2013-04-01

Seed dormancy, a seed status that prohibits germination even in the presence of inductive germination signals, is a poorly understood process. To identify molecular components that regulate seed dormancy, we screened T-DNA insertion lines and identified a mutant designated honsu (hon). HON loss-of-function mutants display deep seed dormancy, whereas HON-overexpressing lines display shallow seed dormancy. HON encodes a seed-specific group A phosphatase 2C (PP2C) and is one of the major negative regulators of seed dormancy among group A PP2Cs. Like other PP2C family members, HON interacts with PYR1/RCAR11 in the presence of ABA. Our analysis indicates that HON inhibits ABA signaling and activates gibberellic acid signaling, and both of these conditions must be satisfied to promote the release of seed dormancy. However, HON mRNA levels are increased in mutants displaying deep seed dormancy or under conditions that deepen seed dormancy, and decreased in mutants displaying shallow seed dormancy or under conditions that promote the release of seed dormancy. Taken together, our results indicate that the expression of HON mRNA is homeostatically regulated by seed dormancy.

E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis.

PubMed

Lin, Kuan-Ho; Kuo, Wei-Wen; Shibu, Marthandam Asokan; Day, Cecilia-Hsuan; Hsieh, You-Liang; Chung, Li-Chin; Chen, Ray-Jade; Wen, Su-Ying; Viswanadha, Vijaya Padma; Huang, Chih-Yang

2017-04-24

Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2), for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs). In addition, protein phosphatase such as protein phosphatase 1 (PP1) and calcineurin (PP2B) are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO)-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

PubMed Central

Du, Haijuan; Massiah, Michael A.

2011-01-01

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1. PMID:22194938

ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase)

PubMed Central

Andrade, Erika C.; Musante, Veronica; Horiuchi, Atsuko; Greengard, Paul; Taylor, Jane R.

2017-01-01

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We find that the protein ARPP-16, which is highly expressed in striatal medium spiny neurons, acts as a selective inhibitor of certain forms of the serine/threonine protein phosphatase, PP2A, when phosphorylated by the kinase, MAST3. Under basal conditions, ARPP-16 is phosphorylated by MAST3 to a very high stoichiometry. However, the actions of MAST3 are antagonized by dopamine and cAMP-regulated signaling leading to disinhibition of ARPP-16 and increased PP2A action. PMID:28167675

ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase).

PubMed

Andrade, Erika C; Musante, Veronica; Horiuchi, Atsuko; Matsuzaki, Hideo; Brody, A Harrison; Wu, Terence; Greengard, Paul; Taylor, Jane R; Nairn, Angus C

2017-03-08

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We find that the protein ARPP-16, which is highly expressed in striatal medium spiny neurons, acts as a selective inhibitor of certain forms of the serine/threonine protein phosphatase, PP2A, when phosphorylated by the kinase, MAST3. Under basal conditions, ARPP-16 is phosphorylated by MAST3 to a very high stoichiometry. However, the actions of MAST3 are antagonized by dopamine and cAMP-regulated signaling leading to disinhibition of ARPP-16 and increased PP2A action. Copyright © 2017 the authors 0270-6474/17/372709-14$15.00/0.

CDC-48/p97 is required for proper meiotic chromosome segregation via controlling AIR-2/Aurora B kinase localization in Caenorhabditis elegans.

PubMed

Sasagawa, Yohei; Higashitani, Atsushi; Urano, Takeshi; Ogura, Teru; Yamanaka, Kunitoshi

2012-08-01

CDC-48/p97 is a AAA (ATPases associated with diverse cellular activities) chaperone involved in protein conformational changes such as the disassembly of protein complexes. We previously reported that Caenorhabditis elegans CDC-48.1 and CDC-48.2 (CDC-48s) are essential for the progression of meiosis I metaphase. Here, we report that CDC-48s are required for proper chromosome segregation during meiosis in C. elegans. In wild-type worms, at the diakinesis phase, phosphorylation of histone H3, one of the known substrates of aurora B kinase (AIR-2), on meiosis I chromatids correlated with AIR-2 localization at the cohesion sites of homologous chromatids. Conversely, depletion of CDC-48s resulted in a significant expansion of signals for AIR-2 and phosphorylated histone H3 over the entire length of meiotic chromosomes, leading to defective chromosome segregation, while the total amount of AIR-2 in lysates was not changed by the depletion of CDC-48s. The defective segregation of meiotic chromosomes caused by the depletion of CDC-48s was suppressed by the simultaneous depletion of AIR-2 and is similar to that observed following the depletion of protein phosphatase 1 (PP1) phosphatases. However, the amount and localization of PP1 were not changed by the depletion of CDC-48s. These results suggest that CDC-48s control the restricted localization of AIR-2 to the cohesion sites of homologous chromatids in meiosis I. Copyright © 2012 Elsevier Inc. All rights reserved.

A protein phosphatase feedback mechanism regulates the basal phosphorylation of Chk2 kinase in the absence of DNA damage.

PubMed

Carlessi, Luigi; Buscemi, Giacomo; Fontanella, Enrico; Delia, Domenico

2010-10-01

The checkpoint kinase Chk2 is an effector component of the ATM-dependent DNA damage response (DDR) pathway. The activation of Chk2 by genotoxic stress involves its phosphorylation on T68 by ATM and additional auto/transphosphorylations. Here we demonstrate that in unperturbed cells, chemical inhibition of Chk2 by VRX0466617 (VRX) enhances the phosphorylation of Chk2-T68 throughout the cell cycle phases. This event, dependent on the presence of ATM and catalytically functional Chk2, is not consequential to DNA damage, as neither gamma-H2AX nuclear foci nor increased ATM activation is detected in VRX-treated cells, suggesting the involvement of other regulatory proteins. As serine/threonine protein phosphatases (PPs) regulate the phosphorylation and deactivation of proteins of the DDR pathway, we analyzed their role in phospho-T68-Chk2 regulation. We found that intracellular inhibition of PP1 and PP2A-like activities by okadaic acid markedly raised the accumulation of Chk2-pT68 without DNA damage induction, and this phenomenon was also seen when PP1-C, PP2A-C, and Wip1/PPM1D were simultaneously knockdown by siRNA. Altogether, these data indicate a novel mechanism in undamaged cells where PPs function to maintain the balance between ATM and its direct substrate Chk2 through a regulatory circuit. Copyright © 2010 Elsevier B.V. All rights reserved.

ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

PubMed

Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

2013-03-01

Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

REDUCED DORMANCY5 Encodes a Protein Phosphatase 2C That Is Required for Seed Dormancy in Arabidopsis[C][W][OPEN

PubMed Central

Xiang, Yong; Nakabayashi, Kazumi; Ding, Jia; He, Fei; Bentsink, Leónie; Soppe, Wim J.J.

2014-01-01

Seed dormancy determines germination timing and contributes to crop production and the adaptation of natural populations to their environment. Our knowledge about its regulation is limited. In a mutagenesis screen of a highly dormant Arabidopsis thaliana line, the reduced dormancy5 (rdo5) mutant was isolated based on its strongly reduced seed dormancy. Cloning of RDO5 showed that it encodes a PP2C phosphatase. Several PP2C phosphatases belonging to clade A are involved in abscisic acid signaling and control seed dormancy. However, RDO5 does not cluster with clade A phosphatases, and abscisic acid levels and sensitivity are unaltered in the rdo5 mutant. RDO5 transcript could only be detected in seeds and was most abundant in dry seeds. RDO5 was found in cells throughout the embryo and is located in the nucleus. A transcriptome analysis revealed that several genes belonging to the conserved PUF family of RNA binding proteins, in particular Arabidopsis PUMILIO9 (APUM9) and APUM11, showed strongly enhanced transcript levels in rdo5 during seed imbibition. Further transgenic analyses indicated that APUM9 reduces seed dormancy. Interestingly, reduction of APUM transcripts by RNA interference complemented the reduced dormancy phenotype of rdo5, indicating that RDO5 functions by suppressing APUM transcript levels. PMID:25415980

Protein phosphatases 2A as well as reactive oxygen species involved in tributyltin-induced apoptosis in mouse livers.

PubMed

Zhang, Yali; Chen, Yonggang; Sun, Lijun; Liang, Jing; Guo, Zonglou; Xu, Lihong

2014-02-01

Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce caspase-3-dependent apoptosis in human amniotic cells through protein phosphatase 2A (PP2A) inhibition and consequent JNK activation. This in vivo study was undertaken to further verify the results derived from our previous in vitro study. Mice were orally dosed with 0, 10, 20, and 60 mg/kg of body weight TBT, and levels of PP2A, reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK), Bax/Bcl-2, and caspase-3 were detected in the mouse livers. Apoptosis was also evaluated using the TUNEL assay. The results showed that PP2A activity was inhibited, ROS levels were elevated, and MAPKs including ERK, JNK, and p38 were activated in mouse livers treated with the highest dose of TBT. Additionally, the ratio of Bax/Bcl-2 was increased, caspase-3 was activated, and apoptosis in mouse livers could be detected in the highest dose group. Therefore, a possible signaling pathway in TBT-induced apoptosis in mouse livers involves PP2A inhibition and ROS elevation serving a pivotal function as upstream activators of MAPKs; activation of MAPKs in turn leads to an increase in the Bax/Bcl-2 ratio, ultimately leading to the activation of caspase-3. The results give a comprehensive and novel description of the mechanism of TBT-induced toxicity. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

Tumor Suppression and Sensitization to Taxol Induces Apoptosis of EIA in Breast Cancer Cells

DTIC Science & Technology

2005-06-01

participated in the regulation of apoptosis induced by ceramide, mistletoe lectin, and 4-hydroxynonenal, an aldehyde product of mem- brane lipid peroxidation... Mistletoe lectin induces apoptosis and telomerase inhibition in hu- man A253 cancer cells through dephosphorylation of Akt. Arch Pharm Res 2004; 27:68-76...participated subunit of protein phosphatase 2A [PP2A (PP2A/C)l enhanced the activity in the regulation of apoptosis induced by ceramide, mistletoe lectin, of

Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

PubMed Central

Frey, Stefan; Reschka, Eva J.; Pöggeler, Stefanie

2015-01-01

The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain. PMID:26418262

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

PubMed

Kang, H; Sayner, S L; Gross, K L; Russell, L C; Chinkers, M

2001-09-04

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

PubMed

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

PubMed

Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

2015-06-01

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

17β-Estradiol and/or estrogen receptor alpha signaling blocks protein phosphatase 1 mediated ISO induced cardiac hypertrophy.

PubMed

Fang, Hsin-Yuan; Hung, Meng-Yu; Lin, Yueh-Min; Pandey, Sudhir; Chang, Chia-Chien; Lin, Kuan-Ho; Shen, Chia-Yao; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-01-01

Earlier studies have shown that estrogen possess protective function against the development of pathological cardiac hypertrophy. However, the molecular mechanisms of estrogens (E2) protective effect are poorly understood. Additionally, abnormal activation of β-adrenergic signaling have been implicated in the development of pathological cardiac remodeling. However, the role of serine/threonine protein phosphatase 1 (PP1) in pathological cardiac remodeling under the influence of β-adrenergic signaling have been sparsely investigated. In this study, we assessed the downstream effects of abnormal activation of PP1 upon isoproterenol (ISO) induced pathological cardiac changes. We found that pre-treatment of 17β-estradiol (E2), tet-on estrogen receptor-α, or both significantly inhibited ISO-induced increase in cell size, hypertrophy marker gene expression and cytosolic calcium accumulation in H9c2 cells. Additionally, treatment with estrogen receptor inhibitor (ICI) reversed those effects, implicating role of E2 in inhibiting pathological cardiac remodeling. However, specific inhibition of ERα using melatonin, reduced ISO-induced PP1c expression and enhanced the level of ser-16 phosphorylated phospholamban (PLB), responsible for regulation of sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Furthermore, hypertrophic effect caused by overexpression of PP1cα was reduced by treatment with specific inhibitor of ERα. Collectively, we found that estrogen and estrogen receptor-α have protective effect against pathological cardiac changes by suppressing PP1 expression and its downstream signaling pathway, which further needs to be elucidated.

Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

PubMed Central

Modderman, P W; von dem Borne, A E; Sonnenberg, A

1994-01-01

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7514867

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Mingning, E-mail: lcuzfy@163.com; Liu, Lei, E-mail: leiliulab@163.com; Chen, Lieqian, E-mail: lieqianchen@163.com

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 andmore » 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.« less

Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

PubMed Central

Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

2013-01-01

Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis

PubMed Central

Chi, Richard J.; Torres, Onaidy T.; Segarra, Verónica A.; Lansley, Tanya; Chang, Ji Suk; Newpher, Thomas M.; Lemmon, Sandra K.

2012-01-01

Summary Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis. In this study we analyzed scd5-PP1Δ2, a mutation causing impaired PP1 binding. scd5-PP1Δ2 caused hyperphosphorylation of several Prk1 endocytic targets. Live-cell imaging of 15 endocytic components in scd5-PP1Δ2 revealed that most factors arriving before the invagination/actin phase of endocytosis had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homolog), whose lifetime was extended nearly fourfold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than twofold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1Δ2 were suppressed by SLA1 overexpression. This was dependent on the LxxQxTG repeats (SR) of Sla1, which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the dephosphorylated state. Without the SR, Sla1ΔSR was still recruited to the cell surface, but was less concentrated in cortical patches than Pan1. sla1ΔSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region the SH3 region of Sla1 causes constitutive negative regulation of Las17 (WASp). These results demonstrate that Scd5/PP1 is important for recycling Prk1 targets to initiate new rounds of endocytosis and provide new mechanistic information on the role of the Sla1 SR domain in regulating progression to the invagination/actin phase of endocytosis. PMID:22825870

Protein Kinase C-δ Mediates Neuronal Apoptosis in the Retinas of Diabetic Rats via the Akt Signaling Pathway

PubMed Central

Kim, Young-Hee; Kim, Yoon-Sook; Park, Chang-Hwan; Chung, In-Yong; Yoo, Ji-Myong; Kim, Jae-Geun; Lee, Byung-Ju; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

2008-01-01

OBJECTIVE—Protein kinase C (PKC)-δ, an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein, we examined the role of PKC-δ in neuronal apoptosis through Akt in the retinas of diabetic rats. RESEARCH DESIGN AND METHODS—We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) nondiabetic rats. To assess whether PKC-δ affects Akt signaling and cell death in OLETF rat retinas, we examined 1) PKC-δ activity and apoptosis; 2) protein levels of phosphatidylinositol 3-kinase (PI 3-kinase) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A); 3) Akt phosphorylation; and 4) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC-δ inhibitor, or small interfering RNAs (siRNAs) for PKC-δ and HSP90. RESULTS—In OLETF retinas from 35-week-old rats, ganglion cell death, PKC-δ and PP2A activity, and Akt-PP2A binding were significantly increased and Akt phosphorylation and Akt-HSP90 binding were decreased compared with retinas from 24-week-old OLETF and LETO rats. Rottlerin and PKC-δ siRNA abrogated these effects in OLETF retinas from 35-week-old rats. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in LETO retinas from 35-week-old rats compared with those from nontreated LETO rats. CONCLUSIONS—PKC-δ activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways. PMID:18443201

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.

Effect of Microcystin-LR on human trophoblast differentiation in vitro

EPA Science Inventory

Background: Microcystin LR is a potent protein phosphatase 2a (PP2a) inhibitor and generates reactive oxygen species (ROS) believed to be an essential component of a toxic effect. Toxicological studies have demonstrated microcystin (MCYST) disruption of cytoskeletal function and...

STRIP1, a core component of STRIPAK complexes, is essential for normal mesoderm migration in the mouse embryo.

PubMed

Bazzi, Hisham; Soroka, Ekaterina; Alcorn, Heather L; Anderson, Kathryn V

2017-12-19

Regulated mesoderm migration is necessary for the proper morphogenesis and organ formation during embryonic development. Cell migration and its dependence on the cytoskeleton and signaling machines have been studied extensively in cultured cells; in contrast, remarkably little is known about the mechanisms that regulate mesoderm cell migration in vivo. Here, we report the identification and characterization of a mouse mutation in striatin-interacting protein 1 ( Strip1 ) that disrupts migration of the mesoderm after the gastrulation epithelial-to-mesenchymal transition (EMT). STRIP1 is a core component of the biochemically defined mammalian striatin-interacting phosphatases and kinase (STRIPAK) complexes that appear to act through regulation of protein phosphatase 2A (PP2A), but their functions in mammals in vivo have not been examined. Strip1 -null mutants arrest development at midgestation with profound disruptions in the organization of the mesoderm and its derivatives, including a complete failure of the anterior extension of axial mesoderm. Analysis of cultured mesoderm explants and mouse embryonic fibroblasts from null mutants shows that the mesoderm migration defect is correlated with decreased cell spreading, abnormal focal adhesions, changes in the organization of the actin cytoskeleton, and decreased velocity of cell migration. The results show that STRIPAK complexes are essential for cell migration and tissue morphogenesis in vivo. Copyright © 2017 the Author(s). Published by PNAS.

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

Dynamic landscape of the local translation at activated synapses.

PubMed

Khlebodarova, T M; Kogai, V V; Trifonova, E A; Likhoshvai, V A

2018-01-01

The mammalian target of rapamycin (mTOR) signaling pathway is the central regulator of cap-dependent translation at the synapse. Disturbances in mTOR pathway have been associated with several neurological diseases, such as autism and epilepsy. RNA-binding protein FMRP, a negative regulator of translation initiation, is one of the key components of the local translation system. Activation and inactivation of FMRP occurs via phosphorylation by S6 kinase and dephosphorylation by PP2A phosphatase, respectively. S6 kinase and PP2A phosphatase are activated in response to mGluR receptor stimulation through different signaling pathways and at different rates. The dynamic aspects of this system are poorly understood. We developed a mathematical model of FMRP-dependent regulation of postsynaptic density (PSD) protein synthesis in response to mGluR receptor stimulation and conducted in silico experiments to study the regulatory circuit functioning. The modeling results revealed the possibility of generating oscillatory (cyclic and quasi-cyclic), chaotic and even hyperchaotic dynamics of postsynaptic protein synthesis as well as the presence of multiple attractors in a wide range of parameters of the local translation system. The results suggest that autistic disorders associated with mTOR pathway hyperactivation may be due to impaired proteome stability associated with the formation of complex dynamic regimes of PSD protein synthesis in response to stimulation of mGluR receptors on the postsynaptic membrane of excitatory synapses on pyramidal hippocampal cells.

PP2A activity is controlled by methylation and regulates oncoprotein expression in melanoma cells: a mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment.

PubMed

Guénin, Samuel; Schwartz, Laurent; Morvan, Daniel; Steyaert, Jean Marc; Poignet, Amandine; Madelmont, Jean Claude; Demidem, Aicha

2008-01-01

Protein phosphatase 2A (PP2A), an Akt pathway inhibitor, is considered to be activated by methylation of its catalytic subunit. Also PP2A downregulation was proposed to take part in carcinogenesis. Recently, PP2A activation was shown to be activated in response to DNA damage. To obtain further information on the role of PP2A in tumors and response to DNA damage, we investigated the relationship between PP2A methylation and activity, cell proliferation, Akt activation, c-Myc expression and PTEN activity in B16 melanoma cells untreated and after chloroethylnitrosourea (CENU) treatment. In untreated cells, okadaic acid, an antagonist of PP2A methylation, inhibited PP2A activity, stimulated cell proliferation, increased Akt activation and c-Myc expression. Xylulose-5-phosphate, an agonist of PP2A methylation, increased PP2A activity, decreased cell proliferation, Akt activation and c-Myc expression. However, both PP2A methylation modulators increased PTEN activity. During the response to CENU treatment, PP2A methylation and activity were strongly increased, Akt activation and c-Myc expression were decreased. However PTEN activity was increased. After tumor cell growth recovery, these modifications were moderately decreased. PP2A methylation was quantified and correlated positively with PP2A activity, and negatively with criteria for cell aggressiveness (cell proliferation, Akt activation, c-Myc expression). Based on these data, PP2A methylation status controls PP2A activity and oncoproteins expression and PP2A is strongly activated after CENU treatment thus partly explaining the growth inhibition in response to this agent. It follows that PP2A promethylating agents are potential candidates for anticancer drugs.

I(2)(PP2A) regulates p53 and Akt correlatively and leads the neurons to abort apoptosis.

PubMed

Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Zhang, Yao; Shi, Hai-Hong; Yin, Jun; Yao, Xiu-Qing; Peng, Cai-Xia; Hu, Juan; Wang, Qun; Li, Hong-Lian; Wang, Jian-Zhi

2012-02-01

A chronic neuron loss is the cardinal pathology in Alzheimer disease (AD), but it is still not understood why most neurons in AD brain do not accomplish apoptosis even though they are actually exposed to an environment with enriched proapoptotic factors. Protein phosphatase-2A inhibitor-2 (I(2)(PP2A)), an endogenous PP2A inhibitor, is significantly increased in AD brain, but the role of I(2)(PP2A) in AD-like neuron loss is elusive. Here, we show that I(2)(PP2A) regulates p53 and Akt correlatively. The mechanisms involve activated transcription and p38 MAPK activities. More importantly, we demonstrate that the simultaneous activation of Akt induced by I(2)(PP2A) counteracts the hyperactivated p53-induced cell apoptosis. Furthermore, I(2)(PP2A), p53 and Akt are all elevated in the brain of mouse model and AD patients. Our results suggest that the increased I(2)(PP2A) may trigger apoptosis by p53 upregulation, but due to simultaneous activation of Akt, the neurons are aborted from the apoptotic pathway. This finding contributes to the understanding of why most neurons in AD brain do not undergo apoptosis. Copyright © 2010. Published by Elsevier Inc.

Structure- and Modeling-based Identification of the Adenovirus E4orf4 Binding Site in the Protein Phosphatase 2A B55α Subunit*

PubMed Central

Horowitz, Ben; Sharf, Rakefet; Avital-Shacham, Meirav; Pechkovsky, Antonina; Kleinberger, Tamar

2013-01-01

The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators. PMID:23530045

Protein Kinases and Phosphatases in the Control of Cell Fate

PubMed Central

Bononi, Angela; Agnoletto, Chiara; De Marchi, Elena; Marchi, Saverio; Patergnani, Simone; Bonora, Massimo; Giorgi, Carlotta; Missiroli, Sonia; Poletti, Federica; Rimessi, Alessandro; Pinton, Paolo

2011-01-01

Protein phosphorylation controls many aspects of cell fate and is often deregulated in pathological conditions. Several recent findings have provided an intriguing insight into the spatial regulation of protein phosphorylation across different subcellular compartments and how this can be finely orchestrated by specific kinases and phosphatases. In this review, the focus will be placed on (i) the phosphoinositide 3-kinase (PI3K) pathway, specifically on the kinases Akt and mTOR and on the phosphatases PP2a and PTEN, and on (ii) the PKC family of serine/threonine kinases. We will look at general aspects of cell physiology controlled by these kinases and phosphatases, highlighting the signalling pathways that drive cell division, proliferation, and apoptosis. PMID:21904669

The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

PubMed

Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

2012-11-01

Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E

PubMed Central

Kim, Yumi; Holland, Andrew J.; Lan, Weijie; Cleveland, Don W.

2010-01-01

Summary Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here we demonstrate that Aurora kinases A and B phosphorylate a single residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-E’s affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes. PMID:20691903

AR-v7 protein expression is regulated by protein kinase and phosphatase

PubMed Central

Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

2015-01-01

Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

Differential dephosphorylation of the insulin receptor and its 160-kDa substrate (pp160) in rat adipocytes.

PubMed

Mooney, R A; Bordwell, K L

1992-07-15

A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of RsbU in Controlling SigB Activity in Staphylococcus aureus following Alkaline Stress▿

PubMed Central

Pané-Farré, Jan; Jonas, Beate; Hardwick, Steven W.; Gronau, Katrin; Lewis, Richard J.; Hecker, Michael; Engelmann, Susanne

2009-01-01

SigB is an alternative sigma factor that controls a large regulon in Staphylococcus aureus. Activation of SigB requires RsbU, a protein phosphatase 2C (PP2C)-type phosphatase. In a closely related organism, Bacillus subtilis, RsbU activity is stimulated upon interaction with RsbT, a kinase, which following an activating stimulus switches from a 25S high-molecular-weight complex, the stressosome, to the N-terminal domain of RsbU. Active RsbU dephosporylates RsbV and thereby triggers the release of SigB from its inhibitory complex with RsbW. While RsbU, RsbV, RsbW, and SigB are conserved in S. aureus, proteins similar to RsbT and the components of the stressosome are not, raising the question of how RsbU activity and hence SigB activity are controlled in S. aureus. We found that in contrast to the case in B. subtilis, the induced expression of RsbU was sufficient to stimulate SigB-dependent transcription in S. aureus. However, activation of SigB-dependent transcription following alkaline stress did not lead to a clear accumulation of SigB and its regulators RsbV and RsbW or to a change in the RsbV/RsbV-P ratio in S. aureus. When expressed in B. subtilis, the S. aureus RsbU displayed a high activity even in the absence of an inducing stimulus. This high activity could be transferred to the PP2C domain of the B. subtilis RsbU protein by a fusion to the N-terminal domain of the S. aureus RsbU. Collectively, the data suggest that the activity of the S. aureus RsbU and hence SigB may be subjected to different regulation in comparison to that in B. subtilis. PMID:19201800

Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells.

PubMed

Nazarenko, Irina; Schäfer, Reinhold; Sers, Christine

2007-04-15

HRSL3 (also known as H-REV107-1) belongs to a class II tumor suppressor gene family and is downregulated in several human tumors including ovarian carcinomas. To unravel the mechanism of HRSL3 tumor suppressor action, we performed a yeast two-hybrid screen and identified the alpha-isoform of the regulatory subunit A of protein phosphatase 2A (PR65alpha) as a new interaction partner of HRSL3. Interaction between HRSL3 and PR65alpha was confirmed in vitro and by co-immunoprecipitation in mammalian cells. We demonstrate that HRSL3 binds to the endogenous PR65alpha, thereby partially sequestering the catalytic subunit PR36 from the PR65 protein complex, and inhibiting PP2A catalytic activity. Furthermore, binding of HRSL3 to PR65 induces apoptosis in ovarian carcinoma cells in a caspase-dependent manner. Using several mutant HRSL3 constructs, we identified the N-terminal proline-rich region within the HRSL3 protein as the domain that is relevant for both binding of PR65alpha and induction of programmed cell death. This suggests that the negative impact of HRSL3 onto PP2A activity is important for the HRSL3 pro-apoptotic function and indicates a role of PP2A in survival of human ovarian carcinomas. The analysis of distinct PP2A target molecules revealed PKCzeta as being involved in HRSL3 action. These data implicate HRSL3 as a signaling regulatory molecule, which is functionally involved in the oncogenic network mediating growth and survival of ovarian cancer cells.

Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana.

PubMed

Benson, Chantel L; Kepka, Michal; Wunschel, Christian; Rajagopalan, Nandhakishore; Nelson, Ken M; Christmann, Alexander; Abrams, Suzanne R; Grill, Erwin; Loewen, Michele C

2015-05-01

Abscisic acid (ABA) is a phytohormone known to mediate numerous plant developmental processes and responses to environmental stress. In Arabidopsis thaliana, ABA acts, through a genetically redundant family of ABA receptors entitled Regulatory Component of ABA Receptor (RCAR)/Pyrabactin Resistant 1 (PYR1)/Pyrabactin Resistant-Like (PYL) receptors comprised of thirteen homologues acting in concert with a seven-member set of phosphatases. The individual contributions of A. thaliana RCARs and their binding partners with respect to specific physiological functions are as yet poorly understood. Towards developing efficacious plant growth regulators selective for specific ABA functions and tools for elucidating ABA perception, a panel of ABA analogs altered specifically on positions around the ABA ring was assembled. These analogs have been used to probe thirteen RCARs and four type 2C protein phosphatases (PP2Cs) and were also screened against representative physiological assays in the model plant Arabidopsis. The 1'-O methyl ether of (S)-ABA was identified as selective in that, at physiologically relevant levels, it regulates stomatal aperture and improves drought tolerance, but does not inhibit germination or root growth. Analogs with the 7'- and 8'-methyl groups of the ABA ring replaced with bulkier groups generally retained the activity and stereoselectivity of (S)- and (R)-ABA, while alteration of the 9'-methyl group afforded an analog that substituted for ABA in inhibiting germination but neither root growth nor stomatal closure. Further in vitro testing indicated differences in binding of analogs to individual RCARs, as well as differences in the enzyme activity resulting from specific PP2Cs bound to RCAR-analog complexes. Ultimately, these findings highlight the potential of a broader chemical genetics approach for dissection of the complex network mediating ABA-perception, signaling and functionality within a given species and modifications in the future design of ABA agonists. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

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

PubMed

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

2002-03-15

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

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

PubMed

Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

2014-06-25

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

Comprehensive Logic Based Analyses of Toll-Like Receptor 4 Signal Transduction Pathway

PubMed Central

Padwal, Mahesh Kumar; Sarma, Uddipan; Saha, Bhaskar

2014-01-01

Among the 13 TLRs in the vertebrate systems, only TLR4 utilizes both Myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adapter interferon-β-inducing Factor (TRIF) adaptors to transduce signals triggering host-protective immune responses. Earlier studies on the pathway combined various experimental data in the form of one comprehensive map of TLR signaling. But in the absence of adequate kinetic parameters quantitative mathematical models that reveal emerging systems level properties and dynamic inter-regulation among the kinases/phosphatases of the TLR4 network are not yet available. So, here we used reaction stoichiometry-based and parameter independent logical modeling formalism to build the TLR4 signaling network model that captured the feedback regulations, interdependencies between signaling kinases and phosphatases and the outcome of simulated infections. The analyses of the TLR4 signaling network revealed 360 feedback loops, 157 negative and 203 positive; of which, 334 loops had the phosphatase PP1 as an essential component. The network elements' interdependency (positive or negative dependencies) in perturbation conditions such as the phosphatase knockout conditions revealed interdependencies between the dual-specific phosphatases MKP-1 and MKP-3 and the kinases in MAPK modules and the role of PP2A in the auto-regulation of Calmodulin kinase-II. Our simulations under the specific kinase or phosphatase gene-deficiency or inhibition conditions corroborated with several previously reported experimental data. The simulations to mimic Yersinia pestis and E. coli infections identified the key perturbation in the network and potential drug targets. Thus, our analyses of TLR4 signaling highlights the role of phosphatases as key regulatory factors in determining the global interdependencies among the network elements; uncovers novel signaling connections; identifies potential drug targets for infections. PMID:24699232

Molecular mechanism of serine/threonine protein phosphatase 1 (PP1cα-PP1r7) in spermatogenesis of Toxocara canis.

PubMed

Ma, Guang Xu; Zhou, Rong Qiong; Song, Zhen Hui; Zhu, Hong Hong; Zhou, Zuo Yong; Zeng, Yuan Qin

2015-09-01

Toxocariasis is one of the most important, but neglected, zoonoses, which is mainly caused by Toxocara canis. To better understand the role of serine/threonine protein phosphatase 1 (PP1) in reproductive processes of male adult T. canis, differential expression analysis was used to reveal the profiles of PP1 catalytic subunit α (PP1cα) gene Tc-stp-1 and PP1 regulatory subunit 7 (PP1r7) gene TcM-1309. Indirect fluorescence immunocytochemistry was carried out to determine the subcellular distribution of PP1cα. Double-stranded RNA interference (RNAi) assays were employed to illustrate the function and mechanism of PP1cα in male adult reproduction. Real-time quantitative PCR (qPCR) showed transcriptional consistency of Tc-stp-1 and TcM-1309 in sperm-producing germline tissues and localization research showed cytoplasmic distribution of PP1cα in sf9 cells, which indicated relevant involvements of PP1cα and PP1r7 in spermatogenesis. Moreover, spatiotemporal transcriptional differences of Tc-stp-1 were determined by gene knockdown analysis, which revealed abnormal morphologies and blocked meiotic divisions of spermatocytes by phenotypic aberration scanning, thereby highlighting the crucial involvement of PP1cα in spermatogenesis. These results revealed a PP1cα-PP1r7 mechanism by which PP1 regulates kinetochore-microtubule interactions in spermatogenesis and provided important clues to identify novel drug or vaccine targets for toxocariasis control. Copyright © 2015 Elsevier B.V. All rights reserved.

Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations

PubMed Central

Liu, Ruijie; Correll, Robert N.; Davis, Jennifer; Vagnozzi, Ronald J.; York, Allen J.; Sargent, Michelle A.; Nairn, Angus C.; Molkentin, Jeffery D.

2015-01-01

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, β and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-oxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1β) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca2+ handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1β isoform in affecting cardiac contractile function. PMID:26334248

EphB4 Receptor Tyrosine Kinase in Prostate Cancer

DTIC Science & Technology

2011-09-01

San Diego, La Jolla, CA, USAAbbreviations: MAP kinase, mitogen-activated prote tidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatid PI3K...okadaic acid (MP Biomedicals, 150 μM stock in DMSO), LY294002, PD98059, rapamycin (see previous section), dasatinib (LC Laboratories; 50 μM stock in DMSO...the phosphatase inhibitor tautomycin, which preferentially inhibits PP1 over PP2A. The cells were stimulated and analyzed as in (D). (F) Okadaic acid

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer

PubMed Central

Golden, Teresa; Aragon, Ileana V.; Rutland, Beth; Tucker, J. Allan; Shevde, Lalita A.; Samant, Rajeev S.; Zhou, Guofei; Amable, Lauren; Skarra, Danalea; Honkanen, Richard E.

2008-01-01

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 overexpression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive overexpression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model overexpression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 overexpression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 overexpression may aid tumor progression. PMID:18280813

Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

PubMed

Chellaiah, Meenakshi A; Schaller, Michael D

2009-08-01

PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

Inhibition of serine/threonine phosphatase PP2A enhances cancer chemotherapy by blocking DNA damage induced defense mechanisms.

PubMed

Lu, Jie; Kovach, John S; Johnson, Francis; Chiang, Jeffrey; Hodes, Richard; Lonser, Russell; Zhuang, Zhengping

2009-07-14

A variety of mechanisms maintain the integrity of the genome in the face of cell stress. Cancer cell response to chemotherapeutic and radiation-induced DNA damage is mediated by multiple defense mechanisms including polo-like kinase 1 (Plk-1), protein kinase B (Akt-1), and/or p53 pathways leading to either apoptosis or cell cycle arrest. Subsequently, a subpopulation of arrested viable cancer cells may remain and recur despite aggressive and repetitive therapy. Here, we show that modulation (activation of Akt-1 and Plk-1 and repression of p53) of these pathways simultaneously results in paradoxical enhancement of the effectiveness of cytotoxic chemotherapy. We demonstrate that a small molecule inhibitor, LB-1.2, of protein phosphatase 2A (PP2A) activates Plk-1 and Akt-1 and decreases p53 abundance in tumor cells. Combined with temozolomide (TMZ; a DNA-methylating chemotherapeutic drug), LB-1.2 causes complete regression of glioblastoma multiforme (GBM) xenografts without recurrence in 50% of animals (up to 28 weeks) and complete inhibition of growth of neuroblastoma (NB) xenografts. Treatment with either drug alone results in only short-term inhibition/regression with all xenografts resuming rapid growth. Combined with another widely used anticancer drug, Doxorubicin (DOX, a DNA intercalating agent), LB-1.2 also causes marked GBM xenograft regression, whereas DOX alone only slows growth. Inhibition of PP2A by LB-1.2 blocks cell-cycle arrest and increases progression of cell cycle in the presence of TMZ or DOX. Pharmacologic inhibition of PP2A may be a general method for enhancing the effectiveness of cancer treatments that damage DNA or disrupt components of cell replication.

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

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth

PubMed Central

Sugimoto, Hiroki; Kondo, Satoshi; Tanaka, Tomoko; Imamura, Chie; Muramoto, Nobuhiko; Hattori, Etsuko; Ogawa, Ken’ichi; Mitsukawa, Norihiro; Ohto, Chikara

2014-01-01

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass. PMID:25038254

PP2A: more than a reset switch to activate pRB proteins during the cell cycle and in response to signaling cues

PubMed Central

Kurimchak, Alison; Graña, Xavier

2015-01-01

In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.

Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum.

PubMed

Miyazaki, S; Koga, R; Bohnert, H J; Fukuhara, T

1999-03-01

Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue specific. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers. Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature flowers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress. Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four full-length transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs. Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.

PP2A inhibition assay using recombinant enzyme for rapid detection of okadaic acid and its analogs in shellfish.

PubMed

Ikehara, Tsuyoshi; Imamura, Shihoko; Yoshino, Atsushi; Yasumoto, Takeshi

2010-01-01

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC(50) values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 μg/g and 0.0611 μg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 μg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097.

Recombinant α- β- and γ-Synucleins Stimulate Protein Phosphatase 2A Catalytic Subunit Activity in Cell Free Assays

PubMed Central

Lek, Sovanarak; Vargas-Medrano, Javier; Villanueva, Ernesto; Marcus, Brian; Godfrey, Wesley; Perez, Ruth G.

2017-01-01

α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo. PMID:28829427

Molecular cloning, expression and single nucleotide polymorphisms of protein phosphatase 1 (PP1) in mandarin fish (Siniperca chuatsi).

PubMed

Cheng, Xiao-Yan; He, Shan; Liang, Xu-Fang; Song, Yi; Yuan, Xiao-Chen; Li, Ling; Wen, Zheng-Yong; Cai, Wen-Jing; Tao, Ya-Xiong

2015-11-01

In the wild, mandarin fish (Siniperca chuatsi) only feed on live prey fish, refusing dead prey. When reared in ponds, training will result in some mandarin fish accepting artificial diets. However, little is currently known about the molecular mechanism of the individual difference. Serine/threonine protein phosphatase 1 (PP1) is a suppressor of learning and long-term memory (LTM) in mammals. In the present study, the relationship between PP1 and the individual difference in acceptance of artificial diets in mandarin fish was investigated. The complete CDS (coding sequence) of four PP1 isoforms (PP1caa, PP1cab, PP1cb and PP1cc) were cloned in mandarin fish. The amino acid sequences of these PP1 isoforms are highly conserved in different species. The mRNA expressions of PP1caa and PP1cb in brain of artificial diet feeders were significantly higher than those in nonfeeders, suggesting the deficiency in the maintenance of long-term memory of its natural food habit (live prey fish). The SNP loci in PP1caa and PP1cb were also found to be associated with the individual difference in acceptance of artificial diets in mandarin fish. These SNPs of PP1caa and PP1cb genes could be useful markers for gene-associated breeding of mandarin fish, which could accept artificial diets. In conclusion, different mRNA expression and SNPs of PP1caa and PP1cb genes in feeders and nonfeeders of artificial diets might contribute to understanding the molecular mechanism of individual difference in acceptance of artificial diets in mandarin fish. Copyright © 2015 Elsevier Inc. All rights reserved.

Pin1At regulates PIN1 polar localization and root gravitropism.

PubMed

Xi, Wanyan; Gong, Ximing; Yang, Qiaoyun; Yu, Hao; Liou, Yih-Cherng

2016-01-21

Root gravitropism allows plants to establish root systems and its regulation depends on polar auxin transport mediated by PIN-FORMED (PIN) auxin transporters. PINOID (PID) and PROTEIN PHOSPHATASE 2A (PP2A) act antagonistically on reversible phosphorylation of PINs. This regulates polar PIN distribution and auxin transport. Here we show that a peptidyl-prolyl cis/trans isomerase Pin1At regulates root gravitropism. Downregulation of Pin1At suppresses root agravitropic phenotypes of pp2aa and 35S:PID, while overexpression of Pin1At affects root gravitropic responses and enhances the pp2aa agravitropic phenotype. Pin1At also affects auxin transport and polar localization of PIN1 in stele cells, which is mediated by PID and PP2A. Furthermore, Pin1At catalyses the conformational change of the phosphorylated Ser/Thr-Pro motifs of PIN1. Thus, Pin1At mediates the conformational dynamics of PIN1 and affects PID- and PP2A-mediated regulation of PIN1 polar localization, which correlates with the regulation of root gravitropism.

Pin1At regulates PIN1 polar localization and root gravitropism

PubMed Central

Xi, Wanyan; Gong, Ximing; Yang, Qiaoyun; Yu, Hao; Liou, Yih-Cherng

2016-01-01

Root gravitropism allows plants to establish root systems and its regulation depends on polar auxin transport mediated by PIN-FORMED (PIN) auxin transporters. PINOID (PID) and PROTEIN PHOSPHATASE 2A (PP2A) act antagonistically on reversible phosphorylation of PINs. This regulates polar PIN distribution and auxin transport. Here we show that a peptidyl-prolyl cis/trans isomerase Pin1At regulates root gravitropism. Downregulation of Pin1At suppresses root agravitropic phenotypes of pp2aa and 35S:PID, while overexpression of Pin1At affects root gravitropic responses and enhances the pp2aa agravitropic phenotype. Pin1At also affects auxin transport and polar localization of PIN1 in stele cells, which is mediated by PID and PP2A. Furthermore, Pin1At catalyses the conformational change of the phosphorylated Ser/Thr-Pro motifs of PIN1. Thus, Pin1At mediates the conformational dynamics of PIN1 and affects PID- and PP2A-mediated regulation of PIN1 polar localization, which correlates with the regulation of root gravitropism. PMID:26791759

Alterations in GluR2 AMPA receptor phosphorylation at serine 880 following group I metabotropic glutamate receptor stimulation in the rat dorsal striatum.

PubMed

Ahn, Sung Min; Choe, Eun Sang

2010-04-01

Phosphorylation of ionotropic glutamate receptors in the brain plays a crucial role in the regulation of synaptic plasticity. In this study, we investigated the regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor phosphorylation by the stimulation of group I metabotropic glutamate receptors (mGluRs) in the dorsal striatum in vivo. The results showed that intrastriatal infusion of the group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG, 250 nmol), enhanced the sensitivity of GluR2 subunit in its phosphorylation at serine 880 (S880) in the dorsal striatum. This enhancement of the sensitivity of GluR2-S880 phosphorylation was reduced by blocking group I mGluRs and N-methyl-D-aspartate (NMDA) receptors. Similar reduction of the enhancement was also induced by inhibiting phospholipase C (PLC), calcium/calmodulin-dependent protein kinase (CaMK), c-Jun N-terminal kinase (JNK), and protein kinase C (PKC). Inhibition of protein phosphatase (PP) 1/2A and calcineurin (PP2B) alone enhanced GluR2-S880 phosphorylation in the dorsal striatum, whereas inhibition of these phosphatases did not further enhance the S880 phosphorylation by DHPG stimulation. In addition, inhibition of PP1/2A or PP2B also enhanced the phosphorylation of CaMKII, JNK and PKC. These data suggest that the phosphorylation of AMPA receptor GluR2 subunit at S880 is subject to the upregulation by the stimulation of group I mGluRs. Interactions among glutamate receptors, protein kinases, and PPs participate in this upregulation. (c) 2009 Wiley-Liss, Inc.

DELAY OF GERMINATION1 requires PP2C phosphatases of the ABA signalling pathway to control seed dormancy.

PubMed

Née, Guillaume; Kramer, Katharina; Nakabayashi, Kazumi; Yuan, Bingjian; Xiang, Yong; Miatton, Emma; Finkemeier, Iris; Soppe, Wim J J

2017-07-13

The time of seed germination is a major decision point in the life of plants determining future growth and development. This timing is controlled by seed dormancy, which prevents germination under favourable conditions. The plant hormone abscisic acid (ABA) and the protein DELAY OF GERMINATION 1 (DOG1) are essential regulators of dormancy. The function of ABA in dormancy is rather well understood, but the role of DOG1 is still unknown. Here, we describe four phosphatases that interact with DOG1 in seeds. Two of them belong to clade A of type 2C protein phosphatases: ABA-HYPERSENSITIVE GERMINATION 1 (AHG1) and AHG3. These phosphatases have redundant but essential roles in the release of seed dormancy epistatic to DOG1. We propose that the ABA and DOG1 dormancy pathways converge at clade A of type 2C protein phosphatases.The DOG1 protein is a major regulator of seed dormancy in Arabidopsis. Here, Née et al. provide evidence that DOG1 can interact with the type 2C protein phosphatases AHG1 and AHG3 and that this represents the convergence point of the DOG1-regulated dormancy pathway and signalling by the plant hormone abscisic acid.

Therapeutic benefits of a component of coffee in a rat model of Alzheimer's disease.

PubMed

Basurto-Islas, Gustavo; Blanchard, Julie; Tung, Yunn Chyn; Fernandez, Jose R; Voronkov, Michael; Stock, Maxwell; Zhang, Sherry; Stock, Jeffry B; Iqbal, Khalid

2014-12-01

A minor component of coffee unrelated to caffeine, eicosanoyl-5-hydroxytryptamide (EHT), provides protection in a rat model for Alzheimer's disease (AD). In this model, viral expression of the phosphoprotein phosphatase 2A (PP2A) endogenous inhibitor, the I2(PP2A), or SET protein in the brains of rats leads to several characteristic features of AD including cognitive impairment, tau hyperphosphorylation, and elevated levels of cytoplasmic amyloid-β protein. Dietary supplementation with EHT for 6-12 months resulted in substantial amelioration of all these defects. The beneficial effects of EHT could be associated with its ability to increase PP2A activity by inhibiting the demethylation of its catalytic subunit PP2Ac. These findings raise the possibility that EHT may make a substantial contribution to the apparent neuroprotective benefits associated with coffee consumption as evidenced by numerous epidemiologic studies indicating that coffee drinkers have substantially lowered risk of developing AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Therapeutic benefits of a component of coffee in a rat model of Alzheimer disease1

PubMed Central

Basurto-Islas, Gustavo; Blanchard, Julie; Tung, Yunn Chyn; Fernandez, Jose R.; Voronkov, Michael; Stock, Maxwell; Zhang, Sherry; Stock, Jeffry B.; Iqbal, Khalid

2014-01-01

A minor component of coffee unrelated to caffeine, eicosanoyl-5-hydroxytryptamide (EHT) provides protection in a rat model for Alzheimer’s disease (AD). In this model, viral expression of the phosphoprotein phosphatase 2A (PP2A) endogenous inhibitor, the I2PP2A or SET protein in the brains of rats leads to several characteristic features of AD including cognitive impairment, tau hyperphosphorylation, and elevated levels of cytoplasmic β-amyloid protein. Dietary supplementation with EHT for 6–12 months resulted in substantial amelioration of all of these defects. The beneficial effects of EHT could be associated with its ability to increase PP2A activity by inhibiting the demethylation of its catalytic subunit PP2Ac. These findings raise the possibility that EHT may make a substantial contribution to the apparent neuroprotective benefits associated with coffee consumption as evidenced by numerous epidemiological studies indicating the coffee drinkers have substantially lowered risk of developing AD. PMID:25034344

Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

PubMed Central

Garbe, David S.; Fang, Yanshan; Zheng, Xiangzhong; Sowcik, Mallory; Anjum, Rana; Gygi, Steven P.; Sehgal, Amita

2013-01-01

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per01 arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period. PMID:24086144

Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

PubMed

Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

2018-05-04

The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity.

PubMed

Mine, Akira; Berens, Matthias L; Nobori, Tatsuya; Anver, Shajahan; Fukumoto, Kaori; Winkelmüller, Thomas M; Takeda, Atsushi; Becker, Dieter; Tsuda, Kenichi

2017-07-11

Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1 , HAI2 , and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato ( Pto ) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

Curcumin Modulates the NMDA Receptor Subunit Composition Through a Mechanism Involving CaMKII and Ser/Thr Protein Phosphatases.

PubMed

Mallozzi, Cinzia; Parravano, Mariacristina; Gaddini, Lucia; Villa, Marika; Pricci, Flavia; Malchiodi-Albedi, Fiorella; Matteucci, Andrea

2018-05-30

Curcumin is one of the major compounds contained in turmeric, the powdered rhizome of Curcuma longa. Results obtained in various experimental models indicate that curcumin has the potential to treat a large variety of neuronal diseases. Excitotoxicity, the toxicity due to pathological glutamate receptors stimulation, has been considered to be involved in several ocular pathologies including ischemia, glaucoma, and diabetic retinopathy. The NMDA receptor (NMDAR), a heteromeric ligand-gated ion channel, is composed of GluN1 and GluN2 subunits. There are four GluN2 subunits (GluN2A-D), which are major determinants of the functional properties of NMDARs. It is widely accepted that GluN2B has a pivotal role in excitotoxicity while the role of GluN2A remains controversial. We previously demonstrated that curcumin is neuroprotective against NMDA-induced excitotoxicity with a mechanism involving an increase of GluN2A subunit activity. In this paper, we investigate the mechanisms involved in curcumin-induced GluN2A increase in retinal cultures. Our results show that curcumin treatment activated CaMKII with a time-course that paralleled those of GluN2A increase. Moreover, KN-93, a CaMKII inhibitor, was able to block the effect of curcumin on GluN2A expression. Finally, in our experimental model, curcumin reduced ser/thr phosphatases activity. Using okadaic acid, a specific PP1 and PP2A blocker, we observed an increase in GluN2A levels in cultures. The ability of okadaic acid to mimic the effect of curcumin on GluN2A expression suggests that curcumin might regulate GluN2A expression through a phosphatase-dependent mechanism. In conclusion, our findings indicate curcumin modulation of CaMKII and/or ser/thr phosphatases activities as a mechanism involved in GluN2A expression and neuroprotection against excitotoxicity.

Inflammatory stimuli promote growth and invasion of pancreatic cancer cells through NF-κB pathway dependent repression of PP2Ac

PubMed Central

Tao, Min; Liu, Lu; Shen, Meng; Zhi, Qiaoming; Gong, Fei-Ran; Zhou, Binhua P.; Wu, Yadi; Liu, Haiyan; Chen, Kai; Shen, Bairong; Wu, Meng-Yao; Shou, Liu-Mei; Li, Wei

2016-01-01

ABSTRACT Previous studies have indicated that inflammatory stimulation represses protein phosphatase 2A (PP2A), a well-known tumor suppressor. However, whether PP2A repression participates in pancreatic cancer progression has not been verified. We used lipopolysaccharide (LPS) and macrophage-conditioned medium (MCM) to establish in vitro inflammation models, and investigated whether inflammatory stimuli affect pancreatic cancer cell growth and invasion PP2A catalytic subunit (PP2Ac)-dependently. Via nude mouse models of orthotopic tumor xenografts and dibutyltin dichloride (DBTC)-induced chronic pancreatitis, we evaluated the effect of an inflammatory microenvironment on PP2Ac expression in vivo. We cloned the PP2Acα and PP2Acβ isoform promoters to investigate the PP2Ac transcriptional regulation mechanisms. MCM accelerated pancreatic cancer cell growth; MCM and LPS promoted cell invasion. DBTC promoted xenograft growth and metastasis, induced tumor-associated macrophage infiltration, promoted angiogenesis, activated the nuclear factor-κB (NF-κB) pathway, and repressed PP2Ac expression. In vitro, LPS and MCM downregulated PP2Ac mRNA and protein. PP2Acα overexpression attenuated JNK, ERK, PKC, and IKK phosphorylation, and impaired LPS/MCM-stimulated cell invasion and MCM-promoted cell growth. LPS and MCM activated the NF-κB pathway in vitro. LPS and MCM induced IKK and IκB phosphorylation, leading to p65/RelA nuclear translocation and transcriptional activation. Overexpression of the dominant negative forms of IKKα attenuated LPS and MCM downregulation of PP2Ac, suggesting inflammatory stimuli repress PP2Ac expression NF-κB pathway–dependently. Luciferase reporter gene assay verified that LPS and MCM downregulated PP2Ac transcription through an NF-κB–dependent pathway. Our study presents a new mechanism in inflammation-driven cancer progression through NF-κB pathway–dependent PP2Ac repression. PMID:26761431

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

PubMed Central

Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A.; De Wever, Veerle; Morrice, Nick A.; Meek, Katheryn; Lees-Miller, Susan P.

2014-01-01

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ. PMID:24844881

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.

[Tartrate-resistant acid phosphatase in free-living Amoeba proteus].

PubMed

Sopina, V A

2002-01-01

Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

X-linked microtubule-associated protein, Mid1, regulates axon development

PubMed Central

Lu, Tingjia; Chen, Renchao; Cox, Timothy C.; Moldrich, Randal X.; Kurniawan, Nyoman; Tan, Guohe; Perry, Jo K.; Ashworth, Alan; Bartlett, Perry F.; Xu, Li; Zhang, Jing; Lu, Bin; Wu, Mingyue; Shen, Qi; Liu, Yuanyuan; Richards, Linda J.; Xiong, Zhiqi

2013-01-01

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS. PMID:24194544

X-linked microtubule-associated protein, Mid1, regulates axon development.

PubMed

Lu, Tingjia; Chen, Renchao; Cox, Timothy C; Moldrich, Randal X; Kurniawan, Nyoman; Tan, Guohe; Perry, Jo K; Ashworth, Alan; Bartlett, Perry F; Xu, Li; Zhang, Jing; Lu, Bin; Wu, Mingyue; Shen, Qi; Liu, Yuanyuan; Richards, Linda J; Xiong, Zhiqi

2013-11-19

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.

Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

PubMed Central

Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

2011-01-01

Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-Å resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases. PMID:22160701

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

PubMed Central

Bavaria, Mitul N.; Jin, Shi; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of protein phosphatase 2A (PP2Ac) formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells. PMID:24253595

Structural basis and functions of abscisic acid receptors PYLs

PubMed Central

Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

2015-01-01

Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

PubMed Central

Davis, Ryan J.; Swanger, Jherek; Hughes, Bridget T.

2017-01-01

ABSTRACT Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic. PMID:28137908

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

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)more » 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 PP2A/MMP-2 induced PCB153-induced dysfunction of occludin. • Disrupted lipid rafts modulated PCB153-induced increase of permeability. • Lipid rafts act as a signaling platform for PCB153-induced dysfunction of occludin.« less

Transcriptome analyses of chronic traumatic encephalopathy show alterations in protein phosphatase expression associated with tauopathy

PubMed Central

Seo, Jeong-Sun; Lee, Seungbok; Shin, Jong-Yeon; Hwang, Yu Jin; Cho, Hyesun; Yoo, Seong-Keun; Kim, Yunha; Lim, Sungsu; Kim, Yun Kyung; Hwang, Eun Mi; Kim, Su Hyun; Kim, Chong-Hyun; Hyeon, Seung Jae; Yun, Ji-Young; Kim, Jihye; Kim, Yona; Alvarez, Victor E; Stein, Thor D; Lee, Junghee; Kim, Dong Jin; Kim, Jong-Il; Kowall, Neil W; Ryu, Hoon; McKee, Ann C

2017-01-01

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder that is associated with repetitive head injury and has distinctive neuropathological features that differentiate this disease from other neurodegenerative diseases. Intraneuronal tau aggregates, although they occur in different patterns, are diagnostic neuropathological features of CTE, but the precise mechanism of tauopathy is not known in CTE. We performed whole RNA sequencing analysis of post-mortem brain tissue from patients with CTE and compared the results to normal controls to determine the transcriptome signature changes associated with CTE. The results showed that the genes related to the MAP kinase and calcium-signaling pathways were significantly downregulated in CTE. The altered expression of protein phosphatases (PPs) in these networks further suggested that the tauopathy observed in CTE involves common pathological mechanisms similar to Alzheimer's disease (AD). Using cell lines and animal models, we also showed that reduced PPP3CA/PP2B phosphatase activity is directly associated with increases in phosphorylated (p)-tau proteins. These findings provide important insights into PP-dependent neurodegeneration and may lead to novel therapeutic approaches to reduce the tauopathy associated with CTE. PMID:28524178

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

PubMed

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

2017-01-01

SET (SE translocation, SET), a constitutive inhibitor of protein phosphatase 2A (PP2A), is a multifunctional oncoprotein involved in DNA replication, histone modification, nucleosome assembly, gene transcription and cell proliferation. It is widely expressed in human tissues including the gonadal system and brain. Intensive studies have shown that overexpressed SET plays an important role in the development of Alzheimer's disease (AD), and may also contribute to the malignant transformation of breast and ovarian cancers. Recent studies indicated that through interaction with PP2A, SET may upregulate androgen biosynthesis and contribute to hyperandrogenism in polycystic ovary syndrome (PCOS) patients. This review article summarizes data concerning the SET expression in ovaries from PCOS and normal women, and analyzes the role/regulatory mechanism of SET for androgen biosynthesis in PCOS, as well as the significance of this action in the development of PCOS. The potential value of SET-triggered pathway as a therapeutic target and the application of anti-SET reagents for treating hyperandrogenism in PCOS patients are also discussed. Copyright © 2016. Published by Elsevier B.V.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

PubMed Central

Levasseur, Mark; Dumollard, Remi; Chambon, Jean-Philippe; Hebras, Celine; Sinclair, Maureen; Whitaker, Michael; McDougall, Alex

2013-01-01

The fertilising sperm triggers a transient Ca2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/Ccdc20 inhibitor, links release from metaphase II arrest with the Ca2+ transient and its degradation is triggered by the Ca2+-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca2+-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates. PMID:24194472

Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

PubMed

Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

Differential effects of phosphotyrosine phosphatase expression on hormone-dependent and independent pp60c-src activity.

PubMed

Way, B A; Mooney, R A

1994-10-26

pp60c-src kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443-23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependent activation of pp60c-src but failed to increase hormone independent (basal) pp60c-src activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60c-src was not detected in response to PDGF or in PTPase+ cells. PDGF increased the intrinsic tyrosine kinase activity of pp60c-src in both control and PTPase+ cells, but the effect was smaller in PTPase+ cells. In an in vitro assay, hormone-stimulated pp60c-src autophosphorylation from PTPase+ cells was decreased 64 +/- 22%, and substrate phosphorylation by pp60c-src was reduced 54 +/- 16% compared to controls. Hormone-independent pp60c-src kinase activity was unchanged by expression of the PTPase. pp60c-src was, however, an in vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition, in vitro dephosphorylation by CD45 increased pp60c-src activity. These findings suggest that the PDGF receptor was an in vivo substrate of CD45 but pp60c-src was not. The lack of activation of pp60c-src in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.

Protein Phosphatase-1 regulates Rift Valley fever virus replication.

PubMed

Baer, Alan; Shafagati, Nazly; Benedict, Ashwini; Ammosova, Tatiana; Ivanov, Andrey; Hakami, Ramin M; Terasaki, Kaori; Makino, Shinji; Nekhai, Sergei; Kehn-Hall, Kylene

2016-03-01

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

PubMed

Sugimoto, Hiroki; Kondo, Satoshi; Tanaka, Tomoko; Imamura, Chie; Muramoto, Nobuhiko; Hattori, Etsuko; Ogawa, Ken'ichi; Mitsukawa, Norihiro; Ohto, Chikara

2014-10-01

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Andrographolide Enhances Nuclear Factor-κB Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells*

PubMed Central

Hsieh, Cheng Y.; Hsu, Ming J.; Hsiao, George; Wang, Yi H.; Huang, Chi W.; Chen, Shiuan W.; Jayakumar, Thanasekaran; Chiu, Pei T.; Chiu, Yi H.; Sheu, Joen R.

2011-01-01

Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs. Andrographolide also inhibited LPS/IFN-γ-induced p65 nuclear translocation, DNA binding activity, p65 Ser536 phosphorylation, and NF-κB reporter activity. However, IKK phosphorylation and downstream inhibitory κBα phosphorylation and degradation were not altered by the presence of andrographolide in LPS/IFN-γ-stimulated VSMCs. These andrographolide inhibitory actions could be prevented by selective inhibition of neutral sphingomyelinase and protein phosphatase 2A (PP2A). Furthermore, andrographolide was demonstrated to increase ceramide formation and PP2A activity in VSMCs and to inhibit neointimal formation in rat carotid injury models. These results suggest that andrographolide caused neutral sphingomyelinase-mediated ceramide formation and PP2A activation to dephosphorylate p65 Ser536, leading to NF-κB inactivation and subsequent inducible nitric-oxide synthase down-regulation in rat VSMCs stimulated by LPS and IFN-γ. PMID:21169355

Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit, RCN1.

PubMed

Larsen, Paul Brian; Cancel, Jesse Daniel

2003-06-01

Ethylene signaling in Arabidopsis begins with a family of five ethylene receptors that regulate the activity of the Raf-like kinase, CTR1. Recent work to identify novel factors required for modulating ethylene signaling resulted in the isolation of enhanced ethylene response 1 (eer1), a mutant that displays both increased sensitivity and increased amplitude of response to ethylene. Molecular cloning of eer1 reveals that its mutant phenotype results from a loss-of-function mutation in the previously characterized RCN1, one of three PP2A A regulatory subunits in Arabidopsis. Our analysis shows that neither RCN1 expression nor PP2A activity is regulated by ethylene. Instead, we found that Arabidopsis PP2A-1C, a PP2A catalytic subunit previously characterized as interacting with RCN1, associates strongly with the kinase domain of CTR1 in vitro. This likely represents a role for PP2A in modulation of CTR1 activity because an in vitro kinase assay did not reveal phosphorylation of either RCN1 or PP2A-1C by CTR1, indicating that neither of them is a substrate for CTR1. PP2A activity is required for Ras-dependent activation of mammalian Raf, with reductions in PP2A activity significantly compromising the effectiveness of this mechanism. Our genetic and biochemical results suggest that a similar requirement for PP2A activity exists for ethylene signaling, with loss-of-function mutations affecting PP2A activity possibly reducing the effectiveness of CTR1 activation, thus lowering the threshold required for manifestation of ethylene response.

PP2A regulates SCF-induced cardiac stem cell migration through interaction with p38 MAPK.

PubMed

Wang, Ying; Xia, Yanli; Kuang, Dong; Duan, Yaqi; Wang, Guoping

2017-12-15

Previous studies have shown that stem cell factor (SCF) induces the migration of cardiac stem cells (CSCs) and helps to repair myocardial infarctions. Earlier studies on the migration mechanism only focused on the activation of kinases; here, we aimed to explore the functional role of protein phosphatase 2A (PP2A) in SCF-induced CSC migration. CSCs were treated with SCF, PP2A enzymatic activity was measured, the phosphorylation levels of PP2A, p38 MAPK and cofilin were evaluated using western blot. Transwell assay was used to determine the migratory ability of CSCs. In vitro, SCF induced the phosphorylation of p38 MAPK and cofilin, leading to the migration of CSCs. Cofilin acted as a downstream signal of p38 MAPK. PP2A was involved in this process. Further studies revealed that PP2A was inactivated via phosphorylation at Tyr307 by SCF and the inactivation/phosphorylation was mediated by activated p38 MAPK, as p38 MAPK inhibitor SB203580 or siRNA prevented SCF-induced inactivation and phosphorylation of PP2A. When CSCs were pretreated with PP2A inhibitor (okadaic acid, OA), SCF-induced CSC migration and the downstream signals were enhanced, and the enhancement was reversed when p38 MAPK was blocked. Additionally, co-immunoprecipitation showed a direct interaction of PP2A with p38 MAPK. Our results indicated that PP2A regulated the SCF-induced activation of p38 MAPK/cofilin signaling pathway and subsequent migration of CSCs by interaction with p38 MAPK. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2013-01-01

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 Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-PKC–induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F 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 Jak2V617F 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 Jak2V617F MPNs. PMID:23926298

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

PubMed

Ren, Dapeng; Fisher, Laura A; Zhao, Jing; Wang, Ling; Williams, Byron C; Goldberg, Michael L; Peng, Aimin

2017-06-16

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target

PubMed Central

Li, Jing; Han, Sufang; Qian, Ziliang; Su, Xinying; Fan, Shuqiong; Fu, Jiangang; Liu, Yuanjie; Yin, Xiaolu; Gao, Zeren; Zhang, Jingchuan; Yu, De-Hua; Ji, Qunsheng

2014-01-01

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs. PMID:24253382

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

Carfilzomib induces leukaemia cell apoptosis via inhibiting ELK1/KIAA1524 (Elk-1/CIP2A) and activating PP2A not related to proteasome inhibition.

PubMed

Liu, Chun-Yu; Hsieh, Feng-Shu; Chu, Pei-Yi; Tsai, Wen-Chun; Huang, Chun-Teng; Yu, Yuan-Bin; Huang, Tzu-Ting; Ko, Po-Shen; Hung, Man-Hsin; Wang, Wan-Lun; Shiau, Chung-Wai; Chen, Kuen-Feng

2017-06-01

Enhancing the tumour suppressive activity of protein phosphatase 2A (PP2A) has been suggested to be an anti-leukaemic strategy. KIAA1524 (also termed CIP2A), an oncoprotein inhibiting PP2A, is associated with disease progression in chronic myeloid leukaemia and may be prognostic in cytogenetically normal acute myeloid leukaemia. Here we demonstrated that the selective proteasome inhibitor, carfilzomib, induced apoptosis in sensitive primary leukaemia cells and in sensitive leukaemia cell lines, associated with KIAA1524 protein downregulation, increased PP2A activity and decreased p-Akt, but not with the proteasome inhibition effect of carfilzomib. Ectopic expression of KIAA1524, or pretreatment with the PP2A inhibitor, okadaic acid, suppressed carfilzomib-induced apoptosis and KIAA1524 downregulation in sensitive cells, whereas co-treatment with the PP2A agonist, forskolin, enhanced carfilzomib-induced apoptosis in resistant cells. Mechanistically, carfilzomib affected KIAA1524 transcription through disturbing ELK1 (Elk-1) binding to the KIAA1524 promoter. Moreover, the drug sensitivity and mechanism of carfilzomib in xenograft mouse models correlated well with the effects of carfilzomib on KIAA1524 and p-Akt expression, as well as PP2A activity. Our data disclosed a novel drug mechanism of carfilzomib in leukaemia cells and suggests the potential therapeutic implication of KIAA1524 in leukaemia treatment. © 2017 John Wiley & Sons Ltd.

[Preparation and activity validation of PP7 bacteriophage-like particles displaying PAP114-128 peptide].

PubMed

Sun, Yanli; Sun, Yanhua

2016-10-01

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

Two Bistable Switches Govern M Phase Entry.

PubMed

Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla

2016-12-19

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Computational study of the covalent bonding of microcystins to cysteine residues--a reaction involved in the inhibition of the PPP family of protein phosphatases.

PubMed

Pereira, Susana R; Vasconcelos, Vítor M; Antunes, Agostinho

2013-01-01

Microcystins (MCs) are cyclic peptides, produced by cyanobacteria, that are hepatotoxic to mammals. The toxicity mechanism involves the potent inhibition of protein phosphatases, as the toxins bind the catalytic subunits of five enzymes of the phosphoprotein phosphatase (PPP) family of serine/threonine-specific phosphatases: Ppp1 (aka PP1), Ppp2 (aka PP2A), Ppp4, Ppp5 and Ppp6. The interaction with the proteins includes the formation of a covalent bond with a cysteine residue. Although this reaction seems to be accessory for the inhibition of PPP enzymes, it has been suggested to play an important part in the biological role of MCs and furthermore is involved in their nonenzymatic conjugation to glutathione. In this study, the molecular interaction of microcystins with their targeted PPP catalytic subunits is reviewed, including the relevance of the covalent bond for overall inhibition. The chemical reaction that leads to the formation of the covalent bond was evaluated in silico, both thermodynamically and kinetically, using quantum mechanical-based methods. As a result, it was confirmed to be a Michael-type addition, with simultaneous abstraction of the thiol hydrogen by a water molecule, transfer of hydrogen from the water to the α,β-unsaturated carbonyl group of the microcystin and addition of the sulfur to the β-carbon of the microcystin moiety. The calculated kinetics are in agreement with previous experimental results that had indicated the reaction to occur in a second step after a fast noncovalent interaction that inhibited the enzymes per se. © 2011 The Authors Journal compilation © 2011 FEBS.

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes

PubMed Central

Cheng, Tsing; Orlow, Seth J.; Manga, Prashiela

2013-01-01

Summary Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of proapoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis[C][W

PubMed Central

Spartz, Angela K.; Ren, Hong; Park, Mee Yeon; Grandt, Kristin N.; Lee, Sang Ho; Murphy, Angus S.; Sussman, Michael R.; Overvoorde, Paul J.; Gray, William M.

2014-01-01

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H+-ATPases (PM H+-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H+-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H+-ATPase function. We find that SAUR19 stimulates PM H+-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H+-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H+-ATPases, and negatively regulate PM H+-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion. PMID:24858935

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

PubMed

Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

PubMed

Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

2015-08-01

The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

Insulin deprivation induces PP2A inhibition and tau hyperphosphorylation in hTau mice, a model of Alzheimer’s disease-like tau pathology

PubMed Central

Gratuze, Maud; Julien, Jacinthe; Petry, Franck R.; Morin, Françoise; Planel, Emmanuel

2017-01-01

Abnormally hyperphosphorylated tau aggregated as intraneuronal neurofibrillary tangles is one of the two neuropathological hallmarks of Alzheimer’s disease (AD). The majority of AD cases are sporadic with numerous environmental, biological and genetic risks factors. Interestingly, insulin dysfunction and hyperglycaemia are both risk factors for sporadic AD. However, how hyperglycaemia and insulin dysfunction affect tau pathology, is not well understood. In this study, we examined the effects of insulin deficiency on tau pathology in transgenic hTau mice by injecting different doses of streptozotocin (STZ), a toxin that destroys insulin-producing cells in the pancreas. One high dose of STZ resulted in marked diabetes, and five low doses led to a milder diabetes. Both groups exhibited brain tau hyperphosphorylation but no increased aggregation. Tau hyperphosphorylation correlated with inhibition of Protein Phosphatase 2A (PP2A), the main tau phosphatase. Interestingly, insulin injection 30 minutes before sacrifice partially restored tau phosphorylation to control levels in both STZ-injected groups. Our results confirm a link between insulin homeostasis and tau phosphorylation, which could explain, at least in part, a higher incidence of AD in diabetic patients. PMID:28402338

Insulin deprivation induces PP2A inhibition and tau hyperphosphorylation in hTau mice, a model of Alzheimer's disease-like tau pathology.

PubMed

Gratuze, Maud; Julien, Jacinthe; Petry, Franck R; Morin, Françoise; Planel, Emmanuel

2017-04-12

Abnormally hyperphosphorylated tau aggregated as intraneuronal neurofibrillary tangles is one of the two neuropathological hallmarks of Alzheimer's disease (AD). The majority of AD cases are sporadic with numerous environmental, biological and genetic risks factors. Interestingly, insulin dysfunction and hyperglycaemia are both risk factors for sporadic AD. However, how hyperglycaemia and insulin dysfunction affect tau pathology, is not well understood. In this study, we examined the effects of insulin deficiency on tau pathology in transgenic hTau mice by injecting different doses of streptozotocin (STZ), a toxin that destroys insulin-producing cells in the pancreas. One high dose of STZ resulted in marked diabetes, and five low doses led to a milder diabetes. Both groups exhibited brain tau hyperphosphorylation but no increased aggregation. Tau hyperphosphorylation correlated with inhibition of Protein Phosphatase 2A (PP2A), the main tau phosphatase. Interestingly, insulin injection 30 minutes before sacrifice partially restored tau phosphorylation to control levels in both STZ-injected groups. Our results confirm a link between insulin homeostasis and tau phosphorylation, which could explain, at least in part, a higher incidence of AD in diabetic patients.

Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres

PubMed Central

Tanno, Yuji; Kitajima, Tomoya S.; Honda, Takashi; Ando, Yasuto; Ishiguro, Kei-ichiro; Watanabe, Yoshinori

2010-01-01

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. We demonstrate that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, we identified the phosphorylation of hSgo2 by Aurora B at the N-terminal coiled-coil region and the middle region, and showed that these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, our study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells. PMID:20889715

Negative Regulation of Abscisic Acid Signaling by the Fagus sylvatica FsPP2C1 Plays A Role in Seed Dormancy Regulation and Promotion of Seed Germination1

PubMed Central

González-García, Mary Paz; Rodríguez, Dolores; Nicolás, Carlos; Rodríguez, Pedro Luis; Nicolás, Gregorio; Lorenzo, Oscar

2003-01-01

FsPP2C1 was previously isolated from beech (Fagus sylvatica) seeds as a functional protein phosphatase type-2C (PP2C) with all the conserved features of these enzymes and high homology to ABI1, ABI2, and PP2CA, PP2Cs identified as negative regulators of ABA signaling. The expression of FsPP2C1 was induced upon abscisic acid (ABA) treatment and was also up-regulated during early weeks of stratification. Furthermore, this gene was specifically expressed in ABA-treated seeds and was hardly detectable in vegetative tissues. In this report, to provide genetic evidence on FsPP2C1 function in seed dormancy and germination, we used an overexpression approach in Arabidopsis because transgenic work is not feasible in beech. Constitutive expression of FsPP2C1 under the cauliflower mosaic virus 35S promoter confers ABA insensitivity in Arabidopsis seeds and, consequently, a reduced degree of seed dormancy. Additionally, transgenic 35S:FsPP2C1 plants are able to germinate under unfavorable conditions, as inhibitory concentrations of mannitol, NaCl, or paclobutrazol. In vegetative tissues, Arabidopsis FsPP2C1 transgenic plants show ABA-resistant early root growth and diminished induction of the ABA-response genes RAB18 and KIN2, but no effect on stomatal closure regulation. Seed and vegetative phenotypes of Arabidopsis 35S:FsPP2C1 plants suggest that FsPP2C1 negatively regulates ABA signaling. The ABA inducibility of FsPP2C1 expression, together with the transcript accumulation mainly in seeds, suggest that it could play an important role modulating ABA signaling in beechnuts through a negative feedback loop. Finally, we suggest that negative regulation of ABA signaling by FsPP2C1 is a factor contributing to promote the transition from seed dormancy to germination during early weeks of stratification. PMID:12970481

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

PubMed Central

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

2013-01-01

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

Atorvastatin Alleviates Experimental Diabetic Cardiomyopathy by Regulating the GSK-3β-PP2Ac-NF-κB Signaling Axis

PubMed Central

Wu, Wen; Luo, Jie; Ye, Peng; Chen, Shao-liang; Hu, Zuo-ying

2016-01-01

Recent studies reported that atorvastatin (ATOR) alleviated progression of experimental diabetic cardiomyopathy (DCM), possibly by protecting against apoptosis. However, the underlying mechanisms of this protective effect remain unclear. Therefore, our study investigated the role of the glycogen synthase kinase (GSK)-3β-protein phosphatase 2A(PP2A)-NF-κB signaling pathway in the anti-apoptotic and cardioprotective effects of ATOR on cardiomyocytes cultured in high glucose (HG) and in DCM. Our results showed that, in HG-cultured cardiomyocytes, phosphorylation of GSK-3β was decreased, while that of the PP2A catalytic subunit C (PP2Ac) and IKK/IкBα was increased, followed by NF-кB nuclear translocation and apoptosis. IKK/IкBα phosphorylation and NF-кB nuclear translocation were also increased by treatment of cells with okadaic acid (OA), a selective PP2A inhibitor, or by silencing PP2Ac expression. The opposite results were obtained by silencing GSK-3β expression, which resulted in PP2Ac activation. Furthermore, IKK/IкBα phosphorylation and NF-кB nuclear translocation were markedly inhibited and apoptosis attenuated in cells treated with ATOR. These effects occurred through inactivation of GSK-3β and subsequent activation of PP2Ac. They were abolished by treatment of cells with OA or PP2Ac siRNA. In mice with type 1 diabetes mellitus, treatment with ATOR, at 10 mg-kg−1-d−1, significantly suppressed GSK-3β activation, IKK/IкBα phosphorylation, NF-кB nuclear translocation and caspase-3 activation, while also activating PP2Ac. Finally, improvements in histological abnormalities, fibrosis, apoptosis and cardiac dysfunction were observed in diabetic mice treated with ATOR. These findings demonstrated that ATOR protected against HG-induced apoptosis in cardiomyocytes and alleviated experimental DCM by regulating the GSK-3β-PP2A-NF-κB signaling pathway. PMID:27851811

Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

PubMed Central

Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

2013-01-01

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

Cloning and expression profiling of the PacSnRK2 and PacPP2C gene families during fruit development, ABA treatment, and dehydration stress in sweet cherry.

PubMed

Shen, Xinjie; Guo, Xiao; Zhao, Di; Zhang, Qiang; Jiang, Yuzhuang; Wang, Yantao; Peng, Xiang; Wei, Yan; Zhai, Zefeng; Zhao, Wei; Li, Tianhong

2017-10-01

Plant SNF1-related protein kinase 2 (SnRK2) and protein phosphatase 2C (PP2C) family members are core components of the ABA signal transduction pathway. SnRK2 and PP2C proteins have been suggested to play crucial roles in fruit ripening and improving plant tolerance to drought stress, but supporting genetic information has been lacking in sweet cherry (Prunus avium L.). Here, we cloned six full-length SnRK2 genes and three full-length PP2C genes from sweet cherry cv. Hong Deng. Quantitative PCR analysis revealed that PacSnRK2.2, PacSnRK2.3, PacSnRK2.6, and PacPP2C1-3 were negatively regulated in fruits in response to exogenous ABA treatment, PacSnRK2.4 and PacSnRK2.5 were upregulated, and PacSnRK2.1 expression was not affected. The ABA treatment also significantly promoted the accumulation of anthocyanins in sweet cherry fruit. The expression of all PacSnRK2 and PacPP2C genes was induced by dehydration stress, which also promoted the accumulation of drought stress signaling molecules in the sweet cherry fruits, including ABA, soluble sugars, and anthocyanin. Furthermore, a yeast two-hybrid analysis demonstrated that PacPP2C1 interacts with all six PacSnRK2s, while PacPP2C3 does not interact with PacSnRK2.5. PacPP2C2 does not interact with PacSnRK2.1 or PacSnRK2.4. These results indicate that PacSnRK2s and PacPP2Cs may play a variety of roles in the sweet cherry ABA signaling pathway and the fruit response to drought stress. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Mechanisms of ROS modulated cell survival during carcinogenesis.

PubMed

Clerkin, J S; Naughton, R; Quiney, C; Cotter, T G

2008-07-18

There is increasing evidence within the literature that the decreased susceptibility of tumour cells to stimuli that induce apoptosis can be linked to their inherently increased redox potential. The review primarily focuses on the PI3-kinase/Akt pathway, and the multiple points along this signalling pathway that may be redox regulated. The PI3-kinase/Akt pathway can influence a cells' sensitivity to death inducing signals, through direct manipulation of apoptosis regulating molecules or by regulating the activity of key transcription factors. Proteins involved in the control of apoptosis that are directly regulated by the PI3-kinase/Akt pathway include caspase-9, Bad and the transcription factor GSK-3beta. Lately, it is becoming increasingly obvious that phosphatases are a major counter balance to the PI3-kinase/Akt pathway. Phosphatases such as PP2A and PP1alpha can dephosphorylate signalling molecules within the PI3-kinase/Akt pathway, blocking their activity. It is the balance between the kinase activity and the phosphatase activity that determines the presence and strength of the PI3-kinase/Akt signal. This is why any protein modifications that hinder dephosphorylation can increase the tumours survival advantage. One such modification is the oxidation of the sulphydryl group in key cysteine residues present within the active site of the phosphatases. This highlights the link between the increased redox stress in tumours with the PI3-kinase/Akt pathway. This review will discuss the various sources of reactive oxygen species within a tumour and the effect of these radicals on the PI3-kinase/Akt pathway.

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers

PubMed Central

Roberts, Kathryn G.; Smith, Amanda M.; McDougall, Fiona; Carpenter, Helen; Horan, Martin; Neviani, Paolo; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Perrotti, Danilo; Sim, Alistair T.R.; Ashman, Leonie K.; Verrills, Nicole M.

2010-01-01

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST), systemic mastocytosis, and some acute myeloid leukemias (AML). Whilst juxtamembrane mutations commonly detected in GIST are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study we show that myeloid cells expressing activated c-KIT mutants that are imatinib-sensitive (V560G) or –resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with reduced expression of PP2A structural (A) and regulatory subunits (B55α; B56α; B56γ and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacological activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential and induced apoptosis of mutant c-KIT+ cells, whilst having no effect on WT c-KIT cells or empty vector controls. FTY720 treatment caused dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5 and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT+ cancers. PMID:20551067

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

PubMed

Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

2016-12-30

G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cre-lox Univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-Like protein phosphatases

PubMed Central

Jia, Fan; Gampala, Srinivas S.L.; Mittal, Amandeep; Luo, Qingjun; Rock, Christopher D.

2009-01-01

The 14,200 available full length Arabidopsis thaliana cDNAs in the Universal Plasmid System (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a “functional map-space” of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities. PMID:19499346

Nitric oxide leads to cytoskeletal reorganization in the retinal pigment epithelium under oxidative stress.

PubMed

Sripathi, Srinivas R; He, Weilue; Um, Ji-Yeon; Moser, Trevor; Dehnbostel, Stevie; Kindt, Kimberly; Goldman, Jeremy; Frost, Megan C; Jahng, Wan Jin

2012-01-01

Light is a risk factor for various eye diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). We aim to understand how cytoskeletal proteins in the retinal pigment epithetlium (RPE) respond to oxidative stress, including light and how these responses affect apoptotic signaling. Previously, proteomic analysis revealed that the expression levels of vimentin and serine/threonine protein phosphatase 2A (PP2A) are significantly increased when mice are exposed under continuous light for 7 days compared to a condition of 12 hrs light/dark cycling exposure using retina degeneration 1 (rd1) model. When melatonin is administered to animals while they are exposed to continuous light, the levels of vimentin and PP2A return to a normal level. Vimentin is a substrate of PP2A that directly binds to vimentin and dephosphorylates it. The current study shows that upregulation of PP2Ac (catalytic subunit) phosphorylation negatively correlates with vimentin phosphorylation under stress condition. Stabilization of vimentin appears to be achieved by decreased PP2Ac phosphorylation by nitric oxide induction. We tested our hypothesis that site-specific modifications of PP2Ac may drive cytoskeletal reorganization by vimentin dephosphorylation through nitric oxide signaling. We speculate that nitric oxide determines protein nitration under stress conditions. Our results demonstrate that PP2A and vimentin are modulated by nitric oxide as a key element involved in cytoskeletal signaling. The current study suggests that external stress enhances nitric oxide to regulate PP2Ac and vimentin phosphorylation, thereby stabilizing or destabilizing vimentin. Phosphorylation may result in depolymerization of vimentin, leading to nonfilamentous particle formation. We propose that a stabilized vimentin might act as an anti-apoptotic molecule when cells are under oxidative stress.

Nitric oxide leads to cytoskeletal reorganization in the retinal pigment epithelium under oxidative stress

PubMed Central

Um, Ji-Yeon; Moser, Trevor; Dehnbostel, Stevie; Kindt, Kimberly; Goldman, Jeremy; Frost, Megan C.; Jahng, Wan Jin

2016-01-01

Light is a risk factor for various eye diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). We aim to understand how cytoskeletal proteins in the retinal pigment epithetlium (RPE) respond to oxidative stress, including light and how these responses affect apoptotic signaling. Previously, proteomic analysis revealed that the expression levels of vimentin and serine/threonine protein phosphatase 2A (PP2A) are significantly increased when mice are exposed under continuous light for 7 days compared to a condition of 12 hrs light/dark cycling exposure using retina degeneration 1 (rd1) model. When melatonin is administered to animals while they are exposed to continuous light, the levels of vimentin and PP2A return to a normal level. Vimentin is a substrate of PP2A that directly binds to vimentin and dephosphorylates it. The current study shows that upregulation of PP2Ac (catalytic subunit) phosphorylation negatively correlates with vimentin phosphorylation under stress condition. Stabilization of vimentin appears to be achieved by decreased PP2Ac phosphorylation by nitric oxide induction. We tested our hypothesis that site-specific modifications of PP2Ac may drive cytoskeletal reorganization by vimentin dephosphorylation through nitric oxide signaling. We speculate that nitric oxide determines protein nitration under stress conditions. Our results demonstrate that PP2A and vimentin are modulated by nitric oxide as a key element involved in cytoskeletal signaling. The current study suggests that external stress enhances nitric oxide to regulate PP2Ac and vimentin phosphorylation, thereby stabilizing or destabilizing vimentin. Phosphorylation may result in depolymerization of vimentin, leading to nonfilamentous particle formation. We propose that a stabilized vimentin might act as an anti-apoptotic molecule when cells are under oxidative stress. PMID:27974994

Differential effects of protein phosphatases in the recycling of metabotropic glutamate receptor 5.

PubMed

Mahato, P K; Pandey, S; Bhattacharyya, S

2015-10-15

The major excitatory neurotransmitter Glutamate acts on both ionotropic and metabotropic glutamate receptors (mGluRs) in the central nervous system. mGluR5, a member of the group I mGluR family is widely expressed throughout the brain and plays important roles in a variety of neuronal processes including various forms of synaptic plasticity. This receptor is also involved in various neuropsychiatric disorders, viz., Fragile X syndrome, autism etc. It has been reported that mGluR5 undergoes desensitization and subsequently internalization on ligand exposure in various cell types. However, the downstream events after the internalization and the molecular players involved in the post-endocytic events of this receptor have not been studied. In the present study, we find that subsequent to internalization mGluR5 enters the recycling compartment. After that the receptor recycles back to the cell surface. We also show here that the recycling of mGluR5 is dependent on protein phosphatases. Our data suggest that mGluR5 recycling is completely dependent on the activity of PP2A whereas, PP2B has partial effect on this process. Thus our study suggests that mGluR5 recycles back to the cell surface after ligand-dependent internalization and protein phosphatases that have been implicated in various forms of synaptic plasticity have differential effects on the recycling of mGluR5. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

The structures of three metabolites of the algal hepatotoxin okadaic acid produced by oxidation with human cytochrome P450

PubMed Central

Liu, Li; Guoa, Fujiang; Crain, Sheila; Quilliam, Michael A.; Wang, Xiaotang; Rein, Kathleen S.

2012-01-01

Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 μg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,β unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A. PMID:22608922

APPL1 Mediates Adiponectin-Induced LKB1 Cytosolic Localization Through the PP2A-PKCζ Signaling Pathway

PubMed Central

Deepa, Sathyaseelan S.; Zhou, Lijun; Ryu, Jiyoon; Wang, Changhua; Mao, Xuming; Li, Cai; Zhang, Ning; Musi, Nicolas; DeFronzo, Ralph A.; Liu, Feng

2011-01-01

We recently found that the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL)1 is essential for mediating adiponectin signal to induce liver kinase B (LKB)1 cytosloic translocation, an essential step for activation of AMP-activated protein kinase (AMPK) in cells. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that treating C2C12 myotubes with adiponectin promoted APPL1 interaction with protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ), leading to the activation of PP2A and subsequent dephosphorylation and inactivation of PKCζ. The adiponectin-induced inactivation of PKCζ results in dephosphorylation of LKB1 at Ser307 and its subsequent translocation to the cytosol, where it stimulates AMPK activity. Interestingly, we found that metformin also induces LKB1 cytosolic translocation, but the stimulation is independent of APPL1 and the PP2A-PKCζ pathway. Together, our study uncovers a new mechanism underlying adiponectin-stimulated AMPK activation in muscle cells and shed light on potential targets for prevention and treatment of insulin resistance and its associated diseases. PMID:21835890

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

PubMed

Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

2013-11-01

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

MIR7-3HG, a MYC-dependent modulator of cell proliferation, inhibits autophagy by a regulatory loop involving AMBRA1.

PubMed

Capizzi, Mariacristina; Strappazzon, Flavie; Cianfanelli, Valentina; Papaleo, Elena; Cecconi, Francesco

2017-03-04

Macroautophagy/autophagy is a tightly regulated intracellular catabolic pathway involving the lysosomal degradation of cytoplasmic organelles and proteins to be recycled into metabolic precursors. AMBRA1 (autophagy and Beclin 1 regulator 1) has a central role in the autophagy signaling network; it acts upstream of MTORC1-dependent autophagy by stabilizing the kinase ULK1 (unc-51 like autophagy activating kinase 1) and by favoring autophagosome core complex formation. AMBRA1 also regulates the cell cycle by modulating the activity of the phosphatase PPP2/PP2A (protein phosphatase 2) and degradation of MYC. Of note, post-transcriptional regulation mediated by noncoding microRNAs (MIRNAs) contributes significantly to control autophagy. Here we describe a new role for the microRNA MIR7-3HG/MIR-7 as a potent autophagy inhibitor. Indeed, MIR7-3HG targets the 3' untranslated region (UTR) of AMBRA1 mRNA, inducing a decrease of both AMBRA1 mRNA and protein levels, and thus causing a block in autophagy. Furthermore, MIR7-3HG, through AMBRA1 downregulation, prevents MYC dephosphorylation, establishing a positive feedback for its own transcription. These data suggest a new and interesting role of MIR7-3HG as an anti-autophagic MIRNA that may affect oncogenesis through the regulation of the tumor suppressor AMBRA1.

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2.

PubMed

Rodriguez, P L; Leube, M P; Grill, E

1998-11-01

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.

Protein Phosphatase 4 Promotes Chromosome Pairing and Synapsis, and Contributes to Maintaining Crossover Competence with Increasing Age

PubMed Central

Sato-Carlton, Aya; Li, Xuan; Crawley, Oliver; Testori, Sarah; Martinez-Perez, Enrique; Sugimoto, Asako; Carlton, Peter M.

2014-01-01

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics. PMID:25340746

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

PubMed

Kondo, Koyuki; Kubo, Takeo; Kunieda, Takekazu

2015-01-01

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

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

The liver glucose-6-phosphatase of intact microsomes is inhibited and displays sigmoid kinetics in the presence of alpha-ketoglutarate-magnesium and oxaloacetate-magnesium chelates.

PubMed

Mithieux, G; Vega, F V; Riou, J P

1990-11-25

We have recently shown that the Ca.EGTA and Mg.EDTA complexes, but not free Ca2+ or Mg2+, inhibit the liver glucose-6-phosphatase (Mithieux, G., Vega, F. V., Beylot, M., and Riou, J. P. (1990) J. Biol. Chem. 265, 7257-7259). In this work, we report that, when complexed with Mg2+, two endogenous dicarboxylic keto acids (alpha-ketoglutarate (alpha-KG) and oxaloacetate (OAA] inhibit the glucose-6-phosphatase activity at low concentrations of substrate. This phenomenon is specific for complexes of Mg2+ with alpha-KG and OAA since 1) the complexes of Mg2+ with a number of other di- or tricarboxylic acids having high structural analogy with alpha-KG and OAA (oxalate, malate, succinate, citrate, aspartate, and glutamate) do not inhibit the glucose-6-phosphatase activity and 2) the Ca.alpha-KG and Ca.OAA chelates do not inhibit the glucose-6-phosphatase activity. In the presence of Mg.alpha-KG or Mg.OAA chelates, the enzyme displays sigmoid kinetics; the Hanes plots deviate from linearity, indicating the positive cooperative dependence of the velocity upon the substrate concentration. Hill coefficients (equal to 1 in the absence of the chelates) of 1.23 and 1.33 have been determined in the presence of Mg.alpha-KG and Mg.OAA complexes, respectively. The disruption of microsomal integrity by detergents abolishes the effect of Mg.alpha-KG and Mg.OAA, suggesting that the magnesium chelates inhibit the translocase component of the glucose-6-phosphatase system.

FTY720 inhibits mesothelioma growth in vitro and in a syngeneic mouse model.

PubMed

Szymiczek, Agata; Pastorino, Sandra; Larson, David; Tanji, Mika; Pellegrini, Laura; Xue, Jiaming; Li, Shuangjing; Giorgi, Carlotta; Pinton, Paolo; Takinishi, Yasutaka; Pass, Harvey I; Furuya, Hideki; Gaudino, Giovanni; Napolitano, Andrea; Carbone, Michele; Yang, Haining

2017-03-15

Malignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet. Cell viability and anchorage-independent growth were evaluated in a panel of MM cell lines and human mesothelial cells (HM) upon FTY720 treatment to assess in vitro anti-tumor efficacy. The mechanism of action of FTY720 in MM was assessed by measuring the activity of phosphatase protein 2A (PP2A)-a major target of FTY720. The binding of the endogenous inhibitor SET to PP2A in presence of FTY720 was evaluated by immunoblotting and immunoprecipitation. Signaling and activation of programmed cell death were evaluated by immunoblotting and flow cytometry. A syngeneic mouse model was used to evaluate anti-tumor efficacy and toxicity profile of FTY720 in vivo. We show that FTY720 significantly suppressed MM cell viability and anchorage-independent growth without affecting normal HM cells. FTY720 inhibited the phosphatase activity of PP2A by displacement of SET protein, which appeared overexpressed in MM, as compared to HM cells. FTY720 promoted AKT dephosphorylation and Bcl-2 degradation, leading to induction of programmed cell death, as demonstrated by caspase-3 and PARP activation, as well as by cytochrome c and AIF intracellular translocation. Moreover, FTY720 administration in vivo effectively reduced tumor burden in mice without apparent toxicity. Our preclinical data indicate that FTY720 is a potentially promising therapeutic agent for MM treatment.

Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex.

PubMed

Fleming, Matthew R; Brown, Maile R; Kronengold, Jack; Zhang, Yalan; Jenkins, David P; Barcia, Gulia; Nabbout, Rima; Bausch, Anne E; Ruth, Peter; Lukowski, Robert; Navaratnam, Dhasakumar S; Kaczmarek, Leonard K

2016-08-30

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Stimulation of Slack K+ channels alters mass at the plasma membrane by triggering dissociation of a phosphatase-regulatory complex

PubMed Central

Fleming, Matthew R.; Brown, Maile R.; Kronengold, Jack; Zhang, Yalan; Jenkins, David P.; Barcia, Gulia; Nabbout, Rima; Bausch, Anne E.; Ruth, Peter; Lukowski, Robert; Navaratnam, Dhasakumar S.; Kaczmarek, Leonard K.

2016-01-01

Summary Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways, and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies. PMID:27545877

Muscle-Specific Deletion of Rictor Impairs Insulin-Stimulated Glucose Transport and Enhances Basal Glycogen Synthase Activity▿

PubMed Central

Kumar, Anil; Harris, Thurl E.; Keller, Susanna R.; Choi, Kin M.; Magnuson, Mark A.; Lawrence, John C.

2008-01-01

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity. PMID:17967879

Transcriptional regulation of SlPYL, SlPP2C, and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress.

PubMed

Sun, Liang; Wang, Yan-Ping; Chen, Pei; Ren, Jie; Ji, Kai; Li, Qian; Li, Ping; Dai, Sheng-Jie; Leng, Ping

2011-11-01

In order to characterize the potential transcriptional regulation of core components of abscisic acid (ABA) signal transduction in tomato fruit development and drought stress, eight SlPYL (ABA receptor), seven SlPP2C (type 2C protein phosphatase), and eight SlSnRK2 (subfamily 2 of SNF1-related kinases) full-length cDNA sequences were isolated from the tomato nucleotide database of NCBI GenBank. All SlPYL, SlPP2C, and SlSnRK2 genes obtained are homologous to Arabidopsis AtPYL, AtPP2C, and AtSnRK2 genes, respectively. Based on phylogenetic analysis, SlPYLs and SlSnRK2s were clustered into three subfamilies/subclasses, and all SlPP2Cs belonged to PP2C group A. Within the SlPYL gene family, SlPYL1, SlPYL2, SlPYL3, and SlPYL6 were the major genes involved in the regulation of fruit development. Among them, SlPYL1 and SlPYL2 were expressed at high levels throughout the process of fruit development and ripening; SlPYL3 was strongly expressed at the immature green (IM) and mature green (MG) stages, while SlPYL6 was expressed strongly at the IM and red ripe (RR) stages. Within the SlPP2C gene family, the expression of SlPP2C, SlPP2C3, and SlPP2C4 increased after the MG stage; SlPP2C1 and SlPP2C5 peaked at the B3 stage, while SlPP2C2 and SlPP2C6 changed little during fruit development. Within the SlSnRK2 gene family, the expression of SlSnRK2.2, SlSnRK2.3, SlSnRK2.4, and SlSnRK2C was higher than that of other members during fruit development. Additionally, most SlPYL genes were down-regulated, while most SlPP2C and SlSnRK2 genes were up-regulated by dehydration in tomato leaf.

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eto, Masumi, E-mail: masumi.eto@jefferson.edu; Kirkbride, Jason A.; Chugh, Rishika

2013-04-26

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentratedmore » in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.« less

Hypothermia mediates age-dependent increase of tau phosphorylation in db/db mice.

PubMed

El Khoury, Noura B; Gratuze, Maud; Petry, Franck; Papon, Marie-Amélie; Julien, Carl; Marcouiller, François; Morin, Françoise; Nicholls, Samantha B; Calon, Frédéric; Hébert, Sébastien S; Marette, André; Planel, Emmanuel

2016-04-01

Accumulating evidence from epidemiological studies suggest that type 2 diabetes is linked to an increased risk of Alzheimer's disease (AD). However, the consequences of type 2 diabetes on AD pathologies, such as tau hyperphosphorylation, are not well understood. Here, we evaluated the impact of type 2 diabetes on tau phosphorylation in db/db diabetic mice aged 4 and 26weeks. We found increased tau phosphorylation at the CP13 epitope correlating with a deregulation of c-Jun. N-terminal kinase (JNK) and Protein Phosphatase 2A (PP2A) in 4-week-old db/db mice. 26-week-old db/db mice displayed tau hyperphosphorylation at multiple epitopes (CP13, AT8, PHF-1), but no obvious change in kinases or phosphatases, no cleavage of tau, and no deregulation of central insulin signaling pathways. In contrast to younger animals, 26-week-old db/db mice were hypothermic and restoration of normothermia rescued phosphorylation at most epitopes. Our results suggest that, at early stages of type 2 diabetes, changes in tau phosphorylation may be due to deregulation of JNK and PP2A, while at later stages hyperphosphorylation is mostly a consequence of hypothermia. These results provide a novel link between diabetes and tau pathology, and underlie the importance of recording body temperature to better understand the relationship between diabetes and AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

PubMed Central

Lammers, Twan; Peschke, Peter; Ehemann, Volker; Debus, Jürgen; Slobodin, Boris; Lavi, Sara; Huber, Peter

2007-01-01

Background PP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis. Results It was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased. Conclusion Based on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer. PMID:17941990

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

PubMed

Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

2014-06-01

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells.

PubMed

Zhang, Jing; Lauf, Peter K; Adragna, Norma C

2005-07-15

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

PubMed

Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

2015-08-01

Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression. © 2015 Wiley Periodicals, Inc.

Microvascular dysfunction with increased vascular leakage response in mice systemically exposed to arsenic.

PubMed

Chen, Shih-Chieh; Huang, Shin-Yin; Lu, Chi-Yu; Hsu, Ya-Hung; Wang, Dean-Chuan

2014-09-01

The mechanisms underlying cardiovascular disease induced by arsenic exposure are not completely understood. The objectives of this study were to investigate whether arsenic-fed mice have an increased vascular leakage response to vasoactive agents and whether enhanced type-2 protein phosphatase (PP2A) activity is involved in mustard oil-induced leakage. ICR mice were fed water or sodium arsenite (20 mg/kg) for 4 or 8 weeks. The leakage response to vasoactive agents was quantified using the Evans blue (EB) technique or vascular labeling with carbon particles. Increased EB leakage and high density of carbon-labeled microvessels were detected in arsenic-fed mice treated with mustard oil. Histamine induced significantly higher vascular leakage in arsenic-fed mice than in water-fed mice. Pretreatment with the PP2A inhibitor okadaic acid or the neurokinin 1 receptor (NK1R) blocker RP67580 significantly reduced mustard oil-induced vascular leakage in arsenic-fed mice. The protein levels of PP2Ac and NK1R were similar in both groups. PP2A activity was significantly higher in the arsenic-fed mice compared with the control group. These findings indicate that microvessels generally respond to vasoactive agents, and that the increased PP2A activity is involved in mustard oil-induced vascular leakage in arsenic-fed mice. Arsenic may initiate endothelial dysfunction, resulting in vascular leakage in response to vasoactive agents.

A Novel Rasopathy Caused by Recurrent De Novo Missense Mutations In PPP1CB Closely Resembles Noonan Syndrome with Loose Anagen Hair

PubMed Central

Gripp, Karen W.; Aldinger, Kimberly A.; Bennett, James T.; Baker, Laura; Tusi, Jessica; Powell-Hamilton, Nina; Stabley, Deborah; Sol-Church, Katia; Timms, Andrew E.; Dobyns, William B.

2016-01-01

Noonan syndrome is a rasopathy caused by mutations in multiple genes encoding components of the RAS/MAPK pathway. Despite its variable phenotype, limited genotype-phenotype correlations exist. Noonan syndrome with loose anagen hair (NS-LAH) is characterized by its distinctive hair anomalies, developmental differences and structural brain abnormalities and is caused by a single recurrent missense SHOC2 mutation. SHOC2 forms a complex with protein phosphatase 1 (PP1C). Protein phosphatases counterbalance kinases and control activation of signaling proteins, such as the mitogen activated protein kinases of the RAS/MAPK pathway. Here we report four patients with de novo missense mutations in protein phosphatase 1 catalytic subunit beta (PPP1CB), sharing a recognizable phenotype. Three individuals had the recurrent PPP1CB c.146G>C, p.Pro49Arg mutation, the fourth had a c.166G>C, p.Ala56Pro change. All had relative or absolute macrocephaly, low-set and posteriorly angulated ears and developmental delay. Slow growing and/or sparse hair and/or an unruly hair texture was present in all. Three individuals had feeding difficulties requiring feeding tubes. One of two males had cryptorchidism, another had pectus excavatum. Short stature was present in three. A female with the recurrent mutation had a Dandy-Walker malformation and optic nerve hypoplasia. Mild ventriculomegaly occurred in all, cerebellar tonsillar ectopia was seen in two and progressed to Chiari 1 malformation in one individual. Based on the combination of phenotypic findings and PPP1CB’s effect on RAF dephosphorylation within the RAS/MAPK pathway, this novel condition can be considered a rasopathy, most similar to NS-LAH. Collectively, these mutations meet the standardized criteria for pathogenicity. PMID:27264673

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition

PubMed Central

Chang, Yi; Li, Jiun-Yi; Jayakumar, Thanasekaran; Hung, Shou-Huang; Lee, Wei-Cheng; Manubolu, Manjunath; Sheu, Joen-Rong; Hsu, Ming-Jen

2017-01-01

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a (pp2a) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs. PMID:29186909

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition.

PubMed

Chang, Yi; Li, Jiun-Yi; Jayakumar, Thanasekaran; Hung, Shou-Huang; Lee, Wei-Cheng; Manubolu, Manjunath; Sheu, Joen-Rong; Hsu, Ming-Jen

2017-11-27

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a ( pp2a ) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs.

2-Substituted 7-trifluoromethyl-thiadiazolopyrimidones as alkaline phosphatase inhibitors. Synthesis, structure activity relationship and molecular docking study.

PubMed

Jafari, Behzad; Ospanov, Meirambek; Ejaz, Syeda Abida; Yelibayeva, Nazym; Khan, Shafi Ullah; Amjad, Sayyeda Tayyeba; Safarov, Sayfidin; Abilov, Zharylkasyn A; Turmukhanova, Mirgul Zh; Kalugin, Sergey N; Ehlers, Peter; Lecka, Joanna; Sévigny, Jean; Iqbal, Jamshed; Langer, Peter

2018-01-20

Alkaline Phosphatases (APs) play a key role in maintaining a ratio of phosphate to inorganic pyrophosphate (P i /PP i ) and thus regulate extracellular matrix calcification during bone formation and growth. Among different isozymes of AP, aberrant increase in the level of tissue non-specific alkaline phosphatase (TNAP) is strongly associated with vascular calcification and end-stage renal diseases. In this context, we synthesized a novel series of fluorinated pyrimidone derivatives, i.e., 2-bromo-7-trifluoromethyl-5-oxo-5H-1,3,4-thiadiazolepyrimidones. The bromine functionality was further used for derivatisation by nucleophilic aromatic substitution using amines as nucleophiles as well as by Palladium catalysed Suzuki-Miyaura reactions. The synthesized derivatives were found potent but non-selective inhibitors of both isozymes of AP. Arylated thiadiazolopyrimidones exhibited stronger inhibitory activities than 2-amino-thiadiazolopyrimidones. The binding modes and possible interactions of the most active inhibitor within the active site of the enzyme were observed by molecular docking studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The PP1 binding code: a molecular-lego strategy that governs specificity.

PubMed

Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

2013-01-01

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

Simplified preparation of a phosphatase inhibitor and further studies of its action.

PubMed

Coburn, S P; Schaltenbrand, W E

1978-05-01

1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.

New pentacoordinated rhodium species as unexpected products during the in situ generation of dimeric diphosphine-rhodium neutral catalysts.

PubMed

Meissner, Antje; König, Anja; Drexler, Hans-Joachim; Thede, Richard; Baumann, Wolfgang; Heller, Detlef

2014-11-03

Dimeric rhodium complexes of the type [Rh(PP)(μ2 -Cl)]2 (PP=diphosphine) are often used as precatalysts and are generated "in situ" from the corresponding diolefin complexes by exchange of the diene with the desired diphosphine. Herein, we report that the "in situ" procedure also leads to unexpected monomeric pentacoordinated neutral complexes of the type [RhCl(PP)(diolefin)], for the first time herein characterized by NMR spectroscopy and X-ray crystallography for the ligands 1,4-bis(diphenylphosphino)propane (DPPP), 1,4-bis(diphenylphosphino)butane (DPPB), and 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP). The pentacoordinated complexes are in equilibrium with the dimeric target compound [Rh(PP)(μ2 -Cl)]2 . The equilibrium is influenced by the rhodium-diolefin precursor, the solvent and the temperature. Based on the results of NMR and UV/Vis spectroscopic analysis (kinetics) it could be shown that the pentacoordinated complex [RhCl(PP)(diolefin)] may arise both from the "in situ"-generated neutral complex [Rh(PP)(μ2 -Cl)] by reaction with the free diolefin and, more surprisingly, directly from [Rh(diolefin)(μ2 -Cl)]2 and the diphosphine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of glycogen synthase kinase 3 beta (GSK3beta) in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA) in MCF-7 breast cancer cells.

PubMed

Alao, John P; Stavropoulou, Alexandra V; Lam, Eric W-F; Coombes, R Charles

2006-10-03

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

Dormancy-Associated MADS-Box (DAM) and the Abscisic Acid Pathway Regulate Pear Endodormancy Through a Feedback Mechanism.

PubMed

Tuan, Pham Anh; Bai, Songling; Saito, Takanori; Ito, Akiko; Moriguchi, Takaya

2017-08-01

In the pear 'Kosui' (Pyrus pyrifolia Nakai), the dormancy-associated MADS-box (PpDAM1 = PpMADS13-1) gene has been reported to play an essential role in bud endodormancy. Here, we found that PpDAM1 up-regulated expression of 9-cis-epoxycarotenoid dioxygenase (PpNCED3), which is a rate-limiting gene for ABA biosynthesis. Transient assays with a dual luciferase reporter system (LUC assay) and electrophoretic mobility shift assay (EMSA) showed that PpDAM1 activated PpNCED3 expression by binding to the CArG motif in the PpNCED3 promoter. PpNCED3 expression was increased toward endodormancy release in lateral flower buds of 'Kosui', which is consistent with the induced levels of ABA, its catabolism (ABA 8'-hydroxylase) and signaling genes (type 2C protein phosphatase genes and SNF1-related protein kinase 2 genes). In addition, we found that an ABA response element (ABRE)-binding transcription factor, PpAREB1, exhibiting high expression concomitant with endodormancy release, bound to three ABRE motifs in the promoter region of PpDAM1 and negatively regulated its activity. Taken together, our results suggested a feedback regulation between PpDAM1 and the ABA metabolism and signaling pathway during endodormancy of pear. This first evidence of an interaction between a DAM and ABA biosynthesis in vitro will provide further insights into bud endodormancy regulatory mechanisms of deciduous trees including pear. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Chapter 2. Calcineurin signaling and the slow oxidative skeletal muscle fiber type.

PubMed

Mallinson, Joanne; Meissner, Joachim; Chang, Kin-Chow

2009-01-01

Calcineurin, also known as protein phosphatase 2B (PP2B), is a calcium-calmodulin-dependent phosphatase. It couples intracellular calcium to dephosphorylate selected substrates resulting in diverse biological consequences depending on cell type. In mammals, calcineurin's functions include neuronal growth, development of cardiac valves and hypertrophy, activation of lymphocytes, and the regulation of ion channels and enzymes. This chapter focuses on the key roles of calcineurin in skeletal muscle differentiation, regeneration, and fiber type conversion to an oxidative state, all of which are crucial to muscle development, metabolism, and functional adaptations. It seeks to integrate the current knowledge of calcineurin signaling in skeletal muscle and its interactions with other prominent regulatory pathways and their signaling intermediates to form a molecular overview that could provide directions for possible future exploitations in human metabolic health.

Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor.

PubMed

Teo, Adrian Kee Keong; Kulkarni, Rohit N

2012-10-17

Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

Localization of phosphorylated forms of Bcl-2 in mitosis: co-localization with Ki-67 and nucleolin in nuclear structures and on mitotic chromosomes.

PubMed

Barboule, Nadia; Truchet, Isabelle; Valette, Annie

2005-04-01

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics.

PubMed

Simão, Ana Maria S; Yadav, Manisha C; Narisawa, Sonoko; Bolean, Mayte; Pizauro, Joao Martins; Hoylaerts, Marc F; Ciancaglini, Pietro; Millán, José Luis

2010-03-05

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen; Yang, Xi-fei

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we exploremore » the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.« less

Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

PubMed Central

2004-01-01

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR. PMID:15461583

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Heteroleptic copper(I) complexes prepared from phenanthroline and bis-phosphine ligands.

PubMed

Kaeser, Adrien; Mohankumar, Meera; Mohanraj, John; Monti, Filippo; Holler, Michel; Cid, Juan-José; Moudam, Omar; Nierengarten, Iwona; Karmazin-Brelot, Lydia; Duhayon, Carine; Delavaux-Nicot, Béatrice; Armaroli, Nicola; Nierengarten, Jean-François

2013-10-21

Preparation of [Cu(NN)(PP)](+) derivatives has been systematically investigated starting from two libraries of phenanthroline (NN) derivatives and bis-phosphine (PP) ligands, namely, (A) 1,10-phenanthroline (phen), neocuproine (2,9-dimethyl-1,10-phenanthroline, dmp), bathophenanthroline (4,7-diphenyl-1,10-phenanthroline, Bphen), 2,9-diphenethyl-1,10-phenanthroline (dpep), and 2,9-diphenyl-1,10-phenanthroline (dpp); (B) bis(diphenylphosphino)methane (dppm), 1,2-bis(diphenylphosphino)ethane (dppe), 1,3-bis(diphenylphosphino)propane (dppp), 1,2-bis(diphenylphosphino)benzene (dppb), 1,1'-bis(diphenylphosphino)ferrocene (dppFc), and bis[(2-diphenylphosphino)phenyl] ether (POP). Whatever the bis-phosphine ligand, stable heteroleptic [Cu(NN)(PP)](+) complexes are obtained from the 2,9-unsubstituted-1,10-phenanthroline ligands (phen and Bphen). By contrast, heteroleptic complexes obtained from dmp and dpep are stable in the solid state, but a dynamic ligand exchange reaction is systematically observed in solution, and the homoleptic/heteroleptic ratio is highly dependent on the bis-phosphine ligand. Detailed analysis revealed that the dynamic equilibrium resulting from ligand exchange reactions is mainly influenced by the relative thermodynamic stability of the different possible complexes. Finally, in the case of dpp, only homoleptic complexes were obtained whatever the bis-phosphine ligand. Obviously, steric effects resulting from the presence of the bulky phenyl rings on the dpp ligand destabilize the heteroleptic [Cu(NN)(PP)](+) complexes. In addition to the remarkable thermodynamic stability of [Cu(dpp)2]BF4, this negative steric effect drives the dynamic complexation scenario toward almost exclusive formation of homoleptic [Cu(NN)2](+) and [Cu(PP)2](+) complexes. This work provides the definitive rationalization of the stability of [Cu(NN)(PP)](+) complexes, marking the way for future developments in this field.

Liver carbohydrates metabolism: A new islet-neogenesis associated protein peptide (INGAP-PP) target.

PubMed

Villagarcía, Hernán Gonzalo; Román, Carolina Lisi; Castro, María Cecilia; González, Luisa Arbeláez; Ronco, María Teresa; Francés, Daniel Eleazar; Massa, María Laura; Maiztegui, Bárbara; Flores, Luis Emilio; Gagliardino, Juan José; Francini, Flavio

2018-03-01

Islet-Neogenesis Associated Protein-Pentadecapeptide (INGAP-PP) increases β-cell mass and enhances glucose and amino acids-induced insulin secretion. Our aim was to demonstrate its effect on liver metabolism. For that purpose, adult male Wistar rats were injected twice-daily (10 days) with saline solution or INGAP-PP (250 μg). Thereafter, serum glucose, triglyceride and insulin levels were measured and homeostasis model assessment (HOMA-IR) and hepatic insulin sensitivity (HIS) were determined. Liver glucokinase and glucose-6-phosphatase (G-6-Pase) expression and activity, phosphoenolpyruvate carboxykinase (PEPCK) expression, phosphofructokinase-2 (PFK-2) protein content, P-Akt/Akt and glycogen synthase kinase-3β (P-GSK3/GSK3) protein ratios and glycogen deposit were also determined. Additionally, glucokinase activity and G-6-Pase and PEPCK gene expression were also determined in isolated hepatocytes from normal rats incubated with INGAP-PP (5 μg/ml). INGAP-PP administration did not modify any of the serum parameters tested but significantly increased activity of liver glucokinase and the protein level of its cytosolic activator, PFK-2. Conversely, INGAP-PP treated rats decreased gene expression and enzyme activity of gluconeogenic enzymes, G-6-Pase and PEPCK. They also showed a higher glycogen deposit and P-GSK3/GSK3 and P-Akt/Akt ratio. In isolated hepatocytes, INGAP-PP increased GK activity and decreased G-6-Pase and PEPCK expression. These results demonstrate a direct effect of INGAP-PP on the liver acting through P-Akt signaling pathway. INGAP-PP enhances liver glucose metabolism and deposit and reduces its production/output, thereby contributing to maintain normal glucose homeostasis. These results reinforce the concept that INGAP-PP might become a useful tool to treat people with impaired islet/liver glucose metabolism as it occurs in T2D. Copyright © 2018 Elsevier Inc. All rights reserved.

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. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1999-06-01

subunits are expressed ubiquitously and appear to be encoded by small and quite homogeneous gene families. In plants , however, A and C subunit gene...1996). In both plants and animals, different B subunit isoforms are encoded by two or more unrelated gene families, some of which are expressed in a...PP2A functions in whole plants and in mammalian tissue culture cells. This genetic system may also prove useful for analyzing interactions between

Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells

PubMed Central

Bouazza, Belaid; Krytska, Kateryna; Debba-Pavard, Manel; Amrani, Yassine; Honkanen, Richard E.; Tran, Jennifer

2012-01-01

Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-α (10 ng/ml) and IFN-γ (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation. PMID:22592921

Function of Protein Phosphatase 2A in Control of Proliferation: Isolation and Analysis of Dominant-Defective Mutants

DTIC Science & Technology

1998-06-01

the Ca gene. In the case of pWZLneo, translation of Ca and the neomycin phosphotransferase (Neo) protein are coupled by an IRES site. In the case of...expression of both the hygromycin resistance (Hyg) and PP2A-C cassettes. In the colony formation assay, cells infected with the retroviral construct are...than hygromycin in selecting for cells expressing high levels of drug resistance (Hanson and Sedivy, 1995). Cells expressing a relatively low level of

Selective ablation of Ppp1cc gene in testicular germ cells causes oligo-teratozoospermia and infertility in mice.

PubMed

Sinha, Nilam; Puri, Pawan; Nairn, Angus C; Vijayaraghavan, Srinivasan

2013-11-01

The four isoforms of serine/threonine phosphoprotein phosphatase 1 (PP1), derived from three genes, are among the most conserved proteins known. The Ppp1cc gene encodes two alternatively spliced variants, PP1 gamma1 (PPP1CC1) and PP1 gamma2 (PPP1CC2). Global deletion of the Ppp1cc gene, which causes loss of both isoforms, results in male infertility due to impaired spermatogenesis. This phenotype was assumed to be due to the loss of PPP1CC2, which is abundant in testis. While PPP1CC2 is predominant, other PP1 isoforms are also expressed in testis. Given the significant homology between the four PP1 isoforms, the lack of compensation by the other PP1 isoforms for loss of one, only in testis, is surprising. Here we document, for the first time, expression patterns of the PP1 isoforms in postnatal developing and adult mouse testis. The timing and sites of testis expression of PPP1CC1 and PPP1CC2 in testis are nonoverlapping. PPP1CC2 is the only one of the four PP1 isoforms not detected in sertoli cells and spermatogonia. Conversely, PPP1CC2 may be the only PP1 isoform expressed in postmeiotic germ cells. Deletion of the Ppp1cc gene in germ cells at the differentiated spermatogonia stage of development and beyond in Stra8 promoter-driven Cre transgenic mice results in oligo-terato-asthenozoospermia and male infertility, thus phenocopying global Ppp1cc null (-/-) mice. Taken together, these results confirm that spermatogenic defects observed in the global Ppp1cc knockout mice and in mice expressing low levels of PPP1CC2 in testis are due to compromised functions of PPP1CC2 in meiotic and postmeiotic germ cells.

Substituent effect of N-benzylated gramine derivatives that prevent the PP2A inhibition and dissipate the neuronal Ca2+ overload, as a multitarget strategy for the treatment of Alzheimer's disease.

PubMed

Gonzalez, Dorleta; Arribas, Raquel L; Viejo, Lucia; Lajarin-Cuesta, Rocio; de Los Rios, Cristobal

2018-05-15

Following the premises of the multitarget-directed ligands approach for the drug R&D against neurodegenerative diseases, where Alzheimer's disease (AD) outstands, we have synthesized and evaluated analogues of the gramine derivative ITH12657 (1-benzyl-5-methyl-3-(piperidin-1-ylmethyl-1H-indole, 2), which had shown important neuroprotective properties, such as blocking effect of voltage-gated Ca 2+ channels (VGCC), and prevention of phosphoprotein phosphatase 2A (PP2A) inhibition. The new analogues present different substitutions at the pending phenyl ring, what slightly modified their pharmacological characteristics. The VGCC blockade was enhanced in derivatives possessing nitro groups, while the pro-PP2A feature was ameliorated by the presence of fluorine. Chlorine atoms supplied good activities over the two biological targets aimed; nevertheless that substitution provoked loss of viability at 100-fold higher concentrations (10 μM), what discards them for a deeper pharmacological study. Overall, the para-fluorine derivative of ITH12657 was the most promising candidate for further preclinical assays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.

PubMed

Álvarez-Fernández, Mónica; Sánchez-Martínez, Ruth; Sanz-Castillo, Belén; Gan, Pei Pei; Sanz-Flores, María; Trakala, Marianna; Ruiz-Torres, Miguel; Lorca, Thierry; Castro, Anna; Malumbres, Marcos

2013-10-22

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death.

PubMed

Marks, A R

2001-04-01

Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

PubMed Central

Chen, Ruming; Rato, Cláudia; Yan, Yahui; Crespillo-Casado, Ana; Clarke, Hanna J; Harding, Heather P; Marciniak, Stefan J; Read, Randy J; Ron, David

2015-01-01

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex. DOI: http://dx.doi.org/10.7554/eLife.04871.001 PMID:25774600

Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores.

PubMed

London, Nitobe; Ceto, Steven; Ranish, Jeffrey A; Biggins, Sue

2012-05-22

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein. Copyright © 2012 Elsevier Ltd. All rights reserved.

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons.

PubMed

Martin, Ludovic; Magnaudeix, Amandine; Wilson, Cornelia M; Yardin, Catherine; Terro, Faraj

2011-11-01

Alterations in glycogen synthase kinase-3β (GSK3β) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6-BIDECO (6-bromoindirubin-3'-[O-(N,N-diethylcarbamyl)-oxime] and 6-BIMYEO (6-bromoindirubin-3'-[O-(2-morpholin-1-ylethyl)-oxime] hydrochloride) on OKA-induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6-BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6-BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6-BIDECO and 6-BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. Copyright © 2011 Wiley-Liss, Inc.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

PubMed

Shah, Ahsan U; Sarwar, Amna; Orabi, Abrahim I; Gautam, Samir; Grant, Wayne M; Park, Alexander J; Shah, Adnan U; Liu, Jun; Mistry, Pramod K; Jain, Dhanpat; Husain, Sohail Z

2009-11-01

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Drosophila Syd-1, Liprin-α, and Protein Phosphatase 2A B′ Subunit Wrd Function in a Linear Pathway to Prevent Ectopic Accumulation of Synaptic Materials in Distal Axons

PubMed Central

Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A.; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J.; Levitan, Edwin S.

2014-01-01

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B′ [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). PMID:24948803

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons.

PubMed

Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J; Levitan, Edwin S; Wu, Chunlai

2014-06-18

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B' [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). Copyright © 2014 the authors 0270-6474/14/348474-14$15.00/0.

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

PubMed Central

Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2014-01-01

Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

1 ALPHA-Hydroxyvitamin D5 as a Chemotherapeutic and Possibly Chemopreventive Agent

DTIC Science & Technology

2004-09-01

with differential, platelets, PT, and PTT. 7.1.4 Serum chemistries : glucose, electrolytes (Na+, K+, Cl-, C0 2), BUN, creatinine, total protein... chemistries : glucose, electrolytes (Na+, K+, Cl-, and C02), BUN, creatinine, total protein, albumin, bilirubin, alkaline phosphatase, LDH, SGOT, SGPT, calcium...Roberts AB, Goodman DS (eds) The Retinoids: Biology, Chemistry , and Medicine. New York: Raven, 1994, pp. 573-596. 3. Hong WK, Itri LM. Retinoids and human

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

PubMed Central

Chia, Karin K. M.; Liu, Chia-Chi; Hamilton, Elisha J.; Garcia, Alvaro; Fry, Natasha A.; Hannam, William; Figtree, Gemma A.

2015-01-01

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current (Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22phox and p47phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. PMID:26084308

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

PubMed

Gomez-Samblas, M; García-Rodríguez, J J; Trelis, M; Bernal, D; Lopez-Jaramillo, F J; Santoyo-Gonzalez, F; Vilchez, S; Espino, A M; Bolás-Fernández, F; Osuna, A

2017-04-01

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa. © 2017 The Authors.

Acute Dietary Restriction Acts via TOR, PP2A, and Myc Signaling to Boost Innate Immunity in Drosophila.

PubMed

Lee, Jung-Eun; Rayyan, Morsi; Liao, Allison; Edery, Isaac; Pletcher, Scott D

2017-07-11

Dietary restriction promotes health and longevity across taxa through mechanisms that are largely unknown. Here, we show that acute yeast restriction significantly improves the ability of adult female Drosophila melanogaster to resist pathogenic bacterial infections through an immune pathway involving downregulation of target of rapamycin (TOR) signaling, which stabilizes the transcription factor Myc by increasing the steady-state level of its phosphorylated forms through decreased activity of protein phosphatase 2A. Upregulation of Myc through genetic and pharmacological means mimicked the effects of yeast restriction in fully fed flies, identifying Myc as a pro-immune molecule. Short-term dietary or pharmacological interventions that modulate TOR-PP2A-Myc signaling may provide an effective method to enhance immunity in vulnerable human populations. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Different designs of kinase-phosphatase interactions and phosphatase sequestration shapes the robustness and signal flow in the MAPK cascade

PubMed Central

2012-01-01

Background The three layer mitogen activated protein kinase (MAPK) signaling cascade exhibits different designs of interactions between its kinases and phosphatases. While the sequential interactions between the three kinases of the cascade are tightly preserved, the phosphatases of the cascade, such as MKP3 and PP2A, exhibit relatively diverse interactions with their substrate kinases. Additionally, the kinases of the MAPK cascade can also sequester their phosphatases. Thus, each topologically distinct interaction design of kinases and phosphatases could exhibit unique signal processing characteristics, and the presence of phosphatase sequestration may lead to further fine tuning of the propagated signal. Results We have built four architecturally distinct types of models of the MAPK cascade, each model with identical kinase-kinase interactions but unique kinases-phosphatases interactions. Our simulations unravelled that MAPK cascade’s robustness to external perturbations is a function of nature of interaction between its kinases and phosphatases. The cascade’s output robustness was enhanced when phosphatases were sequestrated by their target kinases. We uncovered a novel implicit/hidden negative feedback loop from the phosphatase MKP3 to its upstream kinase Raf-1, in a cascade resembling the B cell MAPK cascade. Notably, strength of the feedback loop was reciprocal to the strength of phosphatases’ sequestration and stronger sequestration abolished the feedback loop completely. An experimental method to verify the presence of the feedback loop is also proposed. We further showed, when the models were activated by transient signal, memory (total time taken by the cascade output to reach its unstimulated level after removal of signal) of a cascade was determined by the specific designs of interaction among its kinases and phosphatases. Conclusions Differences in interaction designs among the kinases and phosphatases can differentially shape the robustness and signal response behaviour of the MAPK cascade and phosphatase sequestration dramatically enhances the robustness to perturbations in each of the cascade. An implicit negative feedback loop was uncovered from our analysis and we found that strength of the negative feedback loop is reciprocally related to the strength of phosphatase sequestration. Duration of output phosphorylation in response to a transient signal was also found to be determined by the individual cascade’s kinase-phosphatase interaction design. PMID:22748295

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

PubMed Central

Beyer, Dániel; Tándor, Ildikó; Kónya, Zoltán; Bátori, Róbert; Roszik, Janos; Vereb, György; Erdődi, Ferenc; Vasas, Gábor; M-Hamvas, Márta; Jambrovics, Károly; Máthé, Csaba

2012-01-01

Background and Aims Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Methods Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Key Results Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL−1 MCY-LR, accelerated cell cycle at 10 µg mL−1 MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. Conclusions MCY-LR delayed metaphase–anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation. PMID:22819947

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba.

PubMed

Beyer, Dániel; Tándor, Ildikó; Kónya, Zoltán; Bátori, Róbert; Roszik, Janos; Vereb, György; Erdodi, Ferenc; Vasas, Gábor; M-Hamvas, Márta; Jambrovics, Károly; Máthé, Csaba

2012-09-01

Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL(-1) MCY-LR, accelerated cell cycle at 10 µg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.

PubMed

Liangpunsakul, Suthat; Wou, Sung-Eun; Zeng, Yan; Ross, Ruth A; Jayaram, Hiremagalur N; Crabb, David W

2008-12-01

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

NASA Astrophysics Data System (ADS)

Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

2004-08-01

The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

PubMed

Cheng, Jingwei; Park, Donglim Esther; Berrios, Christian; White, Elizabeth A; Arora, Reety; Yoon, Rosa; Branigan, Timothy; Xiao, Tengfei; Westerling, Thomas; Federation, Alexander; Zeid, Rhamy; Strober, Benjamin; Swanson, Selene K; Florens, Laurence; Bradner, James E; Brown, Myles; Howley, Peter M; Padi, Megha; Washburn, Michael P; DeCaprio, James A

2017-10-01

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.

PubMed

Diril, M Kasim; Bisteau, Xavier; Kitagawa, Mayumi; Caldez, Matias J; Wee, Sheena; Gunaratne, Jayantha; Lee, Sang Hyun; Kaldis, Philipp

2016-09-01

The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.

Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

PubMed Central

Kitagawa, Mayumi; Caldez, Matias J.; Gunaratne, Jayantha; Lee, Sang Hyun

2016-01-01

The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. PMID:27631493

Comparison of Microcystis aeruginosa (PCC7820 and PCC7806) growth and intracellular microcystins content determined by liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay anti-Adda and phosphatase bioassay.

PubMed

Ríos, V; Moreno, I; Prieto, A I; Soria-Díaz, M E; Frías, J E; Cameán, A M

2014-03-01

Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

PubMed Central

Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

2008-01-01

Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

PubMed

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-05-02

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair.

PubMed

Gripp, Karen W; Aldinger, Kimberly A; Bennett, James T; Baker, Laura; Tusi, Jessica; Powell-Hamilton, Nina; Stabley, Deborah; Sol-Church, Katia; Timms, Andrew E; Dobyns, William B

2016-09-01

Noonan syndrome is a rasopathy caused by mutations in multiple genes encoding components of the RAS/MAPK pathway. Despite its variable phenotype, limited genotype-phenotype correlations exist. Noonan syndrome with loose anagen hair (NS-LAH) is characterized by its distinctive hair anomalies, developmental differences, and structural brain abnormalities and is caused by a single recurrent missense SHOC2 mutation. SHOC2 forms a complex with protein phosphatase 1 (PP1C). Protein phosphatases counterbalance kinases and control activation of signaling proteins, such as the mitogen-activated protein kinases of the RAS/MAPK pathway. Here we report four patients with de novo missense mutations in protein phosphatase one catalytic subunit beta (PPP1CB), sharing a recognizable phenotype. Three individuals had the recurrent PPP1CB c.146G>C, p.Pro49Arg mutation, the fourth had a c.166G>C, p.Ala56Pro change. All had relative or absolute macrocephaly, low-set and posteriorly angulated ears, and developmental delay. Slow growing and/or sparse hair and/or an unruly hair texture was present in all. Three individuals had feeding difficulties requiring feeding tubes. One of two males had cryptorchidism, another had pectus excavatum. Short stature was present in three. A female with the recurrent mutation had a Dandy-Walker malformation and optic nerve hypoplasia. Mild ventriculomegaly occurred in all, cerebellar tonsillar ectopia was seen in two and progressed to Chiari 1 malformation in one individual. Based on the combination of phenotypic findings and PPP1CB's effect on RAF dephosphorylation within the RAS/MAPK pathway, this novel condition can be considered a rasopathy, most similar to NS-LAH. Collectively, these mutations meet the standardized criteria for pathogenicity. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Involvement of the K+-Cl- co-transporter KCC2 in the sensitization to morphine-induced hyperlocomotion under chronic treatment with zolpidem in the mesolimbic system.

PubMed

Shibasaki, Masahiro; Masukawa, Daiki; Ishii, Kazunori; Yamagishi, Yui; Mori, Tomohisa; Suzuki, Tsutomu

2013-06-01

Benzodiazepines are commonly used as sedatives, sleeping aids, and anti-anxiety drugs. However, chronic treatment with benzodiazepines is known to induce dependence, which is considered related to neuroplastic changes in the mesolimbic system. This study investigated the involvement of K(+) -Cl(-) co-transporter 2 (KCC2) in the sensitization to morphine-induced hyperlocomotion after chronic treatment with zolpidem [a selective agonist of γ-aminobutyric acid A-type receptor (GABAA R) α1 subunit]. In this study, chronic treatment with zolpidem enhanced morphine-induced hyperlocomotion, which is accompanied by the up-regulation of KCC2 in the limbic forebrain. We also found that chronic treatment with zolpidem induced the down-regulation of protein phosphatase-1 (PP-1) as well as the up-regulation of phosphorylated protein kinase C γ (pPKCγ). Furthermore, PP-1 directly associated with KCC2 and pPKCγ, whereas pPKCγ did not associate with KCC2. On the other hand, pre-treatment with furosemide (a KCC2 inhibitor) suppressed the enhancing effects of zolpidem on morphine-induced hyperlocomotion. These results suggest that the mesolimbic dopaminergic system could be amenable to neuroplastic change through a pPKCγ-PP-1-KCC2 pathway by chronic treatment with zolpidem. © 2013 International Society for Neurochemistry.

Free Fatty Acid-Induced PP2A Hyperactivity Selectively Impairs Hepatic Insulin Action on Glucose Metabolism

PubMed Central

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

Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast.

PubMed

Lucena, Rafael; Alcaide-Gavilán, Maria; Anastasia, Steph D; Kellogg, Douglas R

2017-03-04

Wee1 and Cdc25 are conserved regulators of mitosis. Wee1 is a kinase that delays mitosis via inhibitory phosphorylation of Cdk1, while Cdc25 is a phosphatase that promotes mitosis by removing the inhibitory phosphorylation. Although Wee1 and Cdc25 are conserved proteins, it has remained unclear whether their functions and regulation are conserved across diverse species. Here, we analyzed regulation of Wee1 and Cdc25 in fission yeast. Both proteins undergo dramatic cell cycle-dependent changes in phosphorylation that are dependent upon PP2A associated with the regulatory subunit Pab1. The mechanisms that control Wee1 and Cdc25 in fission yeast appear to share similarities to those in budding yeast and vertebrates, which suggests that there may be common mechanisms that control mitotic entry in all eukaryotic cells.

Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast

PubMed Central

2017-01-01

ABSTRACT Wee1 and Cdc25 are conserved regulators of mitosis. Wee1 is a kinase that delays mitosis via inhibitory phosphorylation of Cdk1, while Cdc25 is a phosphatase that promotes mitosis by removing the inhibitory phosphorylation. Although Wee1 and Cdc25 are conserved proteins, it has remained unclear whether their functions and regulation are conserved across diverse species. Here, we analyzed regulation of Wee1 and Cdc25 in fission yeast. Both proteins undergo dramatic cell cycle-dependent changes in phosphorylation that are dependent upon PP2A associated with the regulatory subunit Pab1. The mechanisms that control Wee1 and Cdc25 in fission yeast appear to share similarities to those in budding yeast and vertebrates, which suggests that there may be common mechanisms that control mitotic entry in all eukaryotic cells. PMID:28103117

Non-motor parkinsonian pathology in aging A53T α-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function.

PubMed

Farrell, Kaitlin F; Krishnamachari, Sesha; Villanueva, Ernesto; Lou, Haiyan; Alerte, Tshianda N M; Peet, Eloise; Drolet, Robert E; Perez, Ruth G

2014-02-01

Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α-synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α-synuclein for behavior and α-synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α-synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α-synuclein. Low adrenal PP2A activity co-occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α-synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α-synuclein pathology associated with pre-motor stages of PD. © 2013 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of The International Society for Neurochemistry.

Interactions of ABA signaling core components (SlPYLs, SlPP2Cs, and SlSnRK2s) in tomato (Solanum lycopersicon).

PubMed

Chen, Pei; Sun, Yu-Fei; Kai, Wen-Bin; Liang, Bin; Zhang, Yu-Shu; Zhai, Xia-Wan; Jiang, Li; Du, Yang-Wei; Leng, Ping

2016-10-20

Abscisic acid (ABA) regulates fruit development and ripening via its signaling. However, the exact role of ABA signaling core components in fruit have not yet been clarified. In this study, we investigated the potential interactions of tomato (Solanum lycopersicon) ABA signaling core components using yeast two-hybrid analysis, with or without ABA at different concentrations. The results showed that among 12 PYR/PYL/RCAR ABA receptors (SlPYLs), SlPYL1, SlPYL2, SlPYL4, SlPYL5, SlPYL 7, SlPYL8, SlPYL9, SlPYL10, SlPYL11, and SlPYL13 were ABA-dependent receptors, while SlPYL3 and SlPYL12 were ABA-independent receptors. Among five SlPP2Cs (type 2C protein phosphatases) and seven SlSnRK2s (subfamily 2 of SNF1-related kinases), all SlSnRK2s could interact with SlPP2C2, while SlSnRK2.8 also interacted with SlPP2C3. SlSnRK2.5 could interact with SlABF2/4 (ABA-responsive element binding factors). Expressions of SlPYL1, SlPYL2, SlPYL8, and SlPYL10 were upregulated under exogenous ABA but downregulated under nordihydroguaiaretic acid (NDGA) at the mature green stage of fruit ripening. The expressions of SlPP2C1, SlPP2C2, SlPP2C3, and SlPP2C5 were upregulated in ABA-treated fruit, but downregulated in NDGA-treated fruit at the mature green stage. The expressions of SlSnRK2.4, SlSnRK2.5, SlSnRK2.6, and SlSnRK2.7 were upregulated by ABA, but downregulated by NDGA. However, SlSnRK2.2 was down regulated by ABA. Expression of SlABF2/3/4 was enhanced by ABA but decreased by NDGA. Based on these results, we concluded that the majority of ABA receptor PYLs interact with SlPP2Cs in an ABA-dependent manner. SlPP2C2 and SlPP2C3 can interact with SlSnRK2s. SlSnRK2.5 could interact with SlABF2/4. Most ABA signaling core components respond to exogenous ABA. Copyright © 2016 Elsevier GmbH. All rights reserved.

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

PubMed Central

Tate, Jennifer J.; Cooper, Terrance G.

2013-01-01

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

Mir-338-3p Mediates Tnf-A-Induced Hepatic Insulin Resistance by Targeting PP4r1 to Regulate PP4 Expression.

PubMed

Dou, Lin; Wang, Shuyue; Sun, Libo; Huang, Xiuqing; Zhang, Yang; Shen, Tao; Guo, Jun; Man, Yong; Tang, Weiqing; Li, Jian

2017-01-01

Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression. © 2017 The Author(s). Published by S. Karger AG, Basel.

FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

PubMed Central

Chen, Jia; Yu, Feng; Liu, Ying; Du, Changqing; Li, Xiushan; Zhu, Sirui; Wang, Xianchun; Lan, Wenzhi; Rodriguez, Pedro L.; Liu, Xuanming; Li, Dongping; Chen, Liangbi; Luan, Sheng

2016-01-01

Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals. PMID:27566404

Pharmacological disruption of the MID1/α4 interaction reduces mutant Huntingtin levels in primary neuronal cultures.

PubMed

Monteiro, Olivia; Chen, Changwei; Bingham, Ryan; Argyrou, Argyrides; Buxton, Rachel; Pancevac Jönsson, Christina; Jones, Emma; Bridges, Angela; Gatfield, Kelly; Krauß, Sybille; Lambert, Jeremy; Langston, Rosamund; Schweiger, Susann; Uings, Iain

2018-04-23

Expression of mutant Huntingtin (HTT) protein is central to the pathophysiology of Huntington's Disease (HD). The E3 ubiquitin ligase MID1 appears to have a key role in facilitating translation of the mutant HTT mRNA suggesting that interference with the function of this complex could be an attractive therapeutic approach. Here we describe a peptide that is able to disrupt the interaction between MID1 and the α4 protein, a regulatory subunit of protein phosphatase 2A (PP2A). By fusing this peptide to a sequence from the HIV-TAT protein we demonstrate that the peptide can disrupt the interaction within cells and show that this results in a decrease in levels of ribosomal S6 phosphorylation and HTT expression in cultures of cerebellar granule neurones derived from Hdh Q111/Q7 mice. This data serves to validate this pathway and paves the way for the discovery of small molecule inhibitors of this interaction as potential therapies for HD. Copyright © 2018 Elsevier B.V. All rights reserved.

The relative hydrolytic reactivities of pyrophosphites and pyrophosphates.

PubMed

Mistry, Dharmit; Powles, Nicholas

2013-09-14

The pH-rate profiles for the hydrolysis of pyrophosphate (PP(V)) and pyrophosphite (PP(III), pyro-di-H-phosphonate) are a complex function of pH, reflecting the different ionic species and their relative reactivities. PP(III) is more reactive than PP(V) at all pHs and only PP(III) shows a hydroxide-ion reaction at high pH, so it is 10(10)-fold more reactive than PP(V) in 0.1 M NaOH. The pK(a2) of PP(III) ~0.44, so the dominant species at pH's > 1 is the di-anion PP(III)(2-). Although there is no observable (NMR or ITC) binding of Mg(2+) to the PP(III) di-anion there is a modest increase in the rate of hydrolysis of PP(III) by Mg(2+). PP(III) is neither a substrate nor an inhibitor of pyrophosphatase, the enzyme that efficiently catalyses the hydrolysis of PP(V).

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

The Neurospora crassa PP2A Regulatory Subunits RGB1 and B56 Are Required for Proper Growth and Development and Interact with the NDR Kinase COT1

PubMed Central

Shomin-Levi, Hila; Yarden, Oded

2017-01-01

COT1 is the founding member of the highly conserved nuclear Dbf2-related (NDR) Ser/Thr kinase family and plays a role in the regulation of polar growth and development in Neurospora crassa and other fungi. Changes in COT1 phosphorylation state have been shown to affect hyphal elongation, branching, and conidiation. The function of NDR protein kinases has been shown to be regulated by type 2A protein phosphatases (PP2As). PP2As are heterotrimers comprised of a catalytic and scaffolding protein along with an interchangeable regulatory subunit involved in determining substrate specificity. Inactivation of the N. crassa PP2A regulatory subunits rgb-1 and b56 conferred severe hyphal growth defects. Partial suppression of defects observed in the rgb-1RIP strain (but not in the Δb56 mutant) was observed in cot-1 phosphomimetic mutants, demonstrating that altering COT1 phosphorylation state can bypass, at least in part, the requirement of a functional RGB1 subunit. The functional fusion proteins RGB1::GFP and B56::GFP predominantly localized to hyphal tips and septa, respectively, indicating that their primary activity is in different cellular locations. COT1 protein forms exhibited a hyperphosphorylated gel migration pattern in an rgb-1RIP mutant background, similar to that observed when the fungus was cultured in the presence of the PP2A inhibitor cantharidin. COT1 was hypophosphorylated in a Δb56 mutant background, suggesting that this regulatory subunit may be involved in determining COT1 phosphorylation state, yet in an indirect manner. Reciprocal co-immunoprecipitation analyses, using tagged COT1, PPH1, RGB1, and B56 subunits established that these proteins physically interact. Taken together, our data determine the presence of a functional and physical link between PP2A and COT1 and show that two of the PP2A regulatory subunits interact with the kinase and determine COT1 phosphorylation state. PMID:28928725

FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome–positive acute lymphocytic leukemia

PubMed Central

Neviani, Paolo; Santhanam, Ramasamy; Oaks, Joshua J.; Eiring, Anna M.; Notari, Mario; Blaser, Bradley W.; Liu, Shujun; Trotta, Rossana; Muthusamy, Natarajan; Gambacorti-Passerini, Carlo; Druker, Brian J.; Cortes, Jorge; Marcucci, Guido; Chen, Ching-Shih; Verrills, Nicole M.; Roy, Denis C.; Caligiuri, Michael A.; Bloomfield, Clara D.; Byrd, John C.; Perrotti, Danilo

2007-01-01

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome–positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190BCR/ABL myeloid and lymphoid cell lines and CML-BCCD34+ and Ph1 ALLCD34+/CD19+ progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190BCR/ABL-driven [including p210/p190BCR/ABL (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients. PMID:17717597

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

PubMed

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

TGF-β1/Smad3 Pathway Targets PP2A-AMPK-FoxO1 Signaling to Regulate Hepatic Gluconeogenesis.

PubMed

Yadav, Hariom; Devalaraja, Samir; Chung, Stephanie T; Rane, Sushil G

2017-02-24

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TGF-β1/Smad3 Pathway Targets PP2A-AMPK-FoxO1 Signaling to Regulate Hepatic Gluconeogenesis*

PubMed Central

Yadav, Hariom; Devalaraja, Samir; Chung, Stephanie T.; Rane, Sushil G.

2017-01-01

Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. PMID:28069811

Copper(II) complexes of methimazole, an anti Grave's disease drug. Synthesis, characterization and its potential biological behavior as alkaline phosphatase inhibitor.

PubMed

Urquiza, Nora M; Manca, Silvia G; Moyano, María A; Dellmans, Raquel Arrieta; Lezama, Luis; Rojo, Teófilo; Naso, Luciana G; Williams, Patricia A M; Ferrer, Evelina G

2010-04-01

Methimazole (MeimzH) is an anti-thyroid drug and the first choice for patients with Grave's disease. Two new copper(II) complexes of this drug: [Cu(MeimzH)(2)(NO(3))(2)]*0.5H(2)O and [Cu(MeimzH)(2)(H(2)O)(2)](NO(3))(2)*H(2)O were synthesized and characterized by elemental analysis, dissolution behavior, thermogravimetric analysis and UV-vis, diffuse reflectance, FTIR and EPR spectroscopies. As it is known that copper(II) cation can act as an inhibitor of alkaline phosphatase (ALP), the inhibitory effect of methimazole and its copper(II) complexes on ALP activity has also been investigated.

Gadd34 Requirement for Normal Hemoglobin Synthesis

PubMed Central

Patterson, Andrew D.; Hollander, M. Christine; Miller, Georgina F.; Fornace, Albert J.

2006-01-01

The protein encoded by growth arrest and DNA damage-inducible transcript 34 (Gadd34) is associated with translation initiation regulation following certain stress responses. Through interaction with the protein phosphatase 1 catalytic subunit (PP1c), Gadd34 recruits PP1c for the removal of an inhibitory phosphate group on the α subunit of elongation initiation factor 2, thereby reversing the shutoff of protein synthesis initiated by stress-inducible kinases. In the absence of stress, the physiologic consequences of Gadd34 function are not known. Initial analysis of Gadd34-null mice revealed several significant findings, including hypersplenism, decreased erythrocyte volume, increased numbers of circulating erythrocytes, and decreased hemoglobin content, resembling some thalassemia syndromes. Biochemical analysis of the hemoglobin-producing reticulocyte (an erythrocyte precursor) revealed that the decreased hemoglobin content in the Gadd34-null erythrocyte is due to the reduced initiation of the globin translation machinery. We propose that an equilibrium state exists between Gadd34/PP1c and the opposing heme-regulated inhibitor kinase during hemoglobin synthesis in the reticulocyte. PMID:16478986

Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.

PubMed

Worby, Carolyn A; Gentry, Matthew S; Dixon, Jack E

2006-10-13

Laforin is the only phosphatase in the animal kingdom that contains a carbohydrate-binding module. Mutations in the gene encoding laforin result in Lafora disease, a fatal autosomal recessive neurodegenerative disorder, which is diagnosed by the presence of intracellular deposits of insoluble complex carbohydrates known as Lafora bodies. We demonstrate that laforin interacts with proteins known to be involved in glycogen metabolism and rule out several of these proteins as potential substrates. Surprisingly, we find that laforin displays robust phosphatase activity against a phosphorylated complex carbohydrate. Furthermore, this activity is unique to laforin, since several other phosphatases are unable to dephosphorylate polysaccharides. Finally, fusing the carbohydrate-binding module of laforin to the dual specific phosphatase VHR does not result in the ability of this phosphatase to dephosphorylate polysaccharides. Therefore, we hypothesize that laforin is unique in its ability to utilize a phosphorylated complex carbohydrate as a substrate and that this function may be necessary for the maintenance of normal cellular glycogen.

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

NASA Technical Reports Server (NTRS)

Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.;

A Recessive Contiguous Gene Deletion of Chromosome 2p16 Associated with Cystinuria and a Mitochondrial Disease

PubMed Central

Parvari, Ruti; Brodyansky, Irena; Elpeleg, Orly; Moses, Shimon; Landau, Daniel; Hershkovitz, Eli

2001-01-01

Deletions ranging from 100 Kb to 1 Mb—too small to be detected under the microscope—may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cβ gene (PP2Cβ), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes’ products, one of which may be essential for the synthesis of mitochondrial encoded proteins. PMID:11524703

A recessive contiguous gene deletion of chromosome 2p16 associated with cystinuria and a mitochondrial disease.

PubMed

Parvari, R; Brodyansky, I; Elpeleg, O; Moses, S; Landau, D; Hershkovitz, E

2001-10-01

Deletions ranging from 100 Kb to 1 Mb--too small to be detected under the microscope--may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cbeta gene (PP2Cbeta), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes' products, one of which may be essential for the synthesis of mitochondrial encoded proteins.

Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1)

PubMed Central

Filter, Joshua J; Williams, Byron C; Eto, Masumi; Shalloway, David; Goldberg, Michael L

2017-01-01

The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact inhibition by unfair competition: MLCP protects pCPI-17 from other phosphatases, while pCPI-17 blocks other substrates from MLCP’s active site. MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation. DOI: http://dx.doi.org/10.7554/eLife.24665.001 PMID:28387646

p21-Activated kinase-1 and its role in integrated regulation of cardiac contractility.

PubMed

Sheehan, Katherine A; Ke, Yunbo; Solaro, R John

2007-09-01

We review here a novel concept in the regulation of cardiac contractility involving variations in the activity of the multifunctional enzyme, p21-activated kinase 1 (Pak1), a member of a family of proteins in the small G protein-signaling pathway that is activated by Cdc42 and Rac1. There is a large body of evidence from studies in noncardiac tissue that Pak1 activity is key in regulation of a number of cellular functions, including cytoskeletal dynamics, cell motility, growth, and proliferation. Although of significant potential impact, the role of Pak1 in regulation of the heart has been investigated in only a few laboratories. In this review, we discuss the structure of Pak1 and its sites of posttranslational modification and molecular interactions. We assemble an overview of the current data on Pak1 signaling in noncardiac tissues relative to similar signaling pathways in the heart, and we identify potential roles of Pak1 in cardiac regulation. Finally, we discuss the current state of Pak1 research in the heart in regard to regulation of contractility through functional myofilament and Ca(2+)-flux modification. An important aspect of this regulation is the modulation of kinase and phosphatase activity. We have focused on Pak1 regulation of protein phosphatase 2A (PP2A), which is abundant in cardiac muscle, thereby mediating dephosphorylation of sarcomeric proteins and sensitizing the myofilaments to Ca(2+). We present a model for Pak1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by PP2A dephosphorylation predominates.

Sds22 regulates aurora B activity and microtubule–kinetochore interactions at mitosis

PubMed Central

Posch, Markus; Khoudoli, Guennadi A.; Swift, Sam; King, Emma M.; DeLuca, Jennifer G.

2010-01-01

We have studied Sds22, a conserved regulator of protein phosphatase 1 (PP1) activity, and determined its role in modulating the activity of aurora B kinase and kinetochore–microtubule interactions. Sds22 is required for proper progression through mitosis and localization of PP1 to mitotic kinetochores. Depletion of Sds22 increases aurora B T-loop phosphorylation and the rate of recovery from monastrol arrest. Phospho–aurora B accumulates at kinetochores in Sds22-depleted cells juxtaposed to critical kinetochore substrates. Sds22 modulates sister kinetochore distance and the interaction between Hec1 and the microtubule lattice and, thus, the activation of the spindle assembly checkpoint. These results demonstrate that Sds22 specifically defines PP1 function and localization in mitosis. Sds22 regulates PP1 targeting to the kinetochore, accumulation of phospho–aurora B, and force generation at the kinetochore–microtubule interface. PMID:20921135

[6]-Gingerol Affects Glucose Metabolism by Dual Regulation via the AMPKα2-Mediated AS160-Rab5 Pathway and AMPK-Mediated Insulin Sensitizing Effects.

PubMed

Lee, Jung Ok; Kim, Nami; Lee, Hye Jeong; Moon, Ji Wook; Lee, Soo Kyung; Kim, Su Jin; Kim, Joong Kwan; Park, Sun Hwa; Kim, Hyeon Soo

2015-07-01

[6]-Gingerol has been used to control diabetes and dyslipidemia; however, its metabolic role is poorly understood. In this study, [6]-gingerol increased adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation in mouse skeletal muscle C2C12 cells. Stimulation of glucose uptake by [6]-gingerol was dependent on AMPKα2. Moreover, both Inhibition and knockdown of AMPKα2 blocked [6]-gingerol-induced glucose uptake. [6]-Gingerol significantly decreased the activity of protein phosphatase 2A (PP2A). Inhibition of PP2A activity with okadaic acid enhanced the phosphorylation of AMPKα2. Moreover, the interaction between AMPKα2 and PP2A was increased by [6]-gingerol, suggesting that PP2A mediates the effect of [6]-gingerol on AMPK phosphorylation. In addition, [6]-gingerol increased the phosphorylation of Akt-substrate 160 (AS160), which is a Rab GTPase-activating protein. Inhibition of AMPKα2 blocked [6]-gingerol-induced AS160 phosphorylation. [6]-gingerol increased the Rab5, and AMPKα2 knockdown blocked [6]-gingerol-induced expression of Rab5, indicating AMPK play as an upstream of Rab5. It also increased glucose transporter 4 (GLUT4) mRNA and protein expression and stimulated GLUT4 translocation. Furthermore, insulin-mediated glucose uptake and Akt phosphorylation were further potentiated by [6]-gingerol treatment. This potentiation was not observed in the presence of AMPK inhibitor compound C. In summary, our results suggest that [6]-gingerol plays an important role in glucose metabolism via the AMPKα2-mediated AS160-Rab5 pathway and through potentiation of insulin-mediated glucose regulation. © 2015 Wiley Periodicals, Inc.

The ‘spiteful’ origins of human cooperation

PubMed Central

Marlowe, Frank W.; Berbesque, J. Colette; Barrett, Clark; Bolyanatz, Alexander; Gurven, Michael; Tracer, David

2011-01-01

We analyse generosity, second-party (‘spiteful’) punishment (2PP), and third-party (‘altruistic’) punishment (3PP) in a cross-cultural experimental economics project. We show that smaller societies are less generous in the Dictator Game but no less prone to 2PP in the Ultimatum Game. We might assume people everywhere would be more willing to punish someone who hurt them directly (2PP) than someone who hurt an anonymous third person (3PP). While this is true of small societies, people in large societies are actually more likely to engage in 3PP than 2PP. Strong reciprocity, including generous offers and 3PP, exists mostly in large, complex societies that face numerous challenging collective action problems. We argue that ‘spiteful’ 2PP, motivated by the basic emotion of anger, is more universal than 3PP and sufficient to explain the origins of human cooperation. PMID:21159680

The 'spiteful' origins of human cooperation.

PubMed

Marlowe, Frank W; Berbesque, J Colette; Barrett, Clark; Bolyanatz, Alexander; Gurven, Michael; Tracer, David

2011-07-22

We analyse generosity, second-party ('spiteful') punishment (2PP), and third-party ('altruistic') punishment (3PP) in a cross-cultural experimental economics project. We show that smaller societies are less generous in the Dictator Game but no less prone to 2PP in the Ultimatum Game. We might assume people everywhere would be more willing to punish someone who hurt them directly (2PP) than someone who hurt an anonymous third person (3PP). While this is true of small societies, people in large societies are actually more likely to engage in 3PP than 2PP. Strong reciprocity, including generous offers and 3PP, exists mostly in large, complex societies that face numerous challenging collective action problems. We argue that 'spiteful' 2PP, motivated by the basic emotion of anger, is more universal than 3PP and sufficient to explain the origins of human cooperation.

Tripodal polyphosphine ligands as inductors of chelate ring-opening processes in mononuclear palladium(II) and platinum(II) compounds. The X-ray crystal structure of two derivatives containing dangling phosphorus.

PubMed

Fernández-Anca, Damián; García-Seijo, M Inés; García-Fernández, M Esther

2010-03-07

The reaction of NP(3) (tris[2-(diphenylphosphino)ethyl]amine and PP(3) (tris[2-(diphenylphosphino)ethyl]phosphine) with the five-coordinate complexes [PdCl(NP(3))]Cl (1) and [MX(PP(3))]X [M = Pd: X = Cl(2), Br(3), I(4); M = Pt: X = Cl(5), Br(6), I(7)], respectively, followed by (31)P{(1)H}NMR when X = Cl, led to the formation of unprecedented four-coordinate halides in a 1 : 2 metal to ligand ratio, [M(AP(3))(2)]X(2) [A = N, M = Pd: X = Cl(8); A = P, M = Pd: X = Cl(9), Br(10), I(11); A = P, M = Pt: X = Cl(12), Br(13), I (14)], containing reactive dangling phosphorus. Given the non characterised precursors [M(ONO(2))(PP(3))](NO(3))], the interaction between the heteronuclear species [MAg(NO(3))(3)(PP(3))] [M = Pd(15), Pt(16)] and PP(3) was explored. It was found that the addition of 1 equivalent of phosphine afforded [MAg(NO(3))(PP(3))(2)](NO(3))(2) [M = Pd(15*), Pt(16*)] containing Ag(I) bound to two dangling phosphorus while the reaction with 2 equivalents led to the complexes [M(PP(3))(2)](NO(3))(2) [M = Pd (17), Pt (18)] in coexistence with [Ag(2)(mu-PP(3))(2)](NO(3))(2). The fate of Ag(I) on the reaction of the mixed metal compounds with excess PP(3) consisted of preventing dissociation, observed in solution for halides, and acting as an assistant for crystallization. Colourless single crystals of 18 and 10, studied by X-ray diffraction, were afforded by reaction of 16 with 4 equivalents of PP(3) and from solutions of 10 in chloroform coexisting with red crystals of 3, respectively. The structures revealed the presence of dications [M(PP(3))(2)](2+) that show two five-membered chelate rings to M(II) in a square-planar arrangement and four uncoordinated phosphine arms with the counter anions being symmetrically placed at 4.431 (Br(-)) and 13.823 (NO(3)(-)) A from M(II) above and below its coordination, MP(4), plane. Complexes 9 and 12 were shown to undergo an interesting reactivity in solution versus group 11 monocations. The reactions consisted of conversions of the two five-membered chelate rings to M into three (structure I) or two (structure II) fused five-membered chelate rings, formation of species where Pt(II) retained its square-planar environment with the two dangling phosphine arms of each PP(3) bound to Cu(I) or Ag(I) (structure III) and complexes bearing distorted square-planar (P(2)MCl(2)) and presumably tetrahedral (AuP(4)+ P(2)AuCl(2)) arrangements (structure IV). The processes with Ag(I) salts also gave mixtures of I+III (chloride and nitrate) or II+III (nitrate).

Target of rapamycin complex 1 and Tap42-associated phosphatases are required for sensing changes in nitrogen conditions in the yeast Saccharomyces cerevisiae.

PubMed

Li, Jinmei; Yan, Gonghong; Liu, Sichi; Jiang, Tong; Zhong, Mingming; Yuan, Wenjie; Chen, Shaoxian; Zheng, Yin; Jiang, Yong; Jiang, Yu

2017-12-01

In yeast target of rapamycin complex 1 (TORC1) and Tap42-associated phosphatases regulate expression of genes involved in nitrogen limitation response and the nitrogen discrimination pathway. However, it remains unclear whether TORC1 and the phosphatases are required for sensing nitrogen conditions. Utilizing temperature sensitive mutants of tor2 and tap42, we examined the role of TORC1 and Tap42 in nuclear entry of Gln3, a key transcription factor in yeast nitrogen metabolism, in response to changes in nitrogen conditions. Our data show that TORC1 is essential for Gln3 nuclear entry upon nitrogen limitation and downshift in nitrogen quality. However, Tap42-associated phosphatases are required only under nitrogen limitation condition. In cells grown in poor nitrogen medium, the nitrogen permease reactivator kinase (Npr1) inhibits TORC1 activity and alters its association with Tap42, rendering Tap42-associated phosphatases unresponsive to nitrogen limitation. These findings demonstrate a direct role for TORC1 and Tap42-associated phosphatases in sensing nitrogen conditions and unveil an Npr1-dependent mechanism that controls TORC1 and the phosphatases in response to changes in nitrogen quality. © 2017 John Wiley & Sons Ltd.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

PubMed Central

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-01-01

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

Eicosanoyl-5-hydroxytryptamide (EHT) prevents Alzheimer's disease-related cognitive and electrophysiological impairments in mice exposed to elevated concentrations of oligomeric beta-amyloid.

PubMed

Asam, Kesava; Staniszewski, Agnieszka; Zhang, Hong; Melideo, Scott L; Mazzeo, Adolfo; Voronkov, Michael; Huber, Kristen L; Pérez, Eduardo; Stock, Maxwell; Stock, Jeffry B; Arancio, Ottavio; Nicholls, Russell E

2017-01-01

Soluble forms of oligomeric beta-amyloid (Aβ) are thought to play a central role in Alzheimer's disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson's disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions.

Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway

PubMed Central

Jeong, Ae Lee; Han, Sora; Lee, Sunyi; Su Park, Jeong; Lu, Yiling; Yu, Shuangxing; Li, Jane; Chun, Kyung-Hee; Mills, Gordon B.; Yang, Young

2016-01-01

Mutation of PPP2R1A has been observed at high frequency in endometrial serous carcinomas but at low frequency in ovarian clear cell carcinoma. However, the biological role of mutation of PPP2R1A in ovarian and endometrial cancer progression remains unclear. In this study, we found that PPP2R1A expression is elevated in high-grade primary tumor patients with papillary serous tumors of the ovary. To determine whether increased levels or mutation of PPP2R1A might contribute to cancer progression, the effects of overexpression or mutation of PPP2R1A on cell proliferation, migration, and PP2A phosphatase activity were investigated using ovarian and endometrial cancer cell lines. Among the mutations, PPP2R1A-W257G enhanced cell migration in vitro through activating SRC-JNK-c-Jun pathway. Overexpression of wild type (WT) PPP2R1A increased its binding ability with B56 regulatory subunits, whereas PPP2R1A-mutations lost the ability to bind to most B56 subunits except B56δ. Total PP2A activity and PPP2R1A-associated PP2Ac activity were significantly increased in cells overexpressing PPP2R1A-WT. In addition, overexpression of PPP2R1A-WT increased cell proliferation in vitro and tumor growth in vivo. PMID:27272709

Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase.

PubMed

Gomes, Pedro; Saito, Tomoaki; Del Corsso, Cris; Alioua, Abderrahmane; Eghbali, Mansoureh; Toro, Ligia; Stefani, Enrico

2008-10-01

Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

Assembly and disassembly of the nucleolus during the cell cycle.

PubMed

Hernandez-Verdun, Danièle

2011-01-01

The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

PubMed

Cervinski, Mark A; Lee, Hong Kee; Martin, Isabella W; Gavrilov, Dimitar K

2015-02-02

Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Cadmium is a potent inhibitor of PPM phosphatases and targets the M1 binding site

PubMed Central

Pan, Chang; Liu, Hong-Da; Gong, Zheng; Yu, Xiao; Hou, Xu-Ben; Xie, Di-Dong; Zhu, Xi-Bin; Li, Hao-Wen; Tang, Jun-Yi; Xu, Yun-Fei; Yu, Jia-Qi; Zhang, Lian-Ying; Fang, Hao; Xiao, Kun-Hong; Chen, Yu-Guo; Wang, Jiang-Yun; Pang, Qi; Chen, Wei; Sun, Jin-Peng

2013-01-01

The heavy metal cadmium is a non-degradable pollutant. By screening the effects of a panel of metal ions on the phosphatase activity, we unexpectedly identified cadmium as a potent inhibitor of PPM1A and PPM1G. In contrast, low micromolar concentrations of cadmium did not inhibit PP1 or tyrosine phosphatases. Kinetic studies revealed that cadmium inhibits PPM phosphatases through the M1 metal ion binding site. In particular, the negative charged D441 in PPM1G specific recognized cadmium. Our results suggest that cadmium is likely a potent inhibitor of most PPM family members except for PHLPPs. Furthermore, we demonstrated that cadmium inhibits PPM1A-regulated MAPK signaling and PPM1G-regulated AKT signaling potently in vivo. Cadmium reversed PPM1A-induced cell cycle arrest and cadmium insensitive PPM1A mutant rescued cadmium induced cell death. Taken together, these findings provide a better understanding of the effects of the toxicity of cadmium in the contexts of human physiology and pathology. PMID:23903585

Histamine H2 receptor trafficking: role of arrestin, dynamin, and clathrin in histamine H2 receptor internalization.

PubMed

Fernandez, Natalia; Monczor, Federico; Baldi, Alberto; Davio, Carlos; Shayo, Carina

2008-10-01

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with beta-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.

The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

PubMed

Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

2009-02-01

The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

Increases of soil phosphatase and urease activities in potato fields by cropping rotation practices

USDA-ARS?s Scientific Manuscript database

Potato yield in Maine has remained relatively constant for over 50 years. To identify and quantify constraints to potato productivity, we established Status Quo (SQ), Soil Conserving (SC), Soil Improving (SI), Disease Suppressive (DS), and Continuous Potato (PP) cropping systems under both rainfed ...

Overlapping functions of bone sialoprotein and pyrophosphate regulators in directing cementogenesis.

PubMed

Ao, M; Chavez, M B; Chu, E Y; Hemstreet, K C; Yin, Y; Yadav, M C; Millán, J L; Fisher, L W; Goldberg, H A; Somerman, M J; Foster, B L

2017-12-01

Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PP i ), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PP i ) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PP i ) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp -/- ) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp -/- mice and Alpl -/- mice (the latter featuring elevated PP i and OPN), prompted us to examine whether BSP is operating by modulating PP i -associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PP i , altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PP i in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PP i by genetic ablation of Ank on the Bsp -/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp -/- ; Ank -/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank -/- mice. From these data, we conclude that the perturbations in PP i metabolism are not solely driving the cementum pathology in Bsp -/- mice, and that PP i is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PP i . We propose that BSP and PP i work in concert to direct mineralization in cementum and likely other mineralized tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Microcystin-LR Induced Reactive Oxygen Species Mediate Cytoskeletal Disruption and Apoptosis of Hepatocytes in Cyprinus carpio L.

PubMed Central

Jiang, Jinlin; Shan, Zhengjun; Xu, Weili; Wang, Xiaorong; Zhou, Junying; Kong, Deyang; Xu, Jing

2013-01-01

Microcystins (MCs) are a group of cyclic hepatotoxic peptides produced by cyanobacteria. Microcystin-LR (MC-LR) contains Leucine (L) and Arginine (R) in the variable positions, and is one of the most common and potently toxic peptides. MC-LR can inhibit protein phosphatase type 1 and type 2A (PP1 and PP2A) activities and induce excessive production of reactive oxygen species (ROS). The underlying mechanism of the inhibition of PP1 and PP2A has been extensively studied. The over-production of ROS is considered to be another main mechanism behind MC-LR toxicity; however, the detailed toxicological mechanism involved in over-production of ROS in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the hydroxyl radical (•OH) was significantly induced in the liver of carp after a relatively short-term exposure to MC-LR. The elevated reactive oxygen species (ROS) production may play an important role in the disruption of microtubule structure. Pre-injection of the antioxidant N-acetyl-cysteine (NAC) provided significant protection to the cytoskeleton, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction. In addition, the elevated ROS formation induced the expression of apoptosis-related genes, including p38, JNKa, and bcl-2. A significant increase in apoptotic cells was observed at 12 - 48 hours. Our study further supports evidence that ROS are involved in MC-LR induced damage to liver cells in carp, and indicates the need for further study of the molecular mechanisms behind MC-LR toxicity. PMID:24376844

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

PubMed Central

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

2011-01-01

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 (Gt), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po2 = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased Gt. 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 (H2O2). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced Gt increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H2O2 during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in Gt. In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in Gt and occludin reduction at the plasma membrane in AEC. PMID:21257729

Natural Variation in the Thermotolerance of Neural Function and Behavior due to a cGMP-Dependent Protein Kinase

PubMed Central

Dawson-Scully, Ken; Armstrong, Gary A.B.; Kent, Clement; Robertson, R. Meldrum; Sokolowski, Marla B.

2007-01-01

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations. PMID:17712421

Multiple Signals Regulate PLC beta 3 in Human Myometrial Cells

PubMed Central

Zhong, Miao; Murtazina, Dilyara A.; Phillips, Jennifer; Ku, Chun-Ying; Sanborn, Barbara M.

2008-01-01

Summary The regulation of PLCB3-Serine1105 phosphorylation by both negative feedback and negative crosstalk facilitates the integration of multiple signaling pathways in myometrial cells. Phospholipase CB3 (PLCB3) Serine1105, a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-Serine1105 phosphorylation (P-S1105) was studied in immortalized and primary human myometrial cells. CPT-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S1105. Relaxin also stimulated P-S1105; this effect was partially blocked by the protein kinase A (PRKA) inhibitor Rp-8-CPT-cAMPS. Oxytocin, which stimulates Gαq-mediated pathways, also rapidly increased P-S1105, as did PGF2α and ATP. Oxytocin-stimulated phosphorylation was blocked by the protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S1105, whereas the reverse was the case for the oxytocin-stimulated increase in P-S1105. PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 shRNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. Oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid but enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S1105A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S1105 dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium. PMID:18322273

An ABA-regulated and Golgi-localized protein phosphatase controls water loss during leaf senescence in Arabidopsis.

PubMed

Zhang, Kewei; Xia, Xiuying; Zhang, Yanyan; Gan, Su-Sheng

2012-02-01

It is known that a senescing leaf loses water faster than a non-senescing leaf and that ABA has an important role in promoting leaf senescence. However, questions such as why water loss is faster, how water loss is regulated, and how ABA functions in leaf senescence are not well understood. Here we report on the identification and functional analysis of a leaf senescence associated gene called SAG113. The RNA blot and GUS reporter analyses all show that SAG113 is expressed in senescing leaves and is induced by ABA in Arabidopsis. The SAG113 expression levels are significantly reduced in aba2 and abi4 mutants. A GFP fusion protein analysis revealed that SAG113 protein is localized in the Golgi apparatus. SAG113 encodes a protein phosphatase that belongs to the PP2C family and is able to functionally complement a yeast PP2C-deficient mutant TM126 (ptc1Δ). Leaf senescence is delayed in the SAG113 knockout mutant compared with that in the wild type, stomatal movement in the senescing leaves of SAG113 knockouts is more sensitive to ABA than that of the wild type, and the rate of water loss in senescing leaves of SAG113 knockouts is significantly reduced. In contrast, inducible over-expression of SAG113 results in a lower sensitivity of stomatal movement to ABA treatment, more rapid water loss, and precocious leaf senescence. No other aspects of growth and development, including seed germination, were observed. These findings suggest that SAG113, a negative regulator of ABA signal transduction, is specifically involved in the control of water loss during leaf senescence. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

PubMed Central

Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

2011-01-01

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

Effect of tibolone pretreatment on kinases and phosphatases that regulate the expression and phosphorylation of Tau in the hippocampus of rats exposed to ozone.

PubMed

Pinto-Almazan, Rodolfo; Segura-Uribe, Julia J; Soriano-Ursúa, Marvin A; Farfán-García, Eunice D; Gallardo, Juan M; Guerra-Araiza, Christian

2018-03-01

Oxidative stress (OS) is a key process in the development of many neurodegenerative diseases, memory disorders, and other pathological processes related to aging. Tibolone (TIB), a synthetic hormone used as a treatment for menopausal symptoms, decreases lipoperoxidation levels, prevents memory impairment and learning disability caused by ozone (O 3 ) exposure. However, it is not clear if TIB could prevent the increase in phosphorylation induced by oxidative stress of the microtubule-associated protein Tau. In this study, the effects of TIB at different times of administration on the phosphorylation of Tau, the activation of glycogen synthase kinase-3β (GSK3β), and the inactivation of Akt and phosphatases PP2A and PTEN induced by O 3 exposure were assessed in adult male Wistar rats. Rats were divided into 10 groups: control group (ozone-free air plus vehicle [C]), control + TIB group (ozone-free air plus TIB 1 mg/kg [C + TIB]); 7, 15, 30, and 60 days of ozone exposure groups [O 3 ] and 7, 15, 30, and 60 days of TIB 1 mg/kg before ozone exposure groups [O 3 + TIB]. The effects of O 3 exposure and TIB administration were assessed by western blot analysis of total and phosphorylated Tau, GSK3β, Akt, PP2A, and PTEN proteins and oxidative stress marker nitrotyrosine, and superoxide dismutase activity and lipid peroxidation of malondialdehyde by two different spectrophotometric methods (Marklund and TBARS, respectively). We observed that O 3 exposure increases Tau phosphorylation, which is correlated with decreased PP2A and PTEN protein levels, diminished Akt protein levels, and increased GSK3β protein levels in the hippocampus of adult male rats. The effects of O 3 exposure were prevented by the long-term treatment (over 15 days) with TIB. Malondialdehyde and nitrotyrosine levels increased from 15 to 60 days of exposure to O 3 in comparison to C group, and superoxide dismutase activity decreased. Furthermore, TIB administration limited the changes induced by O 3 exposure. Our results suggest a beneficial use of hormone replacement therapy with TIB to prevent neurodegeneration caused by O 3 exposure in rats.

Folic Acid Reduces Tau Phosphorylation by Regulating PP2A Methylation in Streptozotocin-Induced Diabetic Mice

PubMed Central

Zheng, Miaoyan; Zou, Chen; Li, Mengyue; Huang, Guowei; Gao, Yuxia; Liu, Huan

2017-01-01

High incidence rate of Alzheimer’s disease (AD) is observed in patients with type 2 diabetes. Aggregated β-amyloid (Aβ) and hyperphosphorylated tau are the hallmarks of AD. Hyperphosphorylated tau has been detected in diabetic animals as well as in diabetic patients. Folates mediate the transfer of one carbon unit, required in various biochemical reactions. The effect of folate on tau phosphorylation in diabetic models still remains unknown. In this study, we investigated the effect and mechanism of folic acid on hyperphosphorylation of tau in streptozotocin (STZ)-induced diabetic mice. Diabetic mice induced by STZ, at the age of 10 weeks, were administered with three levels of folic acid: folic acid-deficient diet, diet with normal folic acid content, and 120 μg/kg folic acid diet for 8 weeks. Levels of serum folate and blood glucose were monitored. Tau phosphorylation, protein phosphatase 2A (PP2A) methylation, and Glycogen synthase kinase 3β (GSK-3β) phosphorylation were detected using Western blot. The S-adenosyl methionine:S-adenosyl homocysteine ratio (SAM:SAH) in brain tissues was also determined. DNA methyltransferase (DNMT) mRNA expression levels were detected using real-time PCR. Folic acid reduced tau hyperphosphorylation at Ser396 in the brain of diabetes mellitus (DM) mice. In addition, PP2A methylation and DNMT1 mRNA expression were significantly increased in DM mice post folic acid treatment. GSK-3β phosphorylation was not regulated by folic acid administration. Folic acid can reduce tau phosphorylation by regulating PP2A methylation in diabetic mice. These results support that folic acid can serve as a multitarget neuronal therapeutic agent for treating diabetes-associated cognitive dysfunction. PMID:28422052

Defects in the acid phosphatase ACPT cause recessive hypoplastic amelogenesis imperfecta.

PubMed

Smith, Claire El; Whitehouse, Laura LE; Poulter, James A; Brookes, Steven J; Day, Peter F; Soldani, Francesca; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2017-08-01

We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

3D model for Cancerous Inhibitor of Protein Phosphatase 2A armadillo domain unveils highly conserved protein-protein interaction characteristics.

PubMed

Dahlström, Käthe M; Salminen, Tiina A

2015-12-07

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structure and function of the PP2A-shugoshin interaction

PubMed Central

Xu, Zheng; Cetin, Bulent; Anger, Martin; Cho, Uhn Soo; Helmhart, Wolfgang; Nasmyth, Kim; Xu, Wenqing

2009-01-01

SUMMARY Accurate chromosome segregation during mitosis and meiosis depends on shugoshin proteins that prevent precocious dissociation of cohesin from centromeres. Shugoshins associate with PP2A, which is thought to de-phosphorylate cohesin and thereby prevent cleavage by separase during meiosis I. A crystal structure of a complex between a fragment of human Sgo1 and an AB’C PP2A holoenzyme reveals that Sgo1 forms a homodimeric parallel coiled-coil that docks simultaneously onto PP2A’s C and B’ subunits. Sgo1 homo-dimerization is a pre-requisite for PP2A binding. While hSgo1 interacts only with the AB’C holoenzymes, its relative Sgo2 interacts with all PP2A forms and may thus lead to dephosphorylation of distinct substrates. Mutant shugoshin proteins defective in the binding of PP2A cannot protect centromeric cohesin from separase during meiosis I or support the spindle assembly checkpoint in yeast. Finally, we provide evidence that PP2A’s recruitment to chromosomes may be sufficient to protect cohesin from separase in mammalian oocytes. PMID:19716788

Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

PubMed

Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

2011-07-10

Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

Cytochemical localization of phosphatases in the germ- and Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae).

PubMed

Fernandes, A P; Báo, S N

1998-08-01

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon, as well as in Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae). Acid phosphatase was found in the acrosome. Thiamine pyrophosphatase was observed in the Golgi cisternae and in the tail spermatozoon surface. Glucose-6-phosphatase was located in the membrane complex of the acrosomal region. Already, in the Sertoli cells acid phosphatase was located in the lysosomes and glucose-6-phosphatase was observed in association with the endoplasmic reticulum and Golgi complex. These observations support the idea that various phosphatases may play some role in spermatid differentiation and in the interactions germ cells--Sertoli cells during spermiogenesis process.

Polyoma small T antigen triggers cell death via mitotic catastrophe

PubMed Central

Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

2014-01-01

Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

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

PubMed Central

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

2015-01-01

Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. PMID:25399018

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase-1 and phosphatase-2A with peptides.

PubMed

Köhn, Maja

2017-10-01

Protein phosphatase-1 and phosphatase-2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide-based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org, the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling.

PubMed

Gürsel, Demirkan B; Banu, Matei A; Berry, Nicholas; Marongiu, Roberta; Burkhardt, Jan-Karl; Kobylarz, Keith; Kaplitt, Michael G; Rafii, Shahin; Boockvar, John A

2015-01-01

Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.

Microcystins Alter Chemotactic Behavior in Caenorhabditis elegans by Selectively Targeting the AWA Sensory Neuron

PubMed Central

Moore, Caroline E.; Lein, Pamela J.; Puschner, Birgit

2014-01-01

Harmful algal blooms expose humans and animals to microcystins (MCs) through contaminated drinking water. While hepatotoxicity following acute exposure to MCs is well documented, neurotoxicity after sub-lethal exposure is poorly understood. We developed a novel statistical approach using a generalized linear model and the quasibinomial family to analyze neurotoxic effects in adult Caenorhabditis elegans exposed to MC-LR or MC-LF for 24 h. Selective effects of toxin exposure on AWA versus AWC sensory neuron function were determined using a chemotaxis assay. With a non-monotonic response MCs altered AWA but not AWC function, and MC-LF was more potent than MC-LR. To probe a potential role for protein phosphatases (PPs) in MC neurotoxicity, we evaluated the chemotactic response in worms exposed to the PP1 inhibitor tautomycin or the PP2A inhibitor okadaic acid for 24 h. Okadaic acid impaired both AWA and AWC function, while tautomycin had no effect on function of either neuronal cell type at the concentrations tested. These findings suggest that MCs alter the AWA neuron at concentrations that do not cause AWC toxicity via mechanisms other than PP inhibition. PMID:24918360

Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias.

PubMed

Haghighi, Kobra; Pritchard, Tracy J; Liu, Guan-Sheng; Singh, Vivek P; Bidwell, Philip; Lam, Chi Keung; Vafiadaki, Elizabeth; Das, Parthib; Ma, Jianyong; Kunduri, Swati; Sanoudou, Despina; Florea, Stela; Vanderbilt, Erica; Wang, Hong-Shang; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2015-12-01

A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/- Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human G109E-Inhibitor-1 Impairs Cardiac Function and Promotes Arrhythmias

PubMed Central

Haghighi, Kobra; Pritchard, Tracy J.; Liu, Guan-Sheng; Singh, Vivek P.; Bidwell, Philip; Lam, Chi Keung; Vafiadaki, Elizabeth; Das, Parthib; Ma, Jianyong; Kunduri, Swati; Sanoudou, Despina; Florea, Stela; Vanderbilt, Erica; Wang, Hong-Shang; Rubinstein, Jack; Hajjar, Roger J.; Kranias, Evangelia G.

2015-01-01

A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2 Hz +/− Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers. PMID:26455482

Sodium pyrophosphate enhances iron bioavailability from bouillon cubes fortified with ferric pyrophosphate.

PubMed

Cercamondi, Colin I; Duchateau, Guus S M J E; Harika, Rajwinder K; van den Berg, Robin; Murray, Peter; Koppenol, Wieneke P; Zeder, Christophe; Zimmermann, Michael B; Moretti, Diego

2016-08-01

Fe fortification of centrally manufactured and frequently consumed condiments such as bouillon cubes could help prevent Fe deficiency in developing countries. However, Fe compounds that do not cause sensory changes in the fortified product, such as ferric pyrophosphate (FePP), exhibit low absorption in humans. Tetra sodium pyrophosphate (NaPP) can form soluble complexes with Fe, which could increase Fe bioavailability. Therefore, the aim of this study was to investigate Fe bioavailability from bouillon cubes fortified with either FePP only, FePP+NaPP, ferrous sulphate (FeSO4) only, or FeSO4+NaPP. We first conducted in vitro studies using a protocol of simulated digestion to assess the dialysable and ionic Fe, and the cellular ferritin response in a Caco-2 cell model. Second, Fe absorption from bouillon prepared from intrinsically labelled cubes (2·5 mg stable Fe isotopes/cube) was assessed in twenty-four Fe-deficient women, by measuring Fe incorporation into erythrocytes 2 weeks after consumption. Fe bioavailability in humans increased by 46 % (P<0·005) when comparing bouillons fortified with FePP only (4·4 %) and bouillons fortified with FePP+NaPP (6·4 %). Fe absorption from bouillons fortified with FeSO4 only and with FeSO4+NaPP was 33·8 and 27·8 %, respectively (NS). The outcome from the human study is in agreement with the dialysable Fe from the in vitro experiments. Our findings suggest that the addition of NaPP could be a promising strategy to increase Fe absorption from FePP-fortified bouillon cubes, and if confirmed by further research, for other fortified foods with complex food matrices as well.

Strong enhancement effect of silver nanowires on fluorescent property of Eu3+-ligand complexes and desired fluorescent iPP composite materials

NASA Astrophysics Data System (ADS)

Zhang, Yu; Wang, Xinzhi; Tang, Jianguo; Wang, Wei; Wang, Jinping; Belfiore, Laurence A.

2017-04-01

In this contribution, we obtained the strong enhancement effect of silver nanowires(AgNWs) on fluorescent property of Eu3+-antenna complexes through the function of the surface plasmon resonance(SPR) effect. The key structural characteristics are: (1) AgNWs are covered by the Eu3+-ligand complex and spaced by SiO2 nano-layer between AgNWs and Eu3+-ligand complex (this structure is marked as AgNWs@SiO2@EuTP), and (2) AgNWs as nano-material with large ratio of length to diameter show their good dispersion and processability in isotactic polypropylene (iPP). We obtained the important data about the optimal spacer thickness of SiO2 is 15 nm that was not found in previous publications. The enhanced intensity of fluorescence of EuTP by AgNWs in AgNWs@SiO2@EuTP is 9 times compared with that of EuTP. All of these outstanding properties and fine structures were characterized by TEM, FT-IR, XRD, and fluorescence spectrophotometer. On the other hand, the desired fluorescent iPP composite material was obtained through blending AgNWs@SiO2@EuTP into iPP host. Very importantly, the enhancement effect of AgNWs on EuTP fluorescence in AgNWs@SiO2@EuTP is refrained from the quenching caused by host polymer of iPP.

Eicosanoyl-5-hydroxytryptamide (EHT) prevents Alzheimer’s disease-related cognitive and electrophysiological impairments in mice exposed to elevated concentrations of oligomeric beta-amyloid

PubMed Central

Asam, Kesava; Staniszewski, Agnieszka; Zhang, Hong; Melideo, Scott L.; Mazzeo, Adolfo; Voronkov, Michael; Huber, Kristen L.; Pérez, Eduardo; Stock, Maxwell; Stock, Jeffry B.; Arancio, Ottavio

2017-01-01

Soluble forms of oligomeric beta-amyloid (Aβ) are thought to play a central role in Alzheimer’s disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson’s disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions. PMID:29253878

Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved.

PubMed

Ramos, Ana Raquel; Elong Edimo, William's; Erneux, Christophe

2018-01-01

Inositol polyphosphate 5-phosphatases or phosphoinositide 5-phosphatases (PI 5-phosphatases) are enzymes that can act on soluble inositol phosphates and/or phosphoinositides (PIs). Several PI 5-phosphatases have been linked to human genetic diseases, in particular the Lowe protein or OCRL which is mutated in the Lowe syndrome. There are 10 different members of this family and 9 of them can use PIs as substrate. One of these substrates, PI(3,4,5)P3 binds to specific PH domains and recruits as effectors specific proteins to signaling complexes. Protein kinase B is one target protein and activation of the kinase will have a major impact on cell proliferation, survival and cell metabolism. Two other PIs, PI(4,5)P2 and PI(3,4)P2, are produced or used as substrates of PI 5-phosphatases (OCRL, INPP5B, SHIP1/2, SYNJ1/2, INPP5K, INPP5J, INPP5E). The inositol lipids may influence many aspects of cytoskeletal organization, lamellipodia formation and F-actin polymerization. PI 5-phosphatases have been reported to control cell migration, adhesion, polarity and cell invasion particularly in cancer cells. In glioblastoma, reducing SHIP2 expression can positively or negatively affect the speed of cell migration depending on the glioblastoma cell type. The two PI 5-phosphatases SHIP2 or SKIP could be localized at the plasma membrane and can reduce either PI(3,4,5)P3 or PI(4,5)P2 abundance. In the glioblastoma 1321 N1 cells, SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

PubMed

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand; Apte, Shree Kumar

2016-08-15

Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

PubMed Central

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand

2016-01-01

ABSTRACT Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. PMID:27287317

EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases.

PubMed

Wang, Yongbo; Liu, Zexian; Cheng, Han; Gao, Tianshun; Pan, Zhicheng; Yang, Qing; Guo, Anyuan; Xue, Yu

2014-01-01

We present here EKPD (http://ekpd.biocuckoo.org), a hierarchical database of eukaryotic protein kinases (PKs) and protein phosphatases (PPs), the key molecules responsible for the reversible phosphorylation of proteins that are involved in almost all aspects of biological processes. As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50,000 PKs and >10,000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript.

Yersinia enterocolitica YopH-Deficient Strain Activates Neutrophil Recruitment to Peyer's Patches and Promotes Clearance of the Virulent Strain.

PubMed

Dave, Mabel N; Silva, Juan E; Eliçabe, Ricardo J; Jeréz, María B; Filippa, Verónica P; Gorlino, Carolina V; Autenrieth, Stella; Autenrieth, Ingo B; Di Genaro, María S

2016-11-01

Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Role of src-family kinases in hypoxic vasoconstriction of rat pulmonary artery

PubMed Central

Knock, Greg A.; Snetkov, Vladimir A.; Shaifta, Yasin; Drndarski, Svetlana; Ward, Jeremy P.T.; Aaronson, Philip I.

2008-01-01

Aims We investigated the role of src-family kinases (srcFKs) in hypoxic pulmonary vasoconstriction (HPV) and how this relates to Rho-kinase-mediated Ca2+ sensitization and changes in intracellular Ca2+ concentration ([Ca2+]i). Methods and results Intra-pulmonary arteries (IPAs) were obtained from male Wistar rats. HPV was induced in myograph-mounted IPAs. Auto-phosphorylation of srcFKs and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and myosin light-chain (MLC20) in response to hypoxia were determined by western blotting. Translocation of Rho-kinase and effects of siRNA knockdown of src and fyn were examined in cultured pulmonary artery smooth muscle cells (PASMCs). [Ca2+]i was estimated in Fura-PE3-loaded IPA. HPV was inhibited by two blockers of srcFKs, SU6656 and PP2. Hypoxia enhanced phosphorylation of three srcFK proteins at Tyr-416 (60, 59, and 54 kDa, corresponding to src, fyn, and yes, respectively) and enhanced srcFK-dependent tyrosine phosphorylation of multiple target proteins. Hypoxia caused a complex, time-dependent enhancement of MYPT-1 and MLC20 phosphorylation, both in the absence and presence of pre-constriction. The sustained component of this enhancement was blocked by SU6656 and the Rho-kinase inhibitor Y27632. In PASMCs, hypoxia caused translocation of Rho-kinase from the nucleus to the cytoplasm, and this was prevented by anti-src siRNA and to a lesser extent by anti-fyn siRNA. The biphasic increases in [Ca2+]i that accompany HPV were also inhibited by PP2. Conclusion Hypoxia activates srcFKs and triggers protein tyrosine phosphorylation in IPA. Hypoxia-mediated Rho-kinase activation, Ca2+ sensitization, and [Ca2+]i responses are depressed by srcFK inhibitors and/or siRNA knockdown, suggesting a central role of srcFKs in HPV. PMID:18682436

Structural Determinants of Substrate Recognition in the HAD Superfamily Member D-glycero-D-manno-Heptose-1,7-bisphosphate Phosphatase (GmhB)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, H.; Wang, L; Huang, H

2010-01-01

The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 {angstrom} resolution), in a complex with Mg{supmore » 2+} and orthophosphate (1.8 {angstrom} resolution), and in a complex with Mg{sup 2+} and D-glycero-D-manno-heptose 1{beta},7-bisphosphate (2.2 {angstrom} resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg{sup 2+} and orthophosphate (1.7 {angstrom} resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.« less

The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes.

PubMed

Bovo, Elisa; Huke, Sabine; Blatter, Lothar A; Zima, Aleksey V

2017-03-01

Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca 2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca 2+ ] and intra-SR [Ca 2+ ] SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10μM) increased Ca 2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca 2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10μM) abolished the cAMP-dependent increase of SR Ca 2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca 2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca 2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca 2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca 2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca 2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca 2+ leak. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evolutionary and functional perspectives on signaling from neuronal surface to nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen, Samuel M.; Li, Boxing; Tsien, Richard W., E-mail: richard.tsien@nyumc.org

2015-04-24

Reliance on Ca{sup 2+} signaling has been well-preserved through the course of evolution. While the complexity of Ca{sup 2+} signaling pathways has increased, activation of transcription factors including CREB by Ca{sup 2+}/CaM-dependent kinases (CaMKs) has remained critical for long-term plasticity. In C. elegans, the CaMK family is made up of only three members, and CREB phosphorylation is mediated by CMK-1, the homologue of CaMKI. CMK-1 nuclear translocation directly regulates adaptation of thermotaxis behavior in response to changes in the environment. In mammals, the CaMK family has been expanded from three to ten members, enabling specialization of individual elements of amore » signal transduction pathway and increased reliance on the CaMKII subfamily. This increased complexity enables private line communication between Ca{sup 2+} sources at the cell surface and specific cellular targets. Using both new and previously published data, we review the mechanism of a γCaMKII-CaM nuclear translocation. This intricate pathway depends on a specific role for multiple Ca{sup 2+}/CaM-dependent kinases and phosphatases: α/βCaMKII phosphorylates γCaMKII to trap CaM; CaN dephosphorylates γCaMKII to dispatch it to the nucleus; and PP2A induces CaM release from γCaMKII so that CaMKK and CaMKIV can trigger CREB phosphorylation. Thus, while certain basic elements have been conserved from C. elegans, evolutionary modifications offer opportunities for targeted communication, regulation of key nodes and checkpoints, and greater specificity and flexibility in signaling.« less

Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP.

PubMed

Bartley, Christopher M; O'Keefe, Rachel A; Blice-Baum, Anna; Mihailescu, Mihaela-Rita; Gong, Xuan; Miyares, Laura; Karaca, Esra; Bordey, Angélique

2016-01-01

The fragile X mental retardation protein (FMRP) is an mRNA-binding regulator of protein translation that associates with 4-6% of brain transcripts and is central to neurodevelopment. Autism risk genes' transcripts are overrepresented among FMRP-binding mRNAs, and FMRP loss-of-function mutations are responsible for fragile X syndrome, the most common cause of monogenetic autism. It is thought that FMRP-dependent translational repression is governed by the phosphorylation of serine residue 499 (S499). However, recent evidence suggests that S499 phosphorylation is not modulated by metabotropic glutamate receptor class I (mGluR-I) or protein phosphatase 2A (PP2A), two molecules shown to regulate FMRP translational repression. Moreover, the mammalian FMRP S499 kinase remains unknown. We found that casein kinase II (CK2) phosphorylates murine FMRP S499. Further, we show that phosphorylation of FMRP S499 permits phosphorylation of additional, nearby residues. Evidence suggests that these nearby residues are modulated by mGluR-I and PP2A pathways. These data support an alternative phosphodynamic model of FMRP that is harmonious with prior studies and serves as a framework for further investigation.

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

PubMed Central

Cundell, Michael J.; Holder, James

2016-01-01

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

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

PubMed Central

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

2017-01-01

ABSTRACT The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR. PMID:28265001

Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

PubMed

Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

2013-04-17

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.