Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

DEFF Research Database (Denmark)

Powell, K A; Valova, V A; Malladi, C S

2000-01-01

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding ...... assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine...... the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus...

ATM phosphorylation of Mdm2 Ser394 regulates the amplitude and duration of the DNA damage response in mice

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Gannon, Hugh S.; Woda, Bruce A.; Jones, Stephen N.

2012-01-01

Summary DNA damage induced by ionizing radiation (IR) activates the ATM kinase, which subsequently stabilizes and activates the p53 tumor suppressor protein. Although phosphorylation of p53 by ATM was found previously to modulate p53 levels and transcriptional activities in vivo, it does not appear to be a major regulator of p53 stability. We have utilized mice bearing altered Mdm2 alleles to demonstrate that ATM phosphorylation of Mdm2 serine 394 is required for robust p53 stabilization and activation after DNA damage. In addition, we demonstrate that dephosphorylation of Mdm2 Ser394 regulates attenuation of the p53-mediated response to DNA damage. Therefore, the phosphorylation status of Mdm2 Ser394 governs p53 protein levels and functions in cells undergoing DNA damage. PMID:22624716

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

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Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

1996-01-01

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

PKCδ-mediated phosphorylation of BAG3 at Ser187 site induces epithelial-mesenchymal transition and enhances invasiveness in thyroid cancer FRO cells.

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Li, N; Du, Z-X; Zong, Z-H; Liu, B-Q; Li, C; Zhang, Q; Wang, H-Q

2013-09-19

Protein kinase C delta (PKCδ) is a serine (Ser)/threonine kinase, which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. In the current study, Chinese hamster ovary cells were transfected with either a constitutively activated PKCδ or a dominant negative PKCδ, phosphoprotein enrichment, two-dimensional difference gel electrophoresis and mass spectrometry was combined to globally identified candidates of PKCδ cascade. We found that Bcl-2 associated athanogene 3 (BAG3) was one of the targets of PKCδ cascade, and BAG3 interacted with PKCδ in vivo. In addition, we clarified that BAG3 was phosphorylate at Ser187 site in a PKCδ-dependent manner in vivo. BAG3 has been implicated in multiple cellular functions, including proliferation, differentiation, apoptosis, migration, invasion, macroautophagy and so on. We generated wild-type (WT)-, Ser187Ala (S187A)- or Ser187Asp (S187D)-BAG3 stably expressing FRO cells, and noticed that phosphorylation state of BAG3 influenced FRO morphology. Finally, for the first time, we showed that BAG3 was implicated in epithelial-mesenchymal transition (EMT) procedure, and phosphorylation state at Ser187 site had a critical role in EMT regulation by BAG3. Collectively, the current study indicates that BAG3 is a novel substrate of PKCδ, and PKCδ-mediated phosphorylation of BAG3 is implicated in EMT and invasiveness of thyroid cancer cells.

Effect of resistance exercise under conditions of reduced blood insulin on AMPKα Ser485/491 inhibitory phosphorylation and AMPK pathway activation.

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Kido, Kohei; Yokokawa, Takumi; Ato, Satoru; Sato, Koji; Fujita, Satoshi

2017-08-01

Insulin stimulates skeletal muscle glucose uptake via activation of the protein kinase B/Akt (Akt) pathway. Recent studies suggest that insulin downregulates AMP-activated protein kinase (AMPK) activity via Ser485/491 phosphorylation of the AMPK α-subunit. Thus lower blood insulin concentrations may induce AMPK signal activation. Acute exercise is one method to stimulate AMPK activation; however, no study has examined the relationship between blood insulin levels and acute resistance exercise-induced AMPK pathway activation. Based on previous findings, we hypothesized that the acute resistance exercise-induced AMPK pathway activation would be augmented by disruptions in insulin secretion through a decrease in AMPKα Ser485/491 inhibitory phosphorylation. To test the hypothesis, 10-wk-old male Sprague-Dawley rats were administered the toxin streptozotocin (STZ; 55 mg/kg) to destroy the insulin secreting β-cells. Three days postinjection, the right gastrocnemius muscle from STZ and control rats was subjected to resistance exercise by percutaneous electrical stimulation. Animals were killed 0, 1, or 3 h later; activation of the Akt/AMPK and downstream pathways in the muscle tissue was analyzed by Western blotting and real-time PCR. Notably, STZ rats showed a significant decrease in basal Akt and AMPKα Ser485/491 phosphorylation, but substantial exercise-induced increases in both AMPKα Thr172 and acetyl-CoA carboxylase (ACC) Ser79 phosphorylation were observed. Although no significant impact on resistance exercise-induced Akt pathway activation or glucose uptake was found, resistance exercise-induced peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 α (PGC-1α) gene expression was augmented by STZ treatment. Collectively, these data suggest that circulating insulin levels may regulate acute resistance exercise-induced AMPK pathway activation and AMPK-dependent gene expression relating to basal AMPKα Ser485/491 phosphorylation. Copyright © 2017

The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

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Fedosejevs, Eric T; Gerdis, Suzanne A; Ying, Sheng; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

2016-10-15

Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca 2+ -dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser 11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca 2+ -dependent Ser 11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca 2+ ions [K 0.5 (Ca 2+ ) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser 11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser 451 , RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca 2+ -signaling and the control of photosynthate partitioning during COS development. © 2016 The Author(s); published by Portland Press Limited on behalf of the

Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

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Reza Saberianfar

Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

C-188 cobalt-60 sealed source integrity: source monitoring

International Nuclear Information System (INIS)

Defalco, G.M.; Shah, V.

1995-01-01

The integrity of C-188 cobalt-60 sealed sources used for radiation processing will be a key factor in the continued industrial acceptance and growth of gamma irradiation technology. Given the public's relatively poor understanding of most nuclear topics and the news media's tendency to sensationalize events, it is appropriate for suppliers and users of gamma technology to be vigilant and conservative regarding the application of cobalt-60 sources to industrial purposes. Nordion's recent decision to extend the optional warranty on its C-188 cobalt-60 sealed source from 15 years to 20 years is based on over 30 years of data generated from its on-going SOURCE SURVEILLANCE PROGRAM. This paper presents an overview of the C-188 SOURCE SURVEILLANCE PROGRAM. (author)

S6K1 is involved in polyploidization through its phosphorylation at Thr421/Ser424.

Science.gov (United States)

Ma, Dongchu; Yu, Huiying; Lin, Di; Sun, Yinghui; Liu, Liping; Liu, Yage; Dai, Bing; Chen, Wei; Cao, Jianping

2009-04-01

Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole-induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole-induced Dami cell model, we found that 4E-BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M-phase in cytoplasm and oscillated in nocodazole-induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E-BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12-myristate 13-acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 --> Gln in PMA-induced Dami cells increased ploidy whereas overexpression of rapamycin-resistant form of S6K1 containing the mutations Thr421 --> Glu and Ser424 --> Asp significantly dephosphorylated 4E-BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA-induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin-resistant form of S6K1 significantly reversed polyploidization of nocodazole-induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole-induced Dami cells. Taken together, these data suggested that S6K1/4E-BP1 pathway may play an important role in polyploidization of megakaryocytes. (c) 2008 Wiley-Liss, Inc.

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

NARCIS (Netherlands)

Wijnker, P.J.M.; Sequeira Oliveira, V.; Witjas-Paalberends, E.R.; Foster, D.B.; dos Remedios, C.G.; Murphy, A.M.; Stienen, G.J.M.; van der Velden, J.

2014-01-01

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

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Hong Wa Yung

2011-03-01

Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

Science.gov (United States)

Yung, Hong Wa

2011-01-01

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling. PMID:21445305

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Science.gov (United States)

Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

2016-02-15

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

Science.gov (United States)

Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

2013-01-01

PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS using composite organic-inorganic nanoparticles (COINs.

Directory of Open Access Journals (Sweden)

Catherine M Shachaf

Full Text Available Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities.To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer. Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701 and Stat6 (Y641, with results comparable to flow cytometry.Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs).

Science.gov (United States)

Shachaf, Catherine M; Elchuri, Sailaja V; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N; Mitchell, Dennis J; Zhang, Jingwu; Swartz, Kenneth B; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P

2009-01-01

Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

Science.gov (United States)

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-06-07

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

Science.gov (United States)

Fang, Ling; Choudhary, Sanjeev; Zhao, Yingxin; Edeh, Chukwudi B; Yang, Chunying; Boldogh, Istvan; Brasier, Allan R.

2014-01-01

Ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol 3 kinase-like kinase family, is a master regulator of the double strand DNA break-repair pathway after genotoxic stress. Here, we found ATM serves as an essential regulator of TNF-induced NF-kB pathway. We observed that TNF exposure of cells rapidly induced DNA double strand breaks and activates ATM. TNF-induced ROS promote nuclear IKKγ association with ubiquitin and its complex formation with ATM for nuclear export. Activated cytoplasmic ATM is involved in the selective recruitment of the E3-ubiquitin ligase β-TrCP to phospho-IκBα proteosomal degradation. Importantly, ATM binds and activates the catalytic subunit of protein kinase A (PKAc), ribosmal S6 kinase that controls RelA Ser 276 phosphorylation. In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased. We further observed decreased binding and recruitment of the transcriptional elongation complex containing cyclin dependent kinase-9 (CDK9; a kinase necessary for triggering transcriptional elongation) to promoters of NF-κB-dependent immediate-early cytokine genes, in ATM knockdown cells. We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation. Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation. PMID:24957606

Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.

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Vichaiwong, Kanokwan; Purohit, Suneet; An, Ding; Toyoda, Taro; Jessen, Niels; Hirshman, Michael F; Goodyear, Laurie J

2010-10-15

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.

Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates

DEFF Research Database (Denmark)

Krintel, Christian; Osmark, Peter; Larsen, Martin Røssel

2008-01-01

Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well...

Stabilization of alanine substituted p53 protein at Ser15, Thr18, and Ser20 in response to ionizing radiation

International Nuclear Information System (INIS)

Yamauchi, Motohiro; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

2004-01-01

Phosphorylation of p53 at Ser15, Thr18, and Ser20 has been thought to be important for p53 stabilization in response to ionizing radiation. In the present study, we examined the X-ray-induced stabilization of Ala-substituted p53 protein at Ser15, Thr18, and Ser20, whose gene expression was controlled under an ecdyson-inducible promoter. We found that all single-, double-, or triple-Ala-substituted p53 at Ser15, Yhr18, and Ser20 were accumulated in the nucleus similarly to wild-type p53 after X-irradiation. These results indicate that the phosphorylation of p53 at Ser15, Thr18, and Ser20 is not necessarily needed for p53 stabilization in response to ionizing radiation

Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

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Giovanna Damia

2001-01-01

Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

Evaluating the relationship between amyloid-β and α-synuclein phosphorylated at Ser129 in dementia with Lewy bodies and Parkinson’s disease

OpenAIRE

Swirski, Marta; Miners, J Scott; de Silva, Rohan; Lashley, Tammaryn; Ling, Helen; Holton, Janice; Revesz, Tamas; Love, Seth

2014-01-01

Introduction Lewy body and Alzheimer-type pathologies often co-exist. Several studies suggest a synergistic relationship between amyloid-β (Aβ) and α-synuclein (α-syn) accumulation. We have explored the relationship between Aβ accumulation and the phosphorylation of α-syn at serine-129 (pSer129 α-syn), in post-mortem human brain tissue and in SH-SY5Y neuroblastoma cells transfected to overexpress human α-syn. Methods We measured levels of Aβ40, Aβ42, α-syn and pSer129 α-syn by sandwich enzyme...

Axin-mediated CKI phosphorylation of beta-catenin at Ser 45

DEFF Research Database (Denmark)

Amit, Sharon; Hatzubai, Ada; Birman, Yaara

2002-01-01

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin...... and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta......-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta...

The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts

NARCIS (Netherlands)

Bruins, Wendy; Bruning, Oskar; Jonker, Martijs J.; Zwart, Edwin; van der Hoeven, Tessa V.; Pennings, Jeroen L. A.; Rauwerda, Han; de Vries, Annemieke; Breit, Timo M.

2008-01-01

Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

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Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

2017-03-31

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G 1 , S, and G 2 ), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

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Ward Michael

2009-11-01

Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

Phosphorylated Akt Protein at Ser473 Enables HeLa Cells to Tolerate Nutrient-Deprived Conditions

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Fathy, Moustafa; Awale, Suresh; Nikaido, Toshio

2017-12-29

Background: Despite angiogenesis, many tumours remain hypovascular and starved of nutrients while continuing to grow rapidly. The specific biochemical mechanisms associated with starvation resistance, austerity, may be new biological characters of cancer that are critical for cancer progression. Objective: This study aim was to investigate the effect of nutrient starvation on HeLa cells and the possible mechanism by which the cells are able to tolerate nutrient-deprived conditions. Methods: Nutrient starvation was achieved by culturing HeLa cells in nutrient-deprived medium (NDM) and cell survival was estimated by using cell counting kit-8. The effect of starvation on cell cycle distribution and the quantitative analysis of apoptotic cells were investigated by flow cytometry using propidium iodide staining. Western blotting was used to detect the expression levels of Akt and phosphorylated Akt at Ser473 (Ser473p-Akt) proteins. Results: HeLa cells displayed extremely long survival when cultured in NDM. The percentage of apoptotic HeLa cells was significantly increased by starvation in a time-dependent manner. A significant increase in the expression of Ser473p-Akt protein after starvation was also observed. Furthermore, it was found that Akt inhibitor III molecule inhibited the cells proliferation in a concentration- and time-dependent manner. Conclusion: Results of the present study provide evidence that Akt activation may be implicated in the tolerance of HeLa cells for nutrient starvation and may help to suggest new therapeutic strategies designed to prevent austerity of cervical cancer cells through inhibition of Akt activation. Creative Commons Attribution License

In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

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Sommerfeld, Mark R; Metzger, Sabine; Stosik, Magdalene; Tennagels, Norbert; Eckel, Jürgen

2004-05-18

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis.

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Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

2014-01-01

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

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Hastie C James

2006-01-01

Full Text Available Abstract Background Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

OpenAIRE

Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

2003-01-01

The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

Hyper-O-GlcNAcylation of YB-1 affects Ser102 phosphorylation and promotes cell proliferation in hepatocellular carcinoma

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Liu, Qingqing [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Tao, Tao [Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Liu, Fang [Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu Province (China); Ni, Runzhou [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Lu, Cuihua, E-mail: lch1516@yeah.net [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Shen, Aiguo, E-mail: shag@ntu.edu.cn [Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu Province (China)

2016-12-10

As an essential post-translational modification, O-GlcNAcylation has been thought to be able to modulate various nuclear and cytoplasmic proteins and is emerging as a key regulator of multiple biological processes, such as transcription, cell growth, signal transduction, and cell motility. Recently, authoritative glycomics analyses have reported extensive crosstalk between O-GlcNAcylation and phosphorylation, which always dynamically interplay with each other and regulate signaling, transcription, and other cellular processes. Also, plentiful studies have shown close correlation between YB-1 phosphorylation and tumorigenesis. Therefore, our study aimed to determine whether YB-1 was O-GlcNAc modified and whether such modification could interact with its phosphorylation during the process of HCC development. Western blot and immunohistochemistry were firstly conducted to reveal obvious up-regulation of YB-1, OGT and O-GlcNAc modification in HCC tissues. What is more, not only YB-1 was identified to be O-GlcNAcylated but hyper-O-GlcNAcylation was demonstrated to facilitate HCC cell proliferation in a YB-1 dependent manner. Moreover, we detected four specific O-GlcNAc sites and confirmed T126A to be the most effective mutant in HCC cell proliferation via close O-GlcNAcylation-phosphorylation interaction. Even more interestingly, we discovered that T126A-induced HCC cell retardation and subdued transcriptional activity of YB-1 could be partially reversed by T126A/S102E mutant. From all above, it is not difficult to find that glycosylated-YB-1 mainly enhanced cell proliferation through congenerous actions with YB-1 phosphorylation and thus played indispensable roles in fine-tuning cell proliferation and procession of HCC. - Highlights: • YB-1 and OGT are associated with HCC prognosis. • YB-1 is O-GlcNAc modified in HCC. • Hyper-O-GlcNAcylation promotes HCC cell proliferation in dependent of YB-1. • The proliferating role of O-GlcNAcylation is based on Ser102

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

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Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

Directory of Open Access Journals (Sweden)

Chang-Ling Li

Full Text Available Megakaryocytes (MKs are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1 at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

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Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

International Nuclear Information System (INIS)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-01

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

Proteasome phosphorylation regulates cocaine-induced sensitization.

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Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

dbPAF: an integrative database of protein phosphorylation in animals and fungi.

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Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

2016-03-24

Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling.

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Barzegar, Mansoureh; Ma, Shuang; Zhang, Chao; Chen, Xin; Gu, Ying; Shang, Chaowei; Jiang, Xiaojuan; Yang, Jiao; Nathan, Cherie-Ann; Yang, Shengyong; Huang, Shile

2017-10-10

Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment. By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target. SKLB188 inhibited HNSCC cell proliferation by inducing G 1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21 Cip1 and p27 Kip1 ), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC 50 =5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose

Exercise increases TBC1D1 phosphorylation in human skeletal muscle

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Jessen, Niels; An, Ding; Lihn, Aina S.; Nygren, Jonas; Hirshman, Michael F.; Thorell, Anders

2011-01-01

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system.

Science.gov (United States)

Slama, Nawel; Leiba, Jade; Eynard, Nathalie; Daffé, Mamadou; Kremer, Laurent; Quémard, Annaïk; Molle, Virginie

2011-09-02

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition. Copyright © 2011 Elsevier Inc. All rights reserved.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor*

Science.gov (United States)

Collier, Mary E. W.; Ettelaie, Camille

2011-01-01

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF. PMID:21310953

Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

International Nuclear Information System (INIS)

Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

2014-01-01

Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

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En-Ju Chou

2016-03-01

Full Text Available CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity.

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

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Ni Zhu

2018-03-01

Full Text Available Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI, a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1, a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a

Rhenium-188: Availability from the W-188/Re-188 Generator and Status of Current Applications

International Nuclear Information System (INIS)

Pillai, M.R.A.; Dash, A.; Knapp, Russ F. Jr.

2012-01-01

Rhenium-188 is one of the most readily available generator derived and useful radionuclides for therapy emitting β-particles (2.12 MeV, 71.1% and 1.965 MeV, 25.6%) and imageable gammas (155 KeV, 15.1%). The 188W/188Re generator is an ideal source for the long term (4-6 months) continuous availability of no carrier added (nca) 188Re suitable for the preparation of radiopharmaceuticals for radionuclide therapy. The challenges associated with the double neutron capture route of production of the parent 188W radionuclide have been a major impediment in the progress of application of 188Re. Tungsten-188 of adequate specific activity can be prepared only in 2-3 of the high flux reactors operating in the World. Several useful technologies have been developed for the preparation of clinical grade 188W/188Re generator. Since the specific activity of 188W used in the generator is relatively low (<5 Ci/g), the eluted 188ReO4- can have low radioactive concentration often insufficient for radiopharmaceutical preparation. However, several efficient post elution concentration techniques have been developed that yield clinically useful 188ReO4-. Rhenium-188 has been used for the preparation of therapeutic radiopharmaceuticals for the management of diseases such as bone metastasis, rheumatoid arthritis and primary cancers. Several early phase clinical studies using radiopharmaceuticals based on 188Re-labeled phosphonates, antibodies, peptides, lipiodol and particulates have been reported. This article reviews the availability, and use of188Re including a discussion of why broader use of 188Re has not progressed as ecpected as a popular radionuclide for therapy.

Neighboring phosphoSer-Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding.

Science.gov (United States)

Rogals, Monique J; Greenwood, Alexander I; Kwon, Jeahoo; Lu, Kun Ping; Nicholson, Linda K

2016-12-01

The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 μm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 μm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1. © 2016 Federation of European

Phosphorylation of p53 at serine 15 in A549 pulmonary epithelial cells exposed to vanadate: Involvement of ATM pathway

International Nuclear Information System (INIS)

Suzuki, Katsura; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato

2007-01-01

When A549 cells were exposed to sodium metavanadate (NaVO 3 ), the pentavalent species of vanadium (vanadate), phosphorylation of p53 protein at Ser15 was found in a time (8-48 h)- and dose (10-200 μM)-dependent manner. After the incubation with 50 or 100 μM NaVO 3 for 48 h, accumulation of p53 protein was accompanied with Ser15 phosphorylation. Among serines in p53 protein immunoprecipitated from A549 cells treated with 100 μM NaVO 3 for 48 h, only Ser15 was markedly phosphorylated. Treatment with other vanadate compounds, sodium orthovanadate (Na 3 VO 4 ) and ammonium metavanadate (NH 4 VO 3 ), also induced Ser15 phosphorylation and accumulation of p53 protein. While phosphorylation of extracellular signal-regulated protein kinase (ERK) was found in cells treated with NaVO 3 , treatment with U0126 did not suppress Ser15 phosphorylation. On the other hand, treatment with wortmannin or caffeine, the inhibitors to phosphatidylinositol 3-kinase related kinases (PIKKs), suppressed both NaVO 3 -induced Ser15 phosphorylation and accumulation of p53 protein. The silencing of ataxia telangiectasia mutated (ATM) expression using short-interference RNA resulted in the marked suppression of Ser15 phosphorylation in A549 cells exposed to NaVO 3 . However, treatment with antioxidants such as catalase and N-acetylcysteine did not suppress NaVO 3 -induced Ser15 phosphorylation. Transcriptional activation of p53 and DNA fragmentation in A549 cells treated with NaVO 3 were suppressed only slightly by S15A mutation, suggesting that Ser15 phosphorylation is not essential for these responses. The present results showed that vanadate induces the phosphorylation of p53 at Ser15 depending on ATM, one of the members of PIKK family, in this human pulmonary epithelial cell line

Tyrosine Phosphorylation in Toll-Like Receptor Signaling

Science.gov (United States)

Chattopadhyay, Saurabh; Sen, Ganes C.

2014-01-01

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis.

Science.gov (United States)

Chou, En-Ju; Hung, Liang-Yi; Tang, Chieh-Ju C; Hsu, Wen-Bin; Wu, Hsin-Yi; Liao, Pao-Chi; Tang, Tang K

2016-03-29

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. Interestingly, the dispersal of the PCM was effectively rescued by ectopic expression of wild-type CPAP or a phospho-mimic CPAP-S467D mutant, but not a non-phosphorylated CPAP-S467A mutant. Finally, we found that CPAP-S467D has a low affinity for microtubule binding but a high affinity for PCM proteins. Together, our results support a model wherein CPAP is required for proper mitotic progression, and phosphorylation of CPAP by Aurora-A is essential for maintaining spindle pole integrity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at C-terminal serine residues

DEFF Research Database (Denmark)

Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B

2014-01-01

heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4....... Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser...

A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

Science.gov (United States)

Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

2012-08-01

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

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Teruya Tamaru

Full Text Available Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

Science.gov (United States)

Copeland, O'Neal; Sadayappan, Sakthivel; Messer, Andrew E; Steinen, Ger J M; van der Velden, Jolanda; Marston, Steven B

2010-12-01

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the

Src kinase regulation by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Roskoski, Robert

2005-01-01

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

Energy Technology Data Exchange (ETDEWEB)

Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Hampton-Smith, Rachel J.; Aloia, Amanda L. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia); Eddes, James S. [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Simpson, Kaylene J. [Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia); The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville (Australia); Hoffmann, Peter [Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia); Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide (Australia); Beard, Michael R. [School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia); Centre for Cancer Biology, SA Pathology, Adelaide (Australia)

2016-04-15

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

In-house Preparation of 188W/188Re Generators

International Nuclear Information System (INIS)

Sriwiang, Wiranee; Minsakorn, Napharat; Wardwilai, Charudej; Yindirum, Chareon; Sangsuriyan, Jatupol

2007-08-01

Full text: Low activity 188W/188Re generators were developed for the purpose of routinely supplying rhenium-188 for laboratory experiments and research works. Chromatography technology was applied in the construction of the generators. The chromatography columns containing 1.74 g alumina (Al2O3) were housed in plastic (PE) shielding inside the 30mm thick lead shield. Three generators were loaded with three different 188W activities; 25mCi (RG1), 25 mCi (RG2) and 50 mCi (RG3). Generator performances were determined in terms of elution yields, radiochemical purity, Al-breakthrough, 188W-breakthrough, and radionuclidic purity of the eluted products. These generators can be used for more than 6 months with average elution yields greater than 90 %. Radiochemical purity of 188ReO4- was high (> 99.7 % by ITLC), low level of 188W -breakthrough and low contamination of 192Ir and 191Os. There were only less than 1 ppm of 186W carrier and less than 3 ppm of Al-breakthrough. Key words: 188W/188Re, Generator, 188Re, Radionuclide

188W→188Re generator for biomedical applications

International Nuclear Information System (INIS)

Kamioki, Hiroshi; Mirzadeh, S.; Lambrecht, R.M.; Knapp, R. Jr.; Dadachova, K.

1994-01-01

An alumina-based 188 W → 188 Re generator was evaluated for 188 Re yield and elution profile and 188 W breakthrough using various reagents for a three-month period. To address the problem of low specific volume, the generator was eluted with reagents which could be easily destroyed by gentle evaporation from acidic solutions (e.g. NH 4 Cl and NH 4 NO 3 ). We also evaluated a proposed ''alumina/anion-exchange'' tandem generator for the preparation of highly purified 188 Re as perrhenic acid. In parallel studies, the adsorption dynamics of tungsten on alumina showed a sharp rise in the tungsten breakthrough at W/Al 2 O 3 ratio of ∼ 120 mg/g corresponding to a distribution constant of ∼ 8400 from nitrate solution at 0.05 M ionic strength. In adsorption on anion exchange resins, carrier-free 188 Re exhibited a behavior very similar to that of macroscopic quantities of Re. These studies further demonstrated the long and useful shelf-life of 188 W → 188 Re generator. (orig.)

Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer.

Science.gov (United States)

Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao

2017-02-24

Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The in vivo phosphorylation sites in multiple isoforms of amphiphysin I from rat brain nerve terminals

DEFF Research Database (Denmark)

Craft, George E; Graham, Mark E; Bache, Nicolai

2008-01-01

: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off......, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline...... that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE....

Phosphorylation coexists with O-GlcNAcylation in a plant virus protein and influences viral infection.

Science.gov (United States)

Martínez-Turiño, Sandra; Pérez, José De Jesús; Hervás, Marta; Navajas, Rosana; Ciordia, Sergio; Udeshi, Namrata D; Shabanowitz, Jeffrey; Hunt, Donald F; García, Juan Antonio

2018-06-01

Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O-GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N-terminus and the beginning of the core region. In contrast with the 'yin-yang' mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O-GlcNAcylated (serines Ser-25, Ser-81, Ser-101 and Ser-118). Our findings show that PPV CP can be concurrently phosphorylated and O-GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser-25 in virions recovered from O-GlcNAcylation-deficient plants, suggesting that crosstalk between O-GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O-GlcNAcylation in the N-proximal segment of CP allows a fine-tuning of protein stability, providing the amount of CP required in each step of viral infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

188W/188Re Generator System and Its Therapeutic Applications

Directory of Open Access Journals (Sweden)

A. Boschi

2014-01-01

Full Text Available The 188Re radioisotope represents a useful radioisotope for the preparation of radiopharmaceuticals for therapeutic applications, particularly because of its favorable nuclear properties. The nuclide decay pattern is through the emission of a principle beta particle having 2.12 MeV maximum energy, which is enough to penetrate and destroy abnormal tissues, and principle gamma rays (Eγ=155 keV, which can efficiently be used for imaging and calculations of radiation dose. 188Re may be conveniently produced by 188W/188Re generator systems. The challenges related to the double neutron capture reaction route to provide only modest yield of the parent 188W radionuclide indeed have been one of the major issues about the use of 188Re in nuclear medicine. Since the specific activity of 188W used in the generator is relatively low (<185 GBq/g, the eluted Re188O4- can have a low radioactive concentration, often ineffective for radiopharmaceutical preparation. However, several efficient postelution concentration techniques have been developed, which yield clinically useful Re188O4- solutions. This review summarizes the technologies developed for the preparation of 188W/188Re generators, postelution concentration of the 188Re perrhenate eluate, and a brief discussion of new chemical strategies available for the very high yield preparation of 188Re radiopharmaceuticals.

Protein Ser/Thr/Tyr phosphorylation in the Archaea.

Science.gov (United States)

Kennelly, Peter J

2014-04-04

The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of "eukaryotic" protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism.

Chromatographic 188W →188Re generator

International Nuclear Information System (INIS)

Khujaev, S.

2005-01-01

Full text: The main purpose of the generator - reception of daughter radioisotope Rhenium-188 from it by periodic elution for a long period of time (more than half-year). It is generally known that Rhenium-188, in the form of its complex connections, is applied in nuclear medicine in treatment and removal of painful syndromes. The generator possesses convenient nuclear-physical characteristics of a daughter radioisotope Rhenium-188. It is a source of (Irradiation with energy 2.12 MeV (98 %) with small contribution soft γ-radiation with energy 0.155 MeV (15 %). The Period of half-life destruction of radioisotope is 17 hours. The 188 W parent radioisotope for the generator is formed by irradiation of 186 W neutrons based on the following reaction: 186 W (n,γ) 187 W (n,γ) 188 W (69 days) → 188 Re (17 hours) + β The following were used as targets for irradiation: 1) Metal Tungsten (powder) of natural structure; 2) Metal Tungsten (plate) of natural structure; 3) Metal Tungsten (wire) of natural structure, d = 12 mm; 4) Metal Tungsten (powder) with enrichment on isotope 186 W - 99.79 %. The irradiated material was exposed to chemical processing with reception of radioactive solution of tungsten-188, from which sorption Tungsten was carried out onto sorbent as poly-wolframate-ions. It is established that Tungsten sorption depends on many factors as there are various chemical forms of Tungsten (VI) in water solutions, ratio of which depends on pH of the solution, concentration of Tungsten in the solution and presence of foreign ions. Tungsten sorption was carried out in static and in dynamic regimes. At dynamic regime the sorbent was placed directly in the generating column. The generator consisted of chromatographic columns with sorbent and radioisotope 188 W, eluting system and radiation protection. Rhenium-188 was taken from the generator as perrhenate sodium by elution of 0.9 % solution of chloride sodium in 10 ml. Technical characteristics of the generator

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation*

OpenAIRE

Nojiri, Mari; Loyet, Kelly M.; Klenchin, Vadim A.; Kabachinski, Gregory; Martin, Thomas F. J.

2009-01-01

CAPS (Ca2+-dependent activator protein for secretion) functions in priming Ca2+-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in...

Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

OpenAIRE

En-Ju Chou; Liang-Yi Hung; Chieh-Ju C. Tang; Wen-Bin Hsu; Hsin-Yi Wu; Pao-Chi Liao; Tang K. Tang

2016-01-01

CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. In...

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

Science.gov (United States)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-08

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

Science.gov (United States)

Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

2016-07-01

In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

Studies of HEDP labelled with 188Re from different generators of 188W /188Re

International Nuclear Information System (INIS)

Marczewski, Barbara Szot

2006-01-01

The widespread interest in 188 Re for therapeutic applications, is due to its attractive 16,9 hours half-life, emission of a β - particle with maximum energy of 2.12 MeV and gamma-ray of 155 keV suitable for imaging. This work presents the radiolabelling of HEDP (etidronate) with 188 Re eluted from alumina-based 188 W/ 188 Re generators and tungstate-based 188 W/ 188 Re gel generators. Dependence of the yield of the 18 '8Re-HEDP on the concentration of the reduction agent, p H, reaction time, temperature and addition of carrier Re 2 O 7 were evaluated. The radiolabelling of 188 Re-HEDP procedure using the optimum conditions resulted a yield >= 98% for liquid and lyophilized kits. This basic formulation contains: 30 mg de HEDP, 7 mg de SnCl 2 , 3 mg de ascorbic acid and addition of 20 mug of Re 2 O 7 . The reactions were carried out with heating in boiling water for 30 minutes followed by 60 minutes of incubation. Another important aspect of this work was the radiochemical quality control comparing the results of PC, TLC and ion chromatography, along with the experiments with HPLC. The biological distribution proved the adequate bone uptake and in vivo stability of 188 Re-HEDP complexes. (author)

Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

DEFF Research Database (Denmark)

Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

2007-01-01

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I

International Nuclear Information System (INIS)

Wang, Hui; Wang, Lin; Song, Li; Zhang, Yan-Wan; Ye, Jue; Xu, Rui-Xia; Shi, Na; Meng, Xian-Min

2013-01-01

The phosphorylation of cardiac troponin I (cTnI) plays an important role in the contractile dysfunction associated with heart failure. Human cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to investigate whether TNNI3K can phosphorylate cTnI at specific sites and to examine whether the phosphorylation of cTnI caused by TNNI3K can regulate cardiac myofilament contractile function. Co-immunoprecipitation was performed to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation was reduced when rTNNI3K was knocked down. To determine the contractile function modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and contribute to the regulation of cardiac myofilament contraction function

Targeting NF-κB RelA/p65 phosphorylation overcomes RITA resistance.

Science.gov (United States)

Bu, Yiwen; Cai, Guoshuai; Shen, Yi; Huang, Chenfei; Zeng, Xi; Cao, Yu; Cai, Chuan; Wang, Yuhong; Huang, Dan; Liao, Duan-Fang; Cao, Deliang

2016-12-28

Inactivation of p53 occurs frequently in various cancers. RITA is a promising anticancer small molecule that dissociates p53-MDM2 interaction, reactivates p53 and induces exclusive apoptosis in cancer cells, but acquired RITA resistance remains a major drawback. This study found that the site-differential phosphorylation of nuclear factor-κB (NF-κB) RelA/p65 creates a barcode for RITA chemosensitivity in cancer cells. In naïve MCF7 and HCT116 cells where RITA triggered vast apoptosis, phosphorylation of RelA/p65 increased at Ser536, but decreased at Ser276 and Ser468; oppositely, in RITA-resistant cells, RelA/p65 phosphorylation decreased at Ser536, but increased at Ser276 and Ser468. A phosphomimetic mutation at Ser536 (p65/S536D) or silencing of endogenous RelA/p65 resensitized the RITA-resistant cells to RITA while the phosphomimetic mutant at Ser276 (p65/S276D) led to RITA resistance of naïve cells. In mouse xenografts, intratumoral delivery of the phosphomimetic p65/S536D mutant increased the antitumor activity of RITA. Furthermore, in the RITA-resistant cells ATP-binding cassette transporter ABCC6 was upregulated, and silencing of ABCC6 expression in these cells restored RITA sensitivity. In the naïve cells, ABCC6 delivery led to RITA resistance and blockage of p65/S536D mutant-induced RITA sensitivity. Taken together, these data suggest that the site-differential phosphorylation of RelA/p65 modulates RITA sensitivity in cancer cells, which may provide an avenue to manipulate RITA resistance. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

International Nuclear Information System (INIS)

O'Hagan, Heather M.; Ljungman, Mats

2004-01-01

It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

DEFF Research Database (Denmark)

Rosenbaek, L L; Assentoft, M; Pedersen, N B

2012-01-01

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antib......The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho......-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and m...

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

Science.gov (United States)

Falk, Shaun P; Weisblum, Bernard

2013-03-01

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival.

Science.gov (United States)

McDonald, Paul C; Oloumi, Arusha; Mills, Julia; Dobreva, Iveta; Maidan, Mykola; Gray, Virginia; Wederell, Elizabeth D; Bally, Marcel B; Foster, Leonard J; Dedhar, Shoukat

2008-03-15

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH(2)- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser(473) phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser(473)P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser(473)P-Akt in the ILK complex. Expression of the NH(2)-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser(473) phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival.

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

Science.gov (United States)

Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

2010-05-15

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

Impairments in site-specific AS160 phosphorylation and effects of exercise training

DEFF Research Database (Denmark)

Consitt, Leslie A; Van Meter, Jessica; Newton, Christopher A

2013-01-01

The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult lifespan (18 to 84 years) and if endurance- and/or strength-oriented exercise training ...... population and that exercise training is an effective intervention for treating these impairments.......The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult lifespan (18 to 84 years) and if endurance- and/or strength-oriented exercise training...... in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666 and phospho-Akt substrate (PAS), but not Ser-318 or Ser-751. Twelve weeks of either endurance- or strength-oriented exercise training increased whole...

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

Science.gov (United States)

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

Directory of Open Access Journals (Sweden)

Laura Civiero

2017-12-01

Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

Science.gov (United States)

Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

2013-03-01

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

International Nuclear Information System (INIS)

Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

2012-01-01

Highlights: ► MG132 induces the phosphorylation of GSK3β Ser9 and, to a lesser extent, of GSK3β Thr390 . ► MG132 induces dephosphorylation of p70S6K Thr389 and phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 dephosphorylates GSK3β Ser9 and phosphorylates GSK3β Thr390 . ► Inactivation of p38 phosphorylates p70S6K Thr389 and increases the phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser 9 and, to a lesser extent, Thr 390 , the dephosphorylation of p70S6K at Thr 389 , and the phosphorylation of p70S6K at Thr 421 and Ser 424 . The specific p38 inhibitor SB203080 reduced the p-GSK3β Ser9 and autophagy through the phosphorylation of p70S6K Thr389 ; however, it augmented the levels of p-ERK, p-GSK3β Thr390 , and p-70S6K Thr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK Ser9 /p70S6K Thr380 and ERK/GSK3β Thr390 /p70S6K Thr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

Science.gov (United States)

Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A; Huganir, Richard L; Tao, Feng

2014-10-08

Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. Copyright © 2014 the authors 0270-6474/14/3413737-10$15.00/0.

Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

International Nuclear Information System (INIS)

Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

2002-01-01

Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

Phosphorylated form of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors

International Nuclear Information System (INIS)

Massias, J.F.; Hardouin, S.; Vieau, D.; Lenne, F.; Bertagna, X.

1994-01-01

Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed the precise study of the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH and their phosphorylated forms on SeR 31 . The authors have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser 31 on various RIA or immuno-radiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immuno-reactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser 31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIA's, nor of the ACTH IRMA. 15 refs., 5 figs., 2 tabs

Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

Directory of Open Access Journals (Sweden)

David Morales-Alamo

2018-03-01

Full Text Available Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH activation and reactive nitrogen and oxygen species (RNOS in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg or room air (PIO2 = 143 mmHg. Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively. However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18, but not after antioxidants ingestion (r = 0.35, P = 0.15. In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

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

Science.gov (United States)

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

Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

DEFF Research Database (Denmark)

Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

2011-01-01

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number...

A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation.

Science.gov (United States)

Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

2016-01-01

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3-AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

Protein kinase CK2 phosphorylates the Fas-associated factor FAF1 in vivo and influences its transport into the nucleus

DEFF Research Database (Denmark)

Olsen, Birgitte B; Jessen, Vibeke; Højrup, Peter

2003-01-01

We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site...... is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison...

Intensive training and reduced volume increases muscle FXYD1 expression and phosphorylation at rest and during exercise in athletes

DEFF Research Database (Denmark)

Thomassen, Martin; Gunnarsson, Thomas Gunnar Petursson; Christensen, Peter Møller

2016-01-01

and phosphorylation were determined by western blotting. Expression of FXYD1 (30%), actin (40%), mTOR (12%), PLN (16%) and CaMKII γ/δ (25%) was higher (P... and during exercise, mainly achieved by an increased FXYD1 ser68 phosphorylation, compared to before the intervention. CaMKII thr287 and eEF2 thr56 phosphorylation at rest and during exercise, overall PKCα/β thr638/641 and mTOR ser2448 phosphorylation during repeated intense exercise as well as resting PLN...

Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

Energy Technology Data Exchange (ETDEWEB)

Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

2012-02-24

Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

International Nuclear Information System (INIS)

Waldron, Richard T.; Whitelegge, Julian P.; Faull, Kym F.; Rozengurt, Enrique

2007-01-01

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic 32 P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains

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

Science.gov (United States)

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.

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

Science.gov (United States)

Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

Science.gov (United States)

Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

2016-01-01

Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

Directory of Open Access Journals (Sweden)

Tachibana Taro

2011-10-01

Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

Science.gov (United States)

Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

2014-05-01

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

International Nuclear Information System (INIS)

Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

2008-01-01

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity

Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

DEFF Research Database (Denmark)

Krintel, Christian; Mörgelin, Matthias; Logan, Derek T

2009-01-01

Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well......, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have...... a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than...

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

Science.gov (United States)

Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

Development of a technology for the preparation of 188W-188Re generators

International Nuclear Information System (INIS)

Oliveira, Alexandre de

2004-01-01

A big interest has recently arisen concerning the use of Rhenium-188 (188Re) for various medical applications. Tumor therapy with antibodies labeled with 188Re is the main application, but it is being studied its application in carcinomas of medullar thyroid, bone pain palliation and radionuclide synovectomy, among others. Rhenium-188 decays 79% to the ground state of stable 188Os (Eβ1max - 2,11 MeV) and 20% to the first excited state (Eβ2max = 1,97 MeV). The deexcitation of this state gives a 155 keV gamma ray (15r%) which can be detected by imaging. Another great advantage is the viability of carrier-free 188Re from the decay of 188W (t 1/2 = 69.4 days) in a generator system. The objective of this work is the development of the technology for the preparation of 188W- 188Re generators. To accomplish this, the steps of the work are: preparation of the targets of W; irradiation of W targets in order to measure the activation and radionuclidic impurities; development of 188W-188Re generators; development of a method for the quality control of 188Re: chemical, radiochemical and radionuclidic purities. The study of alumina-based generators was performed with the irradiation of targets of natural metallic W and W03 and showed that this kind of generator will only be viable with the importation of 188W, due to the low neutron flux of the Reactor IEA-R1 Reactor for the commercial routine production of this radioisotope, but the technology of production and quality control were successful. The gel type chromatographic generators of WZr were produced with natural WO3 targets and showed that, if enriched targets are to be used and with the power upgrade of the IEA-R1 Reactor, they can be produced by the Radiopharmacy Center at IPEN-SP. The quality control methodology were determined and the results were inside the limits given by the Pharmacopoeia. (author)

Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study

Energy Technology Data Exchange (ETDEWEB)

Rajagopal, P.; Klevit, R.E. [Univ. of Washington, Seattle, WA (United States)

1994-12-01

HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residue at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.

IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas tranplant recipient

DEFF Research Database (Denmark)

Bouzakri, K; Karlsson, HRK; Vestergaard, Henrik

2006-01-01

Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied....... In conclusion, peripheral insulin resistance in pancreas-kidney transplant recipients may arise from a negative feedback regulation of the canonical insulin-signaling cascade from excessive serine phosphorylation of IRS-1, possibly as a consequence of immunosuppressive therapy and hyperinsulinemia....... insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal...

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

Science.gov (United States)

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Energy Technology Data Exchange (ETDEWEB)

Freire, Paula Paccielli, E-mail: freirepp@hotmail.com; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil); Leopoldo, Ana Paula Lima; Leopoldo, André Soares [Centro de Educação Física e Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil)

2014-07-15

The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

International Nuclear Information System (INIS)

Freire, Paula Paccielli; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de; Leopoldo, Ana Paula Lima; Leopoldo, André Soares; Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos

2014-01-01

The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Directory of Open Access Journals (Sweden)

Paula Paccielli Freire

2014-07-01

Full Text Available Background: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP, alters the structure of protein kinase A (PKA and leads to phospholamban (PLB phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14, fed with normocaloric diet; and obese (n = 13, fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1, PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16 were assessed by Western blot. Results: Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Conclusion: Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation▿

Science.gov (United States)

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J.; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-01-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis. PMID:20870760

A review on the current status and production technology for 188W-188Re generator system

International Nuclear Information System (INIS)

Kuznetsov, R. A.; Han, H. S.; Cho, W. K.; Park, U. J.; Kim, Y. M.

1998-11-01

The current status of 188 W- 188 Re generator production technology were reviewed in PART 1. Main interests were given to the aspects of 188 W reactor production, irradiated targets reprocessing and generator loading technologies, such as alumina type and gel type generators. In order to develop the more convenient and advanced 188 W- 188 Re generator, further studies must be carried out to get the precise evaluation of production and burn-up cross section of 188 W, the more easily realizable generator loading procedure, and also to optimize the column and generator design to compensate the deterioration of generator performance because of parent radionuclide decay. By irradiation of 186 W enriched sample, 188 W- 188 Re generator production experiments were performed to evaluate the possibility of 188 W- 188 Re generator production using HANARO, and PART 2 describes about the experiments. The experimental results shows the possibility of practical 188 W- 188 Re generator production using of low-specific activity 188 W produced in HANARO. (author). 79 refs., 4 tabs., 26 figs

Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

Science.gov (United States)

Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

2012-01-01

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

EDUCAÇÃO INTEGRAL E ESPIRITUALIDADE: OS BENEFÍCIOS DESSA RELAÇÃO PARA UMA FORMAÇÃO INTEGRAL DO SER HUMANO

Directory of Open Access Journals (Sweden)

Thiago Dutra

2017-07-01

Full Text Available A educação integral com tempo estendido para sete horas diárias e um enfoque multidimensional do ser humano, se coloca como a concepção defendida pela legislação educacional brasileira e por pensadores contemporâneos do processo educacional, tais como Anísio Teixeira e Paulo Freire. O educando deve ser encarado enquanto ser complexo, formado por dimensões física, emocional, mental, social, cultural, e espiritual, sendo necessário que a educação foque no seu desenvolvimento integral. Na sociedade materialista de hoje a dimensão espiritual ficou excluída do ambiente escolar, ou delegada à marginalidade, o artigo procura demonstrar os benefícios e as possibilidades de desenvolvê-la na educação formal, levando em consideração os estudos de diversos autores, tais como Jean Piaget, Carl Jung, Ubiratan D’Ambrosio, entre outros.

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

2014-01-03

Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

International Nuclear Information System (INIS)

Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier; Pipkorn, Rüdiger; Ippel, Hans; Mayo, Kevin H.; Kübler, Dieter; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús

2014-01-01

Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with 15 N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein

Functionalized gold nanostars for label-free detection of PKA phosphorylation using surface-enhanced Raman spectroscopy

Science.gov (United States)

He, Shuai; Kah, James C. Y.

2017-04-01

Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.

Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

International Nuclear Information System (INIS)

Lund, Natalie; Henrion, Daniel; Tiede, Petra; Ziche, Marina; Schunkert, Heribert; Ito, Wulf D.

2010-01-01

The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p Ser239 -VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

Energy Technology Data Exchange (ETDEWEB)

Jang, Deok-Jin; Wang, Daojing

2006-05-26

Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

Issues associated with the use of the Tungsten-188/Rhenium188 generator and concentrator system and preparation of Re-188 HDD: A report

International Nuclear Information System (INIS)

Knapp, F.F.Jr.; Turner, J.H.; Jeong, J.-M.; Padhy, A.K.

2004-01-01

The ready availability of no-carrier-added Rhenium-188 from the Tungsten-188/Rhenium-188 generator represents an important source of a therapeutic radioisotope for a broad range of therapeutic applications in nuclear medicine, oncology, rheumatology and interventional cardiology. The International Atomic Energy Agency (IAEA) is coordinating a clinical trial involving the use of Rhenium188-Lipiodol for therapy of hepatocellular carcinoma. This report summarizes the experience of investigators at ten participating centres associated with the use and performance of the Tungsten-188/Rhenium-188 generators and the preparation and handling of the Re-188 HDD agent. This evaluation has demonstrated the cost effective provision of on-site therapeutic activities of Rhenium-188 and recommendations are made for further development of the next generator prototype in light of this international experience. The high bolus volumes (20-40 ml) of the ORNL generator requires post elution concentration of the Re-188 bolus by passage through the tandem silver cation/anion column system. The high back pressure often encountered during generator elution through the silver cation/anion concentrator system has been identified as a potential problem. The details of a method involving in house preparation of the silver cation columns were provided and implementation of this method for Re-188 bolus concentration is recommended. It is also recommended that ORNL investigators reassess the possibility of increasing Tungsten generator loading capacity and the use of higher specific activity Tungsten-188, with a view to reducing the generator bolus volume. The Re-188 HDD/Lipiodol conjugate?;ate is used in this IAEA trial for radioembolytic therapy of primary liver cancer, and methods for preparation of Re-188 HDD and its extraction into Lipiodol are discussed. Since Re-188 HDD binds to glass surfaces, the recovery yields are variable and can be as low as 40-45%. In an effort to maximize the

PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

Energy Technology Data Exchange (ETDEWEB)

Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

2014-03-10

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

International Nuclear Information System (INIS)

Kewley, Robyn J.; Whitelaw, Murray L.

2005-01-01

The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

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

Science.gov (United States)

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.

Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells.

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Massey, Katherine J; Li, Quanwen; Rossi, Noreen F; Keezer, Susan M; Mattingly, Raymond R; Yingst, Douglas R

2016-02-01

How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser(938) were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K · mg protein(-1) · min(-1) and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser(938) is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.

Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

International Nuclear Information System (INIS)

Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li; Li Junfa

2006-01-01

Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

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Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

2011-01-01

Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

Alternate Phosphorylation/O-GlcNAc Modification on Human Insulin IRSs: A Road towards Impaired Insulin Signaling in Alzheimer and Diabetes

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Zainab Jahangir

2014-01-01

Full Text Available Impaired insulin signaling has been thought of as important step in both Alzheimer’s disease (AD and type 2 diabetes mellitus (T2DM. Posttranslational modifications (PTMs regulate functions and interaction of insulin with insulin receptors substrates (IRSs and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR residues on IRSs. Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine (Ser and threonine (Thr residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated; however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize the risk of AD and T2DM.

LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

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April eReynolds

2014-06-01

Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma

International Nuclear Information System (INIS)

Li, Binbin; Huang, Guoliang; Zhang, Xiangning; Li, Rong; Wang, Jian; Dong, Ziming; He, Zhiwei

2013-01-01

Increased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC). The expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed. Immunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (χ 2 =6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1-induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation. EBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of

Hydroxyapatite based 99Mo-99mTc and 188W-188Re generator systems

International Nuclear Information System (INIS)

Monroy-Guzman, F.; Badillo-Almaraz, V.E.; Flores de la Torre, J.A.; Cosgrove, J.; Knapp, F.F. Jr.

2007-01-01

This paper describes studies evaluating the use of hydroxyapatite as the adsorbent material for both 90M o- 90m Tc and 89W - 188 Re generator systems. Hydroxyapatite is an insoluble solid with anion exchange properties. A study of the sorption behaviour of 90M o, 90m Tc, 188 W and 188 Re on hydroxyapatite in NaCl medium was evaluated by batch experiments. The results demonstrated that while 90M o, 90m Tc and 188 Re are not adsorbed by the hydroxyapatite in NaCl solutions (Kd 89W is strongly adsorbed (Kd >500). On the basis of these measurements, hydroxyapatite 89W - 188 Re generator systems were then constructed and eluted in NaCl solutions. The hydroxyapatite based 89W - 188 Re generator performances are presented. (author)

Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

Science.gov (United States)

Yang, Tao-Tao; Cao, Na; Zhang, Hai-Hui; Wei, Jian-Bo; Song, Xiao-Xia; Yi, Dong-Min; Chao, Shuai-Heng; Zhang, Li-Da; Kong, Ling-Fei; Han, Shuang-Yin; Yang, Yu-Xiu; Ding, Song-Ze

2018-04-15

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased

Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

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Baronian, Grégory; Ginda, Katarzyna; Berry, Laurence; Cohen-Gonsaud, Martin; Zakrzewska-Czerwińska, Jolanta; Jakimowicz, Dagmara; Molle, Virginie

2015-01-01

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

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Grégory Baronian

Full Text Available Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.

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Vonderach, Matthias; Byrne, Dominic P; Barran, Perdita E; Eyers, Patrick A; Eyers, Claire E

2018-06-05

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA c ) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA c - and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA c -regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstract ᅟ.

Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry.

Science.gov (United States)

Czernick, Drew; Liu, Jess; Serge, Dibart; Salih, Erdjan

2013-05-27

Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively. Samples of native phosvitin were subjected to tryptic digestion followed by mass spectrometric analysis: (i) native phosvitin peptides, (ii) after treatment with NaOH, and (iii) after chemical derivatization of P-Ser/P-Thr residues by dithiothreitol under base-catalyzed conditions. A combination of these approaches led to the identification of 68 and 35 phosphopeptides with 89 (81 P-Ser and 8 P-Thr residues) and 62 (57 P-Ser and 5 P-Thr residues) phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. These data for the first time documented on a large scale the major states and sites of phosphorylation of phosvitins with a total of 151 phosphorylation sites. Importantly, the present work also provided the first direct de novo protein amino-acid sequence data for phosvitin 1 protein and evidence for the full expression of vitellogenin I gene. We have for the first time generated a large number of phosphopeptides (~100) and identified 151 phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. Importantly, this study also led to the discovery of a novel phosvitin 1 and provided the first direct de novo protein amino-acid sequence data for the full expression of vitellogenin I gene. There is considerable interest in naturally occurring phosphopeptides/phosphoproteins and their application in biomedical fields and in the food industry because of their molecular characteristics and non-toxic nature, hence, our work opens new avenues to pursue such endeavors. In addition, the results provide

Production of {sup 186}Re and {sup 188}Re, and synthesis of {sup 188}Re-DTPA

Energy Technology Data Exchange (ETDEWEB)

Hashimoto, Kazuyuki; Motoishi, Shoji; Kobayashi, Katsutoshi; Izumo, Mishiroku [Department of Radioisotopes, Japan Atomic Energy Research Institute, Tokai, Ibaraki (Japan); Musdja, Muhammad Yanis

1999-08-01

Production of radioactive rhenium isotopes {sup 186}Re and {sup 188}Re, and synthesis of {sup 188}Re-DTPA have been studied. For {sup 186}Re, a production method by the {sup 185}Re(n, {gamma}) {sup 186}Re reaction in a reactor has been established. For {sup 188}Re, a production method by the double neuron capture reaction of {sup 186}W, which produces a {sup 188}W/{sup 188}Re generator, has been established. For synthesis of {sup 188}Re-DTPA, the optimum conditions, including pH, the amounts of regents and so on, have been determined. (author)

Cotinine improves visual recognition memory and decreases cortical Tau phosphorylation in the Tg6799 mice.

Science.gov (United States)

Grizzell, J Alex; Patel, Sagar; Barreto, George E; Echeverria, Valentina

2017-08-01

Alzheimer's disease (AD) is associated with the progressive aggregation of hyperphosphorylated forms of the microtubule associated protein Tau in the central nervous system. Cotinine, the main metabolite of nicotine, reduced working memory deficits, synaptic loss, and amyloid β peptide aggregation into oligomers and plaques as well as inhibited the cerebral Tau kinase, glycogen synthase 3β (GSK3β) in the transgenic (Tg)6799 (5XFAD) mice. In this study, the effect of cotinine on visual recognition memory and cortical Tau phosphorylation at the GSK3β sites Serine (Ser)-396/Ser-404 and phospho-CREB were investigated in the Tg6799 and non-transgenic (NT) littermate mice. Tg mice showed short-term visual recognition memory impairment in the novel object recognition test, and higher levels of Tau phosphorylation when compared to NT mice. Cotinine significantly improved visual recognition memory performance increased CREB phosphorylation and reduced cortical Tau phosphorylation. Potential mechanisms underlying theses beneficial effects are discussed. Copyright © 2017. Published by Elsevier Inc.

A review on the current status and production technology for {sup 188}W-{sup 188}Re generator system

Energy Technology Data Exchange (ETDEWEB)

Kuznetsov, R. A.; Han, H. S.; Cho, W. K.; Park, U. J.; Kim, Y. M

1998-11-01

The current status of {sup 188}W-{sup 188}Re generator production technology were reviewed in PART 1. Main interests were given to the aspects of {sup 188}W reactor production, irradiated targets reprocessing and generator loading technologies, such as alumina type and gel type generators. In order to develop the more convenient and advanced {sup 188}W-{sup 188}Re generator, further studies must be carried out to get the precise evaluation of production and burn-up cross section of {sup 188}W, the more easily realizable generator loading procedure, and also to optimize the column and generator design to compensate the deterioration of generator performance because of parent radionuclide decay. By irradiation of {sup 186}W enriched sample, {sup 188}W-{sup 188}Re generator production experiments were performed to evaluate the possibility of {sup 188}W-{sup 188}Re generator production using HANARO, and PART 2 describes about the experiments. The experimental results shows the possibility of practical {sup 188}W-{sup 188}Re generator production using of low-specific activity {sup 188}W produced in HANARO. (author). 79 refs., 4 tabs., 26 figs.

Facile fabrication of Ag dendrite-integrated anodic aluminum oxide membrane as effective three-dimensional SERS substrate

Science.gov (United States)

Zhang, Cong-yun; Lu, Ya; Zhao, Bin; Hao, Yao-wu; Liu, Ya-qing

2016-07-01

A novel surface enhanced Raman scattering (SERS)-active substrate has been successfully developed, where Ag-dendrites are assembled on the surface and embedded in the channels of anodic aluminum oxide (AAO) membrane, via electrodeposition in AgNO3/PVP aqueous system. Reaction conditions were systematically investigated to attain the best Raman enhancement. The growth mechanism of Ag dendritic nanostructures has been proposed. The Ag dendrite-integrated AAO membrane with unique hierarchical structures exhibits high SERS activity for detecting rhodamine 6G with a detection limit as low as 1 × 10-11 M. Furthermore, the three-dimensional (3D) substrates display a good reproducibility with the average intensity variations at the major Raman peak less than 12%. Most importantly, the 3D SERS substrates without any surface modification show an outstanding SERS response for the molecules with weak affinity for noble metal surfaces. The potential application for the detection of polycyclic aromatic hydrocarbons (PAHs) was evaluated with fluoranthene as Raman target molecule and a sensitive SERS detection with a limit down to 10-8 M was reached. The 3D SERS-active substrate shows promising potential for rapid detection of trace organic pollutants even weak affinity molecules in the environment.

Development of a high performance {sup 188}W/{sup 188}Re generator by using a synthetic alumina

Energy Technology Data Exchange (ETDEWEB)

Lee, Jun Sig [Korea Atomic Energy Research Institute, 1045 Daeduk-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of)], E-mail: jlee15@kaeri.re.kr; Lee, Jong-Soup; Park, Ul-Jae; Son, Kwang-Jae; Han, Hyon-Soo [Korea Atomic Energy Research Institute, 1045 Daeduk-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of)

2009-07-15

A synthetic alumina functionalized with a sulfate moiety has been developed as the column material of {sup 99}Mo/{sup 99m}Tc and {sup 188}W/{sup 188}Re generators. This material is synthesized by a sol-gel processing. In order to characterize the adsorbent for the {sup 188}W/{sup 188}Re separation, both batch and column contact experiments were conducted. As a result of the experiments, it is found that the maximum capacity of the adsorbent for tungsten is higher than 450 mg/g. Hence it is possible to produce {approx}3 Ci {sup 188}W/{sup 188}Re generator with only 1 g of the adsorbent from {sup 188}W solutions supplied from ORNL, USA or RIAR, Russia. A demonstration study was conducted to show the performance of an {sup 188}W/{sup 188}Re generator column. In this study, 1 Ci of {sup 188}W purchased from RIAR, Russia, is loaded on a 0.9 cm ID column packed with 0.7 g of the adsorbent. Elution of {sup 188}Re is performed every 4-7 days by using the saline solution for more than three months. Nearly 100% of tungsten is loaded by passing 5 ml of the {sup 188}W solution (pH=8) through the dry packed column at a 1 ml/min flow rate. Elution efficiency of {sup 188}Re is 70-90% by using 5 ml of the saline solution. The ratio of {sup 188}W/{sup 188}Re in the eluted solution is 0.002-0.003%. When a Sep-Pak containing 0.26 g of acid alumina is installed as a tandem column, the ratio is decreased to less than 10{sup -3}%. Thin layer chromatography for the eluted {sup 188}Re solution shows 100% radiochemical purity. Also, alumina content in the eluted solution shows less than 10 ppm. Through this study, the performance of this adsorbent was successfully demonstrated. By using the developed adsorbent, minimization of the generator column and consequently the volume of eluant could be possible while maintaining the quality of {sup 188}Re just as much as that available in the market.

Structure of smAKAP and its regulation by PKA-mediated phosphorylation

Science.gov (United States)

Burgers, Pepijn P.; Bruystens, Jessica; Burnley, Rebecca J.; Nikolaev, Viacheslav O.; Keshwani, Malik; Wu, Jian; Janssen, Bert J. C.; Taylor, Susan S.; Heck, Albert J. R.; Scholten, Arjen

2016-01-01

The A-kinase anchoring protein (AKAP) smAKAP has three extraordinary features; it is very small, it is anchored directly to membranes by acyl motifs, and it interacts almost exclusively with the type I regulatory subunits (RI) of cAMP-dependent kinase (PKA). Here, we determined the crystal structure of smAKAP’s A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking (D/D) domain of RIα which reveals an extended hydrophobic interface with unique interaction pockets that drive smAKAP’s high specificity for RI subunits. We also identify a conserved PKA phosphorylation site at Ser66 in the AKB domain which we predict would cause steric clashes and disrupt binding. This correlates with in vivo colocalization and fluorescence polarization studies, where Ser66 AKB phosphorylation ablates RI binding. Hydrogen/deuterium exchange studies confirm that the AKB helix is accessible and dynamic. Furthermore, full-length smAKAP as well as the unbound AKB is predicted to contain a break at the phosphorylation site, and circular dichroism measurements confirm that the AKB domain loses its helicity following phosphorylation. As the active site of PKA’s catalytic subunit does not accommodate α-helices, we predict that the inherent flexibility of the AKB domain enables its phosphorylation by PKA. This represents a novel mechanism, whereby activation of anchored PKA can terminate its binding to smAKAP affecting the regulation of localized cAMP signaling events. PMID:27028580

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

Science.gov (United States)

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

2016-12-16

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae*

Science.gov (United States)

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M.

2016-01-01

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1–77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1–77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. PMID:27834677

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

Science.gov (United States)

Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

2015-05-08

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels

NARCIS (Netherlands)

van Tiel, Claudia M.; Westerman, Jan; Paasman, Marten A.; Hoebens, Martha M.; Wirtz, Karel W. A.; Snoek, Gerry T.

2002-01-01

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165)

PprA phosphorylation by STPK of Deinococcus radiodurans changes its in vitro functions

International Nuclear Information System (INIS)

Rajpurohit, Yogendra S.; Misra, H.S.

2011-01-01

Deinococcus radiodurans shows amazing resistance to both ionizing and non-ionizing radiations. This phenotype is attributed also to its efficient DNA double strand breaks (DSB) repair capability of this bacterium. PprA (pleiotropic protein promoting DNA repair) is unique to D. radiodurans and its role in gamma radiation resistance and DSB repair has been shown in this bacterium. Recombinant PrA protects dsDNA from exonuclease degradation and stimulates the DNA ends joining activity of both T4 DNA ligase and E.coli NAD ligase in vitro. Phosphomotif search showed that PprA has putative phosphorylation site similar to that is characterized for Ser/Thr protein kinases in eukaryotic system. A eukaryotic type Ser/Thr protein kinase (DR2518) of D. radiodurans, could phosphorylate recombinant PprA at Thr amino acid in vitro and the phosphorylation of PprA was also observed in vivo. DR2518 kinase mediated protein phosphorylation of PprA, improves its DNA binding affinity by nearly four fold and stimulated T4 DNA ligase activity more towards intermolecular ligation, as compared to unphosphorylated PprA. Interestingly, the phospho-PprA showed lesser protection of dsDNA than unphospho-PprA when incubated with exonuclease III in solution. The putative Thr of PprA was replaced with Ala (T48A) by site directed mutagenesis, which resulted in significant reduction of PprA phosphorylation by DR2518 kinase. Detailed studies on PprA phosphorylation and its functional significance would be presented. (author)

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

KAUST Repository

Muleya, V.

2016-08-04

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

KAUST Repository

Muleya, V.; Marondedze, Claudius; Wheeler, J. I.; Thomas, Ludivine; Mok, Y.-F.; Griffin, M. D. W.; Manallack, D. T.; Kwezi, L.; Lilley, K. S.; Gehring, Christoph A; Irving, H. R.

2016-01-01

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb

International Nuclear Information System (INIS)

Martinsson, Hanna-Stina; Starborg, Maria; Erlandsson, Fredrik; Zetterberg, Anders

2005-01-01

Single cell analysis allows high resolution investigation of temporal relationships between transition events in G 1 . It has been suggested that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is the molecular mechanism behind passage through the restriction point (R). We performed a detailed single cell study of the temporal relationship between R and pRb phosphorylation in human fibroblasts using time lapse video-microscopy combined with immunocytochemistry. Four principally different criteria for pRb phosphorylation were used, namely (i) phosphorylation of residues Ser 795 and Ser 780 (ii) degree of pRb-association with the nuclear structure, a property that is closely related with pRb phosphorylation status, (iii) release of the transcription factor E2F-1 from pRb, and (iv) accumulation of cyclin E, which is dependent on phosphorylation of pRb. The analyses of individual cells revealed that passage through R preceded phosphorylation of pRb, which occurs in a gradually increasing proportion of cells in late G 1 . Our data clearly suggest that pRb phosphorylation is not the molecular mechanism behind the passage through R. The restriction point and phosphorylation of pRb thus seem to represent two separate check point in G 1

12 CFR 18.8 - Delivery.

Science.gov (United States)

2010-01-01

... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Delivery. 18.8 Section 18.8 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY DISCLOSURE OF FINANCIAL AND OTHER INFORMATION BY NATIONAL BANKS § 18.8 Delivery. Each national bank shall, after receiving a request for an annual...

Protein phosphorylation and its role in archaeal signal transduction

Science.gov (United States)

Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

2016-01-01

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

C1qTNF-related protein 1 improve insulin resistance by reducing phosphorylation of serine 1101 in insulin receptor substrate 1.

Science.gov (United States)

Xin, Yaping; Zhang, Dongming; Fu, Yanqin; Wang, Chongxian; Li, Qingju; Tian, Chenguang; Zhang, Suhe; Lyu, Xiaodong

2017-08-30

C1qTNF-related protein 1 (CTRP1) is independently associated with type 2 diabetes. However, the relationship between CTRP1 and insulin resistance is still not established. This study aimed to explore the role of CTRP1 under the situation of insulin resistance in adipose tissue. Plasma CTRP1 level was investigated in type 2 diabetic subjects (n = 35) and non-diabetic subjects (n = 35). The relationship between CTRP1 and phosphorylation of multi insulin receptor substrate 1 (IRS-1) serine (Ser) sites was further explored. Our data showed that Plasma CTRP1 was higher and negative correlation with insulin resistance in diabetic subjects (r = -0.283, p = 0.018). Glucose utilisation test revealed that the glucose utilisation rate of mature adipocytes was improved by CTRP1 in the presence of insulin. CTRP1 was not only related to IRS-1 protein, but also negatively correlated with IRS-1 Ser1101 phosphorylation (r = -0.398, p = 0.031). Furthermore, Phosphorylation levels of IRS-1 Ser1101 were significantly lower after incubation with 40 ng/mL CTRP1 in mature adipocytes than those with no intervention (p insulin resistance by reducing the phosphorylation of IRS-1 Ser1101, induced in the situation of insulin resistance as a feedback adipokine.

IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas tranplant recipient

DEFF Research Database (Denmark)

Bouzakri, K; Karlsson, HRK; Vestergaard, Henrik

2006-01-01

Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied...... by altered signal transduction, skeletal muscle biopsies were obtained from pancreas-kidney transplant recipients (n = 4), nondiabetic kidney transplant recipients (receiving the same immunosuppressive drugs; n = 5), and healthy subjects (n = 6) before and during a euglycemic-hyperinsulinemic clamp. Basal...... insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal...

IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas transplant recipients

DEFF Research Database (Denmark)

Bouzakri, Karim; Karlsson, Håkan K R; Vestergaard, Henrik

2006-01-01

Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied...... by altered signal transduction, skeletal muscle biopsies were obtained from pancreas-kidney transplant recipients (n = 4), nondiabetic kidney transplant recipients (receiving the same immunosuppressive drugs; n = 5), and healthy subjects (n = 6) before and during a euglycemic-hyperinsulinemic clamp. Basal...... insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal...

Concentration of 188Re-Perrhenate for Therapeutic Radiopharmaceuticals

International Nuclear Information System (INIS)

Bokhari, T.H.; Hina, S.; Ahmad, M.; Iqbal, M.

2013-01-01

Summary: Rhenium-188 (T1/2=16.9h) has great potential for a variety of therapeutic applications, including radionuclide synovectomy, oncology and bone pain palliation. The radioactive concentration of 188Re is dependent upon the specific activity of 188W, which dictates the bed size of the alumina/gel column. Due to the high content of inactive tungsten in neutron irradiated WO3, large columns containing aluminum oxide or gel are needed to prepare to double neutron capture based 188W/188Re generators that results in large elution volumes containing relatively high188W contents and low concentrations of /sup 188/ ReO/sub 4/ This decrease in specific volume of 188ReO/sub 4/ places a limitation because a high radioactive concentration of 188ReO4 - is always needed for filling angioplasty balloons or other therapeutic radiopharmaceuticals like188Re -EHDP 188Re -EDTMP, 188Re - MAG3 and 188Re -DTPA. We report post elution concentration of 188ReO4 - using in- house prepared lead cation exchange and alumina columns. Using these columns high bolus volume (10 mL saline) of 188ReO4 - can conveniently be concentrated in 1 mL of physiological saline for therapeutic use. (author)

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

Science.gov (United States)

Qian, Hai; Patriarchi, Tommaso; Price, Jennifer L; Matt, Lucas; Lee, Boram; Nieves-Cintrón, Madeline; Buonarati, Olivia R; Chowdhury, Dhrubajyoti; Nanou, Evanthia; Nystoriak, Matthew A; Catterall, William A; Poomvanicha, Montatip; Hofmann, Franz; Navedo, Manuel F; Hell, Johannes W

2017-01-24

The L-type Ca 2+ channel Ca v 1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser 1928 in Ca v 1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser 1928 is not required for regulation of cardiac Ca v 1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Ca v 1.2 and enhancement of its activity by the β 2 -adrenergic receptor (β 2 AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Ca v 1.2 by PKA in cardiomyocytes and hippocampal neurons. Copyright © 2017, American Association for the Advancement of Science.

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

Directory of Open Access Journals (Sweden)

Javier Modrego

Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

Science.gov (United States)

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

Stability of rhenium-188 labeled antibody

International Nuclear Information System (INIS)

Lim, B. K.; Jung, J. M.; Jung, J. K.; Lee, D. S.; Lee, M. C.

1999-01-01

For clinical application of beta-emitter labeled antibody, high specific activity is important. Carrier-free Re-188 from W-188/Re-188 generator is an ideal radionuclide for this purpose. However, low stability of Re-188 labeled antibody, especially in high specific activity, due to radiolytic decomposition by high energy (2.1 MeV) beta ray was problem. We studied the stability of Re-188 labeled antibody, and stabilizing effect of several nontoxic radical-quenching agents. Pre-reduced monoclonal antibody (CEA79.4) was labeled with Re-188 by incubating with generator-eluted Re-188-perrhenate in the presence of stannous tartrate for 2 hr at room temperature. Radiochemical purity of each preparation was determined by chromatography (ITLC-SG/acetone, ITLC-SG/Umezawa, Whatman No.1/saline). Human serum albumin was added to the labeled antibodies(2%). Stability of Re-188-CEA79.4 was investigated in the presence of vitamin C, ethanol, or Tween 80 as radical-quenching agents. Specific activities of 4.29∼5.11 MBq/μg were obtained. Labeling efficiencies were 88±4%(n=12). Very low stability after removal of stannous tartrate from the preparation was observed. If stored after purging with N 2 , all the preparations were stable for 10 hr. However, if contacted with air, stability decreased. Perrhenate and Re-188-tartrate was major impurity in declined preparation (12∼47 and 9∼38% each, after 10 hr). Colloid-formation was not a significant problem in all cases. Addition of vitamin C stabilized the labeled antibodies either under N 2 or under air by reducing the formation of perrhenate. High specific activity Re-188 labeled antibody is unstable, especially, in the presence of oxygen. Addition of vitamin C increased the stability

Synthesis of {sup 188}Re-DMSA complex using carrier-free {sup 188}Re

Energy Technology Data Exchange (ETDEWEB)

Hashimoto, Kazuyuki; Izumo, Mishiroku [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment; Islam, M S

1997-03-01

The synthesis of rhenium-DMSA labelled compound using carrier-free {sup 188}Re from the {sup 188}W/{sup 188}Re generator has been carried out. Stannous chloride was used as the reducing agent for reduction of rhenium and ascorbic acid was used as an antioxidant in the reaction media. The dependence of the yield of Re-DMSA complex upon the concentration of reducing agent, pH, reaction time, anti-oxidant, carrier and temperature was investigated. Under optimum conditions, the yield of Re-DMSA complexes were more than 98% for the carrier-free as well as carrier-added {sup 188}Re. The stability of the Re-DMSA complexes at different pH and time were also investigated. It was found that the Re-DMSA complex was very stable and did not undergo any changes or decomposition with the changes of pH from its initial values even after 48 hours of pH change for carrier-free as well as carrier-added complexes. (author)

Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5.

Science.gov (United States)

Roche, Jennifer Virginia; Survery, Sabeen; Kreida, Stefan; Nesverova, Veronika; Ampah-Korsah, Henry; Gourdon, Maria; Deen, Peter M T; Törnroth-Horsefield, Susanna

2017-09-01

The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser 256 , Ser 261 , Ser 264 , and Thr 269 ), of which Ser 256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications. © 2017 by The American Society for

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation.

Science.gov (United States)

García García, Tránsito; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Correia Santos, Sara; Henry, Céline; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise; Poncet, Sandrine

2018-01-01

Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

46 CFR 188.10-37 - Label.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Label. 188.10-37 Section 188.10-37 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS GENERAL PROVISIONS Definition of Terms Used in This Subchapter § 188.10-37 Label. This term means the label required by 49 CFR part 172...

Rhenium-188-Lipiodol therapy of liver cancer: Optimization of conjugate-view imaging of 188Re for patient-specific dosimetry

International Nuclear Information System (INIS)

Chaudakshetrin, P.; Osorio, M.; Padhy, A.K.; Divgi, C.; Zanzonico, P.

2004-01-01

Full text: Intrahepatic artery Lipiodol labeled with generator-produced, β-emitting 188Re (17 hr; Eβ=0.53-0.70 MeV; Range=4 mm) localizes in and may effectively treat inoperable liver tumors. Although 188Re emits an imageable 155-keV γ ray (15%), its 478- and 633-keV β rays (2.3%) complicate imaging. Our objective was to optimize 188Re image quality and conjugate-view accuracy for quantitative imaging-based patient-specific dosimetry for 188Re-Lipiodol. Using an ADAC dual-EpicO-circumflex-detector gamma camera, 188Re intrinsic and extrinsic (with LEGP, MEGP, and HEGP collimation) uniformities using either 99mTc intrinsic or 188Re extrinsic flood corrections were evaluated. 188Re conjugate view count rate vs. activity concentration linearity as a function of scattering/attenuating medium thickness was then evaluated with and without corrections for attenuation (a 188Re transmission image) and for scatter and septal penetration, subtracting a fraction of counts in a lower-energy (109-140 keV)- and a higher-energy (170-201 keV)-window image, respectively, from the 20% (140-171 keV) photopeak image. Our phantom consisted of 5 10-ml vials containing 25 to 400 μCi/ml of 188Re with 0 to 6 cm at different positions (∼5 cm apart) between the detectors (60 cm apart) and with a 0- to 6-cm thickness of non-radioactive water between the vials and each detector. With a 99mTc intrinsic correction, 188Re intrinsic uniformity was excellent ( 10% and 'tubey'), and was poorer the lower the energy rating of the collimation. Uniformity with HEGP collimation and an extrinsic 188Re correction was acceptable ( 0.95) and the slope (cps/pixel/μCi/ml) was constant +25% (vs +10% for 99mTc) for 0- to 6-cm thicknesses of water. Regardless of the fraction of counts subtracted, neither scatter nor septal-penetration correction improved the slope constancy. Conclusion: Downscatter/septal penetration of the 478- and 633-keV 188Re γ-rays complicates imaging of its 155-keV γ-ray. Using HEGP

46 CFR 188.10-51 - Ocean.

Science.gov (United States)

2010-10-01

... Terms Used in This Subchapter § 188.10-51 Ocean. Under this designation shall be included all vessels navigating the waters of any ocean, or the Gulf of Mexico more than 20 nautical miles offshore. ... 46 Shipping 7 2010-10-01 2010-10-01 false Ocean. 188.10-51 Section 188.10-51 Shipping COAST GUARD...

Phosphorylation of KasB Regulates Virulence and Acid-Fastness in Mycobacterium tuberculosis

Science.gov (United States)

Vilchèze, Catherine; Molle, Virginie; Carrère-Kremer, Séverine; Leiba, Jade; Mourey, Lionel; Shenai, Shubhada; Baronian, Grégory; Tufariello, Joann; Hartman, Travis; Veyron-Churlet, Romain; Trivelli, Xavier; Tiwari, Sangeeta; Weinrick, Brian; Alland, David; Guérardel, Yann; Jacobs, William R.; Kremer, Laurent

2014-01-01

Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4–6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase

Phosphorylation of KasB regulates virulence and acid-fastness in Mycobacterium tuberculosis.

Directory of Open Access Journals (Sweden)

Catherine Vilchèze

2014-05-01

Full Text Available Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of

The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

Science.gov (United States)

van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

2002-06-21

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

Science.gov (United States)

Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

The Chemistry of Re-188 Radiopharmaceuticals: Could Re-188 Play the Same Role in Therapy as Tc-99m in Diagnostics?

International Nuclear Information System (INIS)

Duatti, A.

2009-01-01

Radiopharmaceuticals incorporating the β-emitting radionuclide Re-188 are still attracting much interest for their potential application in nuclear medicine as therapeutic agents. There are many advantages of employing this class of radioactive compounds as briefly summarized in the following. (1) Re-188 emits a high-energy β- particle (2.1 MeV) that can be efficiently used to deliver high-dose radiation to the target. (2) Re-188 concomitantly emits a 155-keV γ photon that can be conveniently employed to obtain good-quality SPECT images of the biodistribution of Re-188 radiopharmaceuticals and, ultimately, following in vivo the course of the therapy. (3) Re-188 has a relatively short half-life (17 hours) that may allow multiple treatments of the same patient's disease. (4) Re-188 is a radiometal belonging to the same group of Tc-99m in the transition metal series of the Periodic Table, and shares with its cogener similar (though not identical) chemical properties that could be useful for designing a broad class of Re-188 radiopharmaceuticals having the same biodistribution properties of the corresponding Tc-99m analogues. (5) Similarly to Tc-99m, the radionuclide Re-188 is produced in high-specific activity through the 188 W/ 188 Re transportable generator system. A first challenge encountered in the attempt to develop efficient labeling procedures for Re-188 was related to the low radiochemical yield usually observed in tracer-level preparations of Re-188 radiopharmaceuticals starting from generator-produced [ 188 ReO 4 ]-. This drawback is commonly associated with the low value of the standard reduction potential of the tetraoxo anion as compared to the corresponding Tc-99m pertechnetate anion. In recent years, we reported a simple and efficient procedure for overcoming this problem based on a general chemical principle called 'expansion of the coordination sphere' and involving the addition to the reaction vial of an ancillary ligand (usually, chelating hard

Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

Science.gov (United States)

Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

2016-12-27

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2 S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2 Y393F ) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2 Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2 Y393F/S394A mice and Mdm2 S394A mice display similar phenotypes.

Liquid kit for preparation of 188rhenium-etidronate

International Nuclear Information System (INIS)

Marczewski, Barbara; Dias, Carla Roberta; Moraes, Vanessa; Osso Junior, Joao Alberto

2005-01-01

The aim of this study was the preparation of a liquid kit for radiolabeling of 188 Re-HEDP (hydroxyethylidene diphosphonate). 188 Re was obtained from alumina based 188 W/ 188 Re generators. This paper reports the efficacy of a cold kit stored for more than two weeks, determined by the dependence of the radiolabeling yields of 188 Re-HEDP on the incubation time, reducing agent concentration, the effects of concentration of ligand, the p H of the reaction and the temperature. The cold kits showed a good stability when carrie-free rhenium-188 was added in the reaction mixture. (author)

Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

DEFF Research Database (Denmark)

Manteca, Angel; Ye, Juanying; Sánchez, Jesús

2011-01-01

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one...... events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated...... proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized...

188Re(V) Nitrido Radiopharmaceuticals for Radionuclide Therapy.

Science.gov (United States)

Boschi, Alessandra; Martini, Petra; Uccelli, Licia

2017-01-19

The favorable nuclear properties of rhenium-188 for therapeutic application are described, together with new methods for the preparation of high yield and stable 188 Re radiopharmaceuticals characterized by the presence of the nitride rhenium core in their final chemical structure. 188 Re is readily available from an 188 W/ 188 Re generator system and a parallelism between the general synthetic procedures applied for the preparation of nitride technetium-99m and rhenium-188 theranostics radiopharmaceuticals is reported. Although some differences between the chemical characteristics of the two metallic nitrido fragments are highlighted, it is apparent that the same general procedures developed for the labelling of biologically active molecules with technetium-99m can be applied to rhenium-188 with minor modification. The availability of these chemical strategies, that allow the obtainment, in very high yield and in physiological condition, of 188 Re radiopharmaceuticals, gives a new attractive prospective to employ this radionuclide for therapeutic applications.

188Re(V) Nitrido Radiopharmaceuticals for Radionuclide Therapy

Science.gov (United States)

Boschi, Alessandra; Martini, Petra; Uccelli, Licia

2017-01-01

The favorable nuclear properties of rhenium-188 for therapeutic application are described, together with new methods for the preparation of high yield and stable 188Re radiopharmaceuticals characterized by the presence of the nitride rhenium core in their final chemical structure. 188Re is readily available from an 188W/188Re generator system and a parallelism between the general synthetic procedures applied for the preparation of nitride technetium-99m and rhenium-188 theranostics radiopharmaceuticals is reported. Although some differences between the chemical characteristics of the two metallic nitrido fragments are highlighted, it is apparent that the same general procedures developed for the labelling of biologically active molecules with technetium-99m can be applied to rhenium-188 with minor modification. The availability of these chemical strategies, that allow the obtainment, in very high yield and in physiological condition, of 188Re radiopharmaceuticals, gives a new attractive prospective to employ this radionuclide for therapeutic applications. PMID:28106830

Aquaporins Contribute to ABA-Triggered Stomatal Closure through OST1-Mediated Phosphorylation

Science.gov (United States)

Grondin, Alexandre; Rodrigues, Olivier; Verdoucq, Lionel; Merlot, Sylvain; Leonhardt, Nathalie; Maurel, Christophe

2015-01-01

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (Pf) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the Pf of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121. PMID:26163575

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

Directory of Open Access Journals (Sweden)

Kyungjoon Park

Full Text Available Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal or in a context distinct from the conditioning and extinction contexts (ABC renewal. We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM, a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S; thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements

ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

Science.gov (United States)

Park, Kyungjoon; Song, Beomjong; Kim, Jeongyeon; Hong, Ingie; Song, Sangho; Lee, Junuk; Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C; Lee, Sukwon; Choi, Sukwoo

2014-01-01

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the

Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

International Nuclear Information System (INIS)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-01-01

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO

Toward practical SERS sensing

Science.gov (United States)

Zhao, Yiping

2012-06-01

Since its discovery more than 30 years ago, surface-enhanced Raman scattering (SERS) has been recognized as a highly sensitive detection technique for chemical and biological sensing and medical diagnostics. However, the practical application of this remarkably sensitive technique has not been widely accepted as a viable diagnostic method due to the difficulty in preparing robust and reproducible substrates that provide maximum SERS enhancement. Here, we demonstrate that the aligned silver nanorod (AgNR) array substrates engineered by the oblique angle deposition method are capable of providing extremely high SERS enhancement factors (>108). The substrates are large area, uniform, reproducible, and compatible with general microfabrication process. The enhancement factor depends strongly on the length and shape of the Ag nanorods and the underlying substrate coating. By optimizing AgNR SERS substrates, we show that SERS is able to detect trace amount of toxins, virus, bacteria, or other chemical and biological molecules, and distinguish different viruses/bacteria and virus/bacteria strains. The substrate can be tailored into a multi-well chip for high throughput screening, integrated into fiber tip for portable sensing, incorporated into fluid/microfluidic devices for in situ real-time monitoring, fabricated onto a flexible substrate for tracking and identification, or used as on-chip separation device for ultra-thin layer chromatography and diagnostics. By combining the unique SERS substrates with a handheld Raman system, it can become a practical and portable sensor system for field applications. All these developments have demonstrated that AgNR SERS substrates could play an important role in the future for practical clinical, industrial, defense, and security sensing applications.

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation

Directory of Open Access Journals (Sweden)

Tránsito García García

2018-03-01

Full Text Available Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

Directory of Open Access Journals (Sweden)

Sebastian König

Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

46 CFR 188.10-11 - Chemistry laboratory.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Chemistry laboratory. 188.10-11 Section 188.10-11 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS GENERAL PROVISIONS Definition of Terms Used in This Subchapter § 188.10-11 Chemistry laboratory. This term includes...

Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino- and carboxyl-terminal end domains.

Science.gov (United States)

Ando, S; Tokui, T; Yano, T; Inagaki, M

1996-04-05

We reported earlier that phosphorylation in vitro of keratin filaments reconstituted from rat type I keratin 18 and type II keratin 8 by cAPM-dependent protein kinase induces disassembly of the keratin filament structure. Keratin 8 rather than keratin 18 was the major target of the kinase. We have now identified the sites on rat keratin 8 for cAMP-dependent protein kinase. Sequential analysis of the purified phosphoropeptides, together with the known primary sequence, revealed that four major sites, Ser-12, Ser-23, Ser-36, and Ser-50, and three minor sites, Ser-8, Ser-33, Ser-42, are located in the amino-terminal head domain, while three minor sites, Ser-416, Ser-423 and Ser-425 locate in the carboxyl-terminal tail domain.

Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins

Science.gov (United States)

Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F.; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J.; Camiña, Jesús P.

2016-01-01

The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser362, Ser363 and Thr366 residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr350 and Ser349 are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

P-Ser-HPr-a link between carbon metabolism and the virulence of some pathogenic bacteria

DEFF Research Database (Denmark)

Mijakovic, Ivan

2005-01-01

HPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain...... pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of Prf...... is preceded by an operator site, which serves as target for the CcpA/P-Ser-HPr complex. Numerous Gram-negative pathogens also contain hprK, which is often organised in an operon with transcription regulators necessary for the development of virulence, indicating that in these organisms P-Ser-HPr also plays...

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

Directory of Open Access Journals (Sweden)

Jun Sumaoka

2016-01-01

Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

Absolute counting of 188Re radiopharmaceuticals

International Nuclear Information System (INIS)

Ravindra, Anuradha; Kulkarni, D.B.; Joseph, Leena; Kulkarni, M.S.

2018-01-01

Rhenium-188 is radiopharmaceutical that belongs to the group of strong beta-weak gamma emitters. It emits high energy beta particles, (E β m ax = 2.12MeV) and weak gamma rays (E γ = 155 keV) hence makes it suitable for wide variety of therapeutic as well as diagnostic applications. Therapeutic applications include therapy of tumors, radionuclide synovectomy, bone pain palliation, intra vascular radiation therapy etc. 188 Re-labeled medicines have been employed increasingly in the therapy of tumors and vascular restenosis. To ensure that patient receives the appropriate radiation dose during the treatment, both the activity standardization and the determination of sensitivity coefficient of the secondary standard for 188 Re have become important tasks. This paper presents the methods and results obtained for the following measurements a) Standardisation of the 188 Re by using the 4π proportional counter (4πPC)-gamma extrapolation method b) Determination of sensitivity coefficient (pA/MBq) of the secondary standard ionization chamber type Centronic IG12, 20A for 188 Re

Monoclonal Antibodies Radiolabeling with Rhenium-188 for Radioimmunotherapy

Science.gov (United States)

Martini, Petra; Pasquali, Micol

2017-01-01

Rhenium-188, obtained from an alumina-based tungsten-188/rhenium-188 generator, is actually considered a useful candidate for labeling biomolecules such as antibodies, antibody fragments, peptides, and DNAs for radiotherapy. There is a widespread interest in the availability of labeling procedures that allow obtaining 188Re-labeled radiopharmaceuticals for various therapeutic applications, in particular for the rhenium attachment to tumor-specific monoclonal antibodies (Mo)Abs for immunotherapy. Different approaches have been developed in order to obtain 188Re-radioimmunoconjugates in high radiochemical purity starting from the generator eluted [188Re]ReO4−. The aim of this paper is to provide a short overview on 188Re-labeled (Mo)Abs, focusing in particular on the radiolabeling methods, quality control of radioimmunoconjugates, and their in vitro stability for radioimmunotherapy (RIT), with particular reference to the most important contributions published in literature in this topic. PMID:28951872

46 CFR 188.10-3 - Approved container.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Approved container. 188.10-3 Section 188.10-3 Shipping... PROVISIONS Definition of Terms Used in This Subchapter § 188.10-3 Approved container. This term means a container which is properly labeled, marked and approved by DOT for the commodity which it contains. [CGFR...

Phosphorylation of ARD1 by IKKβ contributes to its destabilization and degradation

International Nuclear Information System (INIS)

Kuo, Hsu-Ping; Lee, Dung-Fang; Xia, Weiya; Lai, Chien-Chen; Li, Long-Yuan; Hung, Mien-Chie

2009-01-01

IκB kinase β (IKKβ), a major kinase downstream of various proinflammatory signals, mediates multiple cellular functions through phosphorylation and regulation of its substrates. On the basis of protein sequence analysis, we identified arrest-defective protein 1 (ARD1), a protein involved in apoptosis and cell proliferation processes in many human cancer cells, as a new IKKβ substrate. We provided evidence showing that ARD1 is indeed a bona fide substrate of IKKβ. IKKβ physically associated with ARD1 and phosphorylated it at Ser209. Phosphorylation by IKKβ destabilized ARD1 and induced its proteasome-mediated degradation. Impaired growth suppression was observed in ARD1 phosphorylation-mimic mutant (S209E)-transfected cells as compared with ARD1 non-phosphorylatable mutant (S209A)-transfected cells. Our findings of molecular interactions between ARD1 and IKKβ may enable further understanding of the upstream regulation mechanisms of ARD1 and of the diverse functions of IKKβ.

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

Science.gov (United States)

Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

2013-05-30

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation

Directory of Open Access Journals (Sweden)

Yuanyuan Cheng

2016-01-01

Full Text Available The opening of mitochondrial permeability transition pore (mPTP is a major cause of cell death in ischemia reperfusion injury. Based on our pilot experiments, plant natural product formononetin enhanced the survival of rat cardiomyocyte H9c2 cells during oxygen glucose deprivation (OGD and reoxygenation. For mechanistic studies, we focused on two major cellular factors, namely, reactive oxygen species (ROS and glycogen synthase kinase 3β (GSK-3β, in the regulation of mPTP opening. We found that formononetin suppressed the formation of ROS and superoxide in a concentration-dependent manner. Formononetin also rescued OGD/reoxygenation-induced loss of mitochondrial membrane integrity. Further studies suggested that formononetin induced Akt activation and GSK-3β (Ser9 phosphorylation, thereby reducing GSK-3β activity towards mPTP opening. PI3K and PKC inhibitors abolished the effects of formononetin on mPTP opening and GSK-3β phosphorylation. Immunoprecipitation experiments further revealed that formononetin increased the binding of phosphor-GSK-3β to adenine nucleotide translocase (ANT while it disrupted the complex of ANT with cyclophilin D. Moreover, immunofluorescence revealed that phospho-GSK-3β (Ser9 was mainly deposited in the space between mitochondria and cell nucleus. Collectively, these results indicated that formononetin protected cardiomyocytes from OGD/reoxygenation injury via inhibiting ROS formation and promoting GSK-3β phosphorylation.

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

Energy Technology Data Exchange (ETDEWEB)

Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

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 concentrated 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.

Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

Directory of Open Access Journals (Sweden)

Sebastian Gehlert

Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

Science.gov (United States)

Choi, Sung E; Kwon, Sanghoon; Seok, Sunmi; Xiao, Zhen; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Shinoda, Kosaku; Kajimura, Shingo; Kemper, Byron; Kemper, Jongsook Kim

2017-08-01

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases. Copyright © 2017 American Society for Microbiology.

Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

Energy Technology Data Exchange (ETDEWEB)

Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

2013-07-12

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

International Nuclear Information System (INIS)

Jo, Hye-Ryeong; Kim, Yong-Seok; Son, Hyeon

2016-01-01

Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

Energy Technology Data Exchange (ETDEWEB)

Jo, Hye-Ryeong [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Kim, Yong-Seok [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of); Son, Hyeon, E-mail: hyeonson@hanyang.ac.kr [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of)

2016-01-29

Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

Human herpesvirus 6B induces phosphorylation of p53 in its regulatory domain by a CK2- and p38-independent pathway

DEFF Research Database (Denmark)

Øster, Bodil; Bundgaard, B; Hupp, T R

2008-01-01

Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although...

26 CFR 1.188-1 - Amortization of certain expenditures for qualified on-the-job training and child care facilities.

Science.gov (United States)

2010-04-01

... qualified on-the-job training and child care facilities. 1.188-1 Section 1.188-1 Internal Revenue INTERNAL... qualified on-the-job training and child care facilities. (a) Allowance of deduction—(1) In general. Under... operation of a qualified on-the-job training or child care facility or are integrally related facilities...

Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

Directory of Open Access Journals (Sweden)

Carmen Clapp

2011-07-01

Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

Affinity of hydroxyapatite by radionuclides parent/child in 188Re/188W generator for radiotherapy

International Nuclear Information System (INIS)

Carrera D, A. A.; Badillo A, V.; Badillo A, V. E.; Monroy G, F.

2009-10-01

To assess the feasibility of using apatites as matrices of 188 W/ 188 Re generator is essential to obtain the distribution coefficients as much of parent radionuclide as child radionuclide in apatite, that is to say to know their affinity for the solid. It was selected the mineral species more representative as adsorbent, the hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 it is known for its great capacity of ions retention and by presenting a large affinity for anionic species in their surface. In this paper we use a synthetic hydroxyapatite marketed by Bio-Rad. This paper presents the preliminary results regarding the affinity of hydroxyapatite for the anionic species tungstates (WO 4 2- ) and perrhenates (ReO 4 - in EDTA, as background electrolyte expressed as distribution coefficients between two immiscible phases obtained with the help of radioactive tracers 187 W and 188 Re respectively. The retention measures of these ions, traces show that Bio-Gel hydroxyapatite presents moderate values of distribution coefficients for anionic species of W(Vi) in EDTA 0.01 mol/L that are in the range p H 5 to 6.5; the parent radionuclide of generator 188 Re/ 188 W is fixed but not enough to consider it a good absorbent. By contrast, the fixation of perrhenate ions is virtually wiped as may be easily removed from a hydroxyapatite column packed with a saline solution. The influence of this saline solution in the removal of perrhenate ions is null practically. (Author)

PCTAIRE1 phosphorylates p27 and regulates mitosis in cancer cells.

Science.gov (United States)

Yanagi, Teruki; Krajewska, Maryla; Matsuzawa, Shu-ichi; Reed, John C

2014-10-15

PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target. ©2014 American Association for Cancer Research.

Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor.

Science.gov (United States)

Bischoff, Markus; Brelle, Solène; Minatelli, Sabrina; Molle, Virginie

2016-05-13

The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus ΔyabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_Ala) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression/activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

Energy Technology Data Exchange (ETDEWEB)

Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; Lee-Oesterreich, Paula J.; Pogge von Strandmann, Lisa; Gritsenko, Marina A.; Jacobs, Jon M.; Moore, Patrick S.; Chang, Yuan

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitory mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.

SERS sensors for DVD platform

DEFF Research Database (Denmark)

Brøgger, Anna Line

This Ph.D. thesis explores the engineering of a portable sensor system for detection of rare and small molecules. The Ph.D. project is part of the research project 'Multi-Sensor DVD platform' (MUSE), aiming to integrate different sensors on a rotating disc. The sensors are chosen to complement each...... other, creating more reliable and stable results for the end user. The rotating disc comprises microfluidic channels, which can be utilized for handling and manipulating liquid samples such as blood or water. The focus of this Ph.D. thesis, is on the integration of one specific sensor on a rotating disc....... The sensor is based upon surface enhanced Raman spectroscopy (SERS), which detects molecular vibrations. The aim of this thesis is to cover the different aspects of the sensor system. SERS substrates, consisting of nanopillars with gold or silver caps on top, have been fabricated by standard micro and nano...

Liquid kit for preparation of {sup 188}rhenium-etidronate

Energy Technology Data Exchange (ETDEWEB)

Marczewski, Barbara; Dias, Carla Roberta; Moraes, Vanessa; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: baszot@gmail.com

2005-10-15

The aim of this study was the preparation of a liquid kit for radiolabeling of {sup 188} Re-HEDP (hydroxyethylidene diphosphonate). {sup 188} Re was obtained from alumina based {sup 188} W/{sup 188} Re generators. This paper reports the efficacy of a cold kit stored for more than two weeks, determined by the dependence of the radiolabeling yields of {sup 188} Re-HEDP on the incubation time, reducing agent concentration, the effects of concentration of ligand, the p H of the reaction and the temperature. The cold kits showed a good stability when carrie-free rhenium-188 was added in the reaction mixture. (author)

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

Science.gov (United States)

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

2004-05-01

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

Science.gov (United States)

Leiba, Jade; Carrère-Kremer, Séverine; Blondiaux, Nicolas; Dimala, Martin Moune; Wohlkönig, Alexandre; Baulard, Alain; Kremer, Laurent; Molle, Virginie

2014-04-18

Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb. Copyright © 2014 Elsevier Inc. All rights reserved.

46 CFR 188.10-59 - Recognized classification society.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Recognized classification society. 188.10-59 Section 188... VESSELS GENERAL PROVISIONS Definition of Terms Used in This Subchapter § 188.10-59 Recognized classification society. This term means the American Bureau of Shipping or other classification society...

Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

Directory of Open Access Journals (Sweden)

Sudharsana R Ande

Full Text Available Prohibitin (PHB or PHB1 is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114 and tyrosine 259 (Tyr259 in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121 and threonine 258 (Thr258 respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s or known tyrosine phosphorylation site(s revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

Science.gov (United States)

Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

2012-02-01

Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

Integration of protein phosphorylation, acetylation, and methylation data sets to outline lung cancer signaling networks.

Science.gov (United States)

Grimes, Mark; Hall, Benjamin; Foltz, Lauren; Levy, Tyler; Rikova, Klarisa; Gaiser, Jeremiah; Cook, William; Smirnova, Ekaterina; Wheeler, Travis; Clark, Neil R; Lachmann, Alexander; Zhang, Bin; Hornbeck, Peter; Ma'ayan, Avi; Comb, Michael

2018-05-22

Protein posttranslational modifications (PTMs) have typically been studied independently, yet many proteins are modified by more than one PTM type, and cell signaling pathways somehow integrate this information. We coupled immunoprecipitation using PTM-specific antibodies with tandem mass tag (TMT) mass spectrometry to simultaneously examine phosphorylation, methylation, and acetylation in 45 lung cancer cell lines compared to normal lung tissue and to cell lines treated with anticancer drugs. This simultaneous, large-scale, integrative analysis of these PTMs using a cluster-filtered network (CFN) approach revealed that cell signaling pathways were outlined by clustering patterns in PTMs. We used the t-distributed stochastic neighbor embedding (t-SNE) method to identify PTM clusters and then integrated each with known protein-protein interactions (PPIs) to elucidate functional cell signaling pathways. The CFN identified known and previously unknown cell signaling pathways in lung cancer cells that were not present in normal lung epithelial tissue. In various proteins modified by more than one type of PTM, the incidence of those PTMs exhibited inverse relationships, suggesting that molecular exclusive "OR" gates determine a large number of signal transduction events. We also showed that the acetyltransferase EP300 appears to be a hub in the network of pathways involving different PTMs. In addition, the data shed light on the mechanism of action of geldanamycin, an HSP90 inhibitor. Together, the findings reveal that cell signaling pathways mediated by acetylation, methylation, and phosphorylation regulate the cytoskeleton, membrane traffic, and RNA binding protein-mediated control of gene expression. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Half-life measurements in doubly-odd sup(186,188,190)Au nuclei and the 188Hg -> Au decay

International Nuclear Information System (INIS)

Abreu, M.C.; Berg, V.; Fransson, K.; Hoeglund, A.; Oms, J.; Porquet, M.G.

1985-01-01

A level scheme has been established for 188 Hg -> Au decay which was studied with the online isotope separator ISOCELE. Precise conversion-electron measurements were performed with a semicircular magnetic spectrograph. The half-lives of the 16.0, 82.7 and 114.8 keV levels in 188 Au were measured with a lens electron spectrometer and reduced transition rates were deduced. Similarly the half-lives of the 36.1, 113.9, 227.7, 251.5, 288.0 and 363.6 keV levels in 186 Au and of the 28.9 and 171.5 keV levels in 190 Au were measured. Comparison of the reduced e.m. transition rates shows that a 1 + and a 2 - state have respectively the same structure in sup(186,) sup(188,) sup(190,) 192 Au, the same conclusion holding for the 1 - sub(g.s.) of sup(188,) sup(190,) 192 Au. Additional measurements in 189 , 191 Hg and 185 , 187 Au together with data from the literature enable us to interpret them as a coupling of certain quasiparticle states of neighbouring odd-A nuclei corresponding to oblate shapes. Indications are given that some other negative- and positive-parity states in 188 Au also belong to an oblate system. The nucleus 188 Au appears as the last of a series of doubly-odd gold nuclei where no shape coexistence has, as yet, been observed. (orig.)

Bone uptake by di and tetraphosphonates labeled with Rhenium-188

International Nuclear Information System (INIS)

Faintuch, B.L.; Osso, J.A. Jr.; Muramoto, E.; Faintuch, S.

2002-01-01

MDP (methylenediphosphonate) and HEDP (hydroxyethylidenediphosphonate), both diphosphonates, and EDTMP (ethylenediamine tetramethylene phosphonic acid) a tetraphosphonate ligand, have been labeled with 188 Re for use in metastatic bone-pain palliation. The aim of this study was a comparison between the three complexes 188 Re-MDP, 188 Re-HEDP and 188 Re-EDTMP concerning the complexation conditions, in order to achieve maximum yield, stability and bone uptake. Methods: MDP was dissolved in water and HEDP and EDTMP were dissolved in NaOH 1N followed by decreasing pH with HCl 1N. To all mixtures stannous chloride and 188 ReO 4 were added in a nitrogen atmosphere. The preparations were heated in a boiling water bath for 15 min. The yields as well as the radiochemical stability were estimated by ITLC. Different concentrations of phosphonates and stannous chloride were evaluated. Biodistribution studies in swiss mice were done for the three 188 Re-phosphonates that presented the best radiochemical yield. Results: For 188 Re-MDP and 188 Re-HEDP the optimal ligand concentration for maximum complexation was 30 mg whereas for 188 Re-EDTMP, it was 40 mg. The best amount of SnCl 2 .2H 2 O was 2 mg/mL for MDP, 3 mg/mL for HEDP and 1 mg/mL for EDTMP. In these conditions the three complexes showed a complexation yield above 95%. All of them presented 4-hour radiochemical stability without the need for ascorbic acid solution, but for 24 hours this stability existed only in the presence of that substance otherwise re-oxidation of 188 Re occurred. All products showed a great uptake by the kidneys. 188 Re-EDTMP had the greatest uptake by the bone (3.13 ± 0.18% ID/g) followed by 188 Re-MDP (1.18 ± 0.05%ID/g) and 188 Re-HEDP (1.03 ± 0.12 %ID/g), 4 hour postinjection. 188 Re-EDTMP displayed a bone/muscle ratio of 28.5, 188 Re-MDP 4.9 and 188 Re-HEDP 4.9. Conclusion: 188 Re-EDTMP demonstrated the best potential as a radiopharmaceutical for bone cancer pain relief, encouraging further

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells.

Science.gov (United States)

Kim, Jong-Sik; Baek, Seung Joon; Bottone, Frank G; Sali, Tina; Eling, Thomas E

2005-09-01

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.

Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death

DEFF Research Database (Denmark)

Reimer, Lasse; Lund, Louise Buur; Betzer, Cristine

2018-01-01

, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead...... on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease...

Metformin reduces hyper-reactivity of platelets from patients with polycystic ovary syndrome by improving mitochondrial integrity.

Science.gov (United States)

Randriamboavonjy, Voahanginirina; Mann, W Alexander; Elgheznawy, Amro; Popp, Rüdiger; Rogowski, Paul; Dornauf, Imke; Dröse, Stefan; Fleming, Ingrid

2015-08-31

Polycystic ovary syndrome (PCOS) is associated with decreased fertility, insulin resistance and an increased risk of developing cardiovascular disease. Treating PCOS patients with metformin improves fertility and decreases cardiovascular complications. Given that platelet activation contributes to both infertility and cardiovascular disease development, we assessed platelet reactivity in PCOS patients and the consequences of metformin treatment. Compared to washed platelets from healthy donors, platelets from PCOS patients demonstrated enhanced reactivity and impaired activation of the AMP-activated kinase (AMPK). PCOS platelets also demonstrated enhanced expression of mitochondrial proteins such as the cytochrome c reductase, ATP synthase and the voltage-dependent anion channel-1. However, mitochondrial function was impaired as demonstrated by a decreased respiration rate. In parallel, the phosphorylation of dynamin-related protein-1 (Drp-1) on Ser616 was increased while that on Ser637 decreased. The latter changes were accompanied by decreased mitochondrial size. In insulin-resistant PCOS patients (HOMA-IR> 2) metformin treatment (1.7 g per day for 4 weeks to 6 months) improved insulin sensitivity, restored mitochondrial integrity and function and normalised platelet aggregation. Treatment was without effect in PCOS patients with HOMA-IRtreatment of megakaryocytes with metformin enhanced mitochondrial content and in the same cells metformin enhanced the phosphorylation of the Drp-1 on Ser637 via an AMPKα1-dependent mechanism. In conclusion, the improvement of mitochondrial integrity and platelet reactivity may contribute to the beneficial effects of metformin on cardiovascular disease.

Compartmental and dosimetric studies of anti-CD20 labelled with {sup 188}Re; Estudo compartimental e dosimetrico do Anti-CD20 marcado com {sup 188}Re

Energy Technology Data Exchange (ETDEWEB)

Kuramoto, Graciela Barrio

2016-10-01

}Luanti- CD20, the γ emitter {sup 99m}Tc-Anti-CD20 and α emitter {sup 211}At-Anti-CD20 presented a elimination constant of approximately 0.05 hours{sup -1} in the animal's blood. The dosimetric evaluation of {sup 188}Re-Anti-CD20 was performed using two methodologies: the Monte Carlo method and the use of a point source β{sup -}by Formula Loevinger by Excel program. In the Formula Loevinger, there was a validation of the Monte Carlo method for dosimetry of {sup 188}Re-Anti-CD20 and other products. The doses and dose rates obtained by the two methods were evaluated in comparison with {sup 90}Y-Anti-CD20, {sup 131}I-Anti-CD20 and {sup 177}Lu-Anti-CD20 dosimetry, obtained by the same methodology. The dose study was conducted using mathematical models considering a nude mouse 25 g, simulating different tumor sizes and different forms of distribution of the product within the animal. According to the results, the energy emission β{sup -}, {sup 188}Re- Anti-CD20 has a higher energy deposition for large tumors when compared to other products evaluated. In a simulation with 100% of the product uptake by tumor, 89% of the total dose remained absorbed by the tumor, while preserving the integrity of critical ógãos as heart (2%), lung (5%), column (4%), liver (0.014%) and kidneys 9 (0.0007%). In a simulation where there is a biodistribution of the product in the animal organism, 38% of the total dose absorbed by the tumor and >3% is absorbed by the column. In this situation closer to reality, the extrapolation of the data for a 70kg human, showed that the absorbed dose to tumor corresponds to about 33%; In column 7% and the heart would receive a dose of 35% of the total. The compartmental analysis and dosimetric presented in this work, performed through use of an animal model for the {sup 188}Re-anti-CD20 shows that the product developed and presented in the literature is promising candidate for RIT. (author)

Affinity of hydroxyapatite by radionuclides parent/child in {sup 188}Re/{sup 188}W generator for radiotherapy; Afinidad de la hidroxiapatita por los radionuclidos padre/hijo en el generador {sup 188}Re/{sup 188}W para radioterapia

Energy Technology Data Exchange (ETDEWEB)

Carrera D, A. A. [Universidad Autonoma de Zacatecas, Unidad Academica de Ciencias Quimicas, Campus Universitario Siglo XXI, Ejido La Escondida, Carretera a Guadalajara Km. 6 (Mexico); Badillo A, V. [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Calle Cipres No. 10, Fracc. La Penuela 98068, Zacatecas (Mexico); Badillo A, V. E.; Monroy G, F. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)], e-mail: ana_carrera7@hotmail.com

2009-10-15

To assess the feasibility of using apatites as matrices of {sup 188}W/{sup 188}Re generator is essential to obtain the distribution coefficients as much of parent radionuclide as child radionuclide in apatite, that is to say to know their affinity for the solid. It was selected the mineral species more representative as adsorbent, the hydroxyapatite Ca{sub 10} (PO{sub 4}){sub 6}(OH){sub 2} it is known for its great capacity of ions retention and by presenting a large affinity for anionic species in their surface. In this paper we use a synthetic hydroxyapatite marketed by Bio-Rad. This paper presents the preliminary results regarding the affinity of hydroxyapatite for the anionic species tungstates (WO{sub 4}{sup 2-}) and perrhenates (ReO{sub 4}{sup -} in EDTA, as background electrolyte expressed as distribution coefficients between two immiscible phases obtained with the help of radioactive tracers {sup 187}W and {sup 188}Re respectively. The retention measures of these ions, traces show that Bio-Gel hydroxyapatite presents moderate values of distribution coefficients for anionic species of W(Vi) in EDTA 0.01 mol/L that are in the range p H 5 to 6.5; the parent radionuclide of generator {sup 188}Re/{sup 188}W is fixed but not enough to consider it a good absorbent. By contrast, the fixation of perrhenate ions is virtually wiped as may be easily removed from a hydroxyapatite column packed with a saline solution. The influence of this saline solution in the removal of perrhenate ions is null practically. (Author)

Ultra-thin layer chromatography with integrated silver colloid-based SERS detection.

Science.gov (United States)

Wallace, Ryan A; Lavrik, Nickolay V; Sepaniak, Michael J

2017-01-01

Simplified lab-on-a-chip techniques are desirable for quick and efficient detection of analytes of interest in the field. The following work involves the use of deterministic pillar arrays on the micro-scale as a platform to separate compounds, and the use of Ag colloid within the arrays as a source of increased signal via surface enhanced Raman spectroscopy (SERS). One problem traditionally seen with SERS surfaces containing Ag colloid is oxidation; however, our platforms are superhydrophobic, reducing the amount of oxidation taking place on the surface of the Ag colloid. This work includes the successful separation and SERS detection of a fluorescent dye compounds (resorufin and sulforhodamine 640), fluorescent anti-tumor drugs (Adriamycin and Daunomycin), and purine and pyrimidine bases (adenine, cytosine, guanine, hypoxanthine, and thymine). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Point-of-care detection and real-time monitoring of intravenously delivered drugs via tubing with an integrated SERS sensor.

Science.gov (United States)

Wu, Hsin-Yu; Cunningham, Brian T

2014-05-21

We demonstrate an approach for detection, identification, and kinetic monitoring of drugs flowing within tubing, through the use of a plasmonic nanodome array (PNA) surface. The PNA structures are fabricated using a low-cost nanoreplica molding process upon a flexible plastic substrate that is subsequently integrated with a flow cell that connects in series with ordinary intravenous (IV) drug delivery tubing. To investigate the potential clinical applications for point-of-care detection and real-time monitoring, we perform SERS detection of ten pharmaceutical compounds (hydrocodone, levorphanol, morphine, oxycodone, methadone, phenobarbital, dopamine, diltiazem, promethazine, and mitoxantrone). We demonstrate dose-dependent SERS signal magnitude, resulting in detection limits (ng ml(-1)) well below typical administered dosages (mg ml(-1)). Further, we show that the detected drugs are not permanently attached to the PNA surface, and thus our approach is capable of performing continuous monitoring of drug delivery as materials flow through IV tubing that is connected in series with the sensor. Finally, we demonstrate the potential co-detection of multiple drugs when they are mixed together, and show excellent reproducibility and stability of SERS measurements for periods extending at least five days. The capabilities reported here demonstrate the potential to use PNA SERS surfaces for enhancing the safety of IV drug delivery.

Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

Science.gov (United States)

Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

2009-06-01

Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

Compartmental and dosimetric studies of anti-CD20 labelled with 188Re

International Nuclear Information System (INIS)

Kuramoto, Graciela Barrio

2016-01-01

-1 in the animal's blood. The dosimetric evaluation of 188 Re-Anti-CD20 was performed using two methodologies: the Monte Carlo method and the use of a point source β - by Formula Loevinger by Excel program. In the Formula Loevinger, there was a validation of the Monte Carlo method for dosimetry of 188 Re-Anti-CD20 and other products. The doses and dose rates obtained by the two methods were evaluated in comparison with 90 Y-Anti-CD20, 131 I-Anti-CD20 and 177 Lu-Anti-CD20 dosimetry, obtained by the same methodology. The dose study was conducted using mathematical models considering a nude mouse 25 g, simulating different tumor sizes and different forms of distribution of the product within the animal. According to the results, the energy emission β - , 188 Re- Anti-CD20 has a higher energy deposition for large tumors when compared to other products evaluated. In a simulation with 100% of the product uptake by tumor, 89% of the total dose remained absorbed by the tumor, while preserving the integrity of critical ógãos as heart (2%), lung (5%), column (4%), liver (0.014%) and kidneys 9 (0.0007%). In a simulation where there is a biodistribution of the product in the animal organism, 38% of the total dose absorbed by the tumor and >3% is absorbed by the column. In this situation closer to reality, the extrapolation of the data for a 70kg human, showed that the absorbed dose to tumor corresponds to about 33%; In column 7% and the heart would receive a dose of 35% of the total. The compartmental analysis and dosimetric presented in this work, performed through use of an animal model for the 188 Re-anti-CD20 shows that the product developed and presented in the literature is promising candidate for RIT. (author)

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

Science.gov (United States)

Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

2016-01-01

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338

Caloric restriction mimetic 2-deoxyglucose maintains cytoarchitecture and reduces tau phosphorylation in primary culture of mouse hippocampal pyramidal neurons.

Science.gov (United States)

Bele, M S; Gajare, K A; Deshmukh, A A

2015-06-01

Typical form of neurons is crucially important for their functions. This is maintained by microtubules and associated proteins like tau. Hyperphosphorylation of tau is a major concern in neurodegenerative diseases. Glycogen synthase kinase3β (GSK3β) and cyclin-dependent protein kinase 5 (Cdk5) are the enzymes that govern tau phosphorylation. Currently, efforts are being made to target GSK3β and Cdk5 as possible therapeutic avenues to control tau phosphorylation and treat neurodegenerative diseases related to taupathies. In a number of studies, caloric restriction mimetic 2-deoxyglucose (C6H12O5) was found to be beneficial in improving the brain functions. However, no reports are available on the effect of 2-deoxyglucose 2-DG on tau phosphorylation. In the present study, hippocampal pyramidal neurons from E17 mouse embryos were isolated and cultured on poly-L-lysine-coated coverslips. Neurons from the experimental group were treated with 10 mM 2-deoxyglucose. The treatment of 2-DG resulted in healthier neuronal morphology in terms of significantly lower number of cytoplasmic vacuoles, little or no membrane blebbings, maintained axon hillock and intact neurites. There were decreased immunofluorescence signals for GSK3β, pTau at Ser262, Cdk5 and pTau at Ser235 suggesting decreased tau phosphorylation, which was further confirmed by Western blotting. The results indicate the beneficial effects of 2-DG in controlling the tau phosphorylation and maintaining the healthy neuronal cytoarchitecture.

Study of different absorbent materials for the preparation of generator systems of 99Mo - 99mTc and 188W-188Re

International Nuclear Information System (INIS)

Lopes, Paula Regina Corain; Osso Junior, Joao Alberto

2009-01-01

Amongst some currently radioisotopes, 99m Tc and 188 Re present adequate decay properties for use in Nuclear Medicine. In the current times the most diagnosis examinations are performed with 99m Tc and 188 Re is one radioisotope of potential use in therapy techniques. The objective of this work consists of determining the capacity of some adsorbent materials for retention of molybdenum and tungsten, aiming the optimization of generator systems of 99 Mo- 99m Tc and 188 W- 188 Re with suitable characteristics for application in Nuclear Medicine. Known amounts, in mass, of molybdenum and tungsten were percolated through different chromatographic columns containing different commercial adsorbent materials such as: PZC (poly-zirconium compound), acid alumina and calcinated alumina used in the routine preparation of 99 Mo- 99m Tc generators at IPEN. The tungsten ( 188 W), as well the PZC used in this project, supplied by Russia and Japan, respectively, through the International Atomic Energy Agency (IAEA) and used without any previous preparation. Also trace amounts of 99 Mo and 188 W were added to the initial solutions and the generators were assembled. The 99 Mo- 99m Tc generators were then eluted with known volumes of 0.9% NaCl solution every 24 hours whereas the 188 W- 188 Re generators were eluted every 48 hours. The eluted samples were analyzed by Gamma Spectroscopy and later submitted to quality control evaluation. The results showed that the PZC presents superior retention capacity for Mo of 97.50mg Mo/gPZC, higher than in acid and calcinated alumina, however the elution efficiency at lower pHs is not so high. With regards to the experiments carried out with 188 W and alumina, it was verified that the elution efficiency of 188 Re was not reproducible and the retention capacity of W was 90.23mgW/gAl 2 O 3 at pH 7. (author)

AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

Energy Technology Data Exchange (ETDEWEB)

Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Hyung Soon; Kang, Gum-Yong; Bang, Joo Young [Center for Biomedical Mass Spectrometry, Diatech Korea Co., Ltd., Seoul (Korea, Republic of); Cho, Eun-Jung [National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence and Technology, Seoul National University, Seoul (Korea, Republic of)

2013-02-01

Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

Energy Technology Data Exchange (ETDEWEB)

Pan, Xiao [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Whitten, Douglas A. [Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Wu, Ming [Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Chan, Christina [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wilkerson, Curtis G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Pestka, James J., E-mail: pestka@msu.edu [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States)

2013-04-15

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

International Nuclear Information System (INIS)

Pan, Xiao; Whitten, Douglas A.; Wu, Ming; Chan, Christina; Wilkerson, Curtis G.; Pestka, James J.

2013-01-01

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

Inhibitory Effects of Cytosolic Ca2+ Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets

Directory of Open Access Journals (Sweden)

Hyuk-Woo Kwon

2015-01-01

Full Text Available Intracellular Ca2+ ([Ca2+]i is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro, an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI (Ser1756 to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756 by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa, indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756 phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway.

Science.gov (United States)

Kiranmayi, Malapaka; Chirasani, Venkat R; Allu, Prasanna K R; Subramanian, Lakshmi; Martelli, Elizabeth E; Sahu, Bhavani S; Vishnuprabu, Durairajpandian; Kumaragurubaran, Rathnakumar; Sharma, Saurabh; Bodhini, Dhanasekaran; Dixit, Madhulika; Munirajan, Arasambattu K; Khullar, Madhu; Radha, Venkatesan; Mohan, Viswanathan; Mullasari, Ajit S; Naga Prasad, Sathyamangla V; Senapati, Sanjib; Mahapatra, Nitish R

2016-08-01

Catestatin (CST), an endogenous antihypertensive/antiadrenergic peptide, is a novel regulator of cardiovascular physiology. Here, we report case-control studies in 2 geographically/ethnically distinct Indian populations (n≈4000) that showed association of the naturally-occurring human CST-Gly364Ser variant with increased risk for hypertension (age-adjusted odds ratios: 1.483; P=0.009 and 2.951; P=0.005). Consistently, 364Ser allele carriers displayed elevated systolic (up to ≈8 mm Hg; P=0.004) and diastolic (up to ≈6 mm Hg; P=0.001) blood pressure. The variant allele was also found to be in linkage disequilibrium with other functional single-nucleotide polymorphisms in the CHGA promoter and nearby coding region. Functional characterization of the Gly364Ser variant was performed using cellular/molecular biological experiments (viz peptide-receptor binding assays, nitric oxide [NO], phosphorylated extracellular regulated kinase, and phosphorylated endothelial NO synthase estimations) and computational approaches (molecular dynamics simulations for structural analysis of wild-type [CST-WT] and variant [CST-364Ser] peptides and docking of peptide/ligand with β-adrenergic receptors [ADRB1/2]). CST-WT and CST-364Ser peptides differed profoundly in their secondary structures and showed differential interactions with ADRB2; although CST-WT displaced the ligand bound to ADRB2, CST-364Ser failed to do the same. Furthermore, CST-WT significantly inhibited ADRB2-stimulated extracellular regulated kinase activation, suggesting an antagonistic role towards ADRB2 unlike CST-364Ser. Consequently, CST-WT was more potent in NO production in human umbilical vein endothelial cells as compared with CST-364Ser. This NO-producing ability of CST-WT was abrogated by ADRB2 antagonist ICI 118551. In conclusion, CST-364Ser allele enhanced the risk for hypertension in human populations, possibly via diminished endothelial NO production because of altered interactions of CST-364Ser

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

Science.gov (United States)

Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth

2012-08-07

Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs. Copyright © 2012 Elsevier Ltd. All rights reserved.

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

Science.gov (United States)

Sharpe, Richard M; Koepke, Tyson; Harper, Artemus; Grimes, John; Galli, Marco; Satoh-Cruz, Mio; Kalyanaraman, Ananth; Evans, Katherine; Kramer, David; Dhingra, Amit

2016-01-01

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

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

Directory of Open Access Journals (Sweden)

Nagib eAhsan

2012-07-01

Full Text Available The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1α 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 Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform scanning mutagenesis of the residues flanking the site of phosphorylation. Consistent with the results from phylogenetic analysis of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

Complexation of 188Re-phosphonates: in vitro and in vivo studies

International Nuclear Information System (INIS)

Faintuch, B.L.; Muramoto, E.; Faintuch, S.

2003-01-01

MDP (methylenediphosphonate) and HEDP (hydroxyethylidene diphosphonate), both disphosphonates, and EDTMP (ethylenediamine tetramethylene phosphonic acid), a tetraphosphonate ligand, have been previously labeled with 188 Re for use in metastatic bone-pain palliation. The aim of this study was a comparison between the three complexes 188 Re-MDP, 188 Re-HEDP and 188 Re-EDTMP concerning the complexation conditions, in order to achieve maximum yield, stability and bone uptake. Methods: MDP was dissolved in water and HEDP and EDTMP were dissolved in NaOH 1 N followed by reduction of pH with HCl 1 N. To all mixtures stannous chloride and 188 ReO 4 - were added in a nitrogen atmosphere. The preparations were heated in boiling water bath for 15 min. Yield as well as radiochemical stability was estimated by ITLC. Different concentrations of phosphonates and stannous chloride were evaluated. Biodistribution studies in Swiss mice were done for the three 188 Re-phosphonates that presented the best radiochemical yield. The optimal ligand concentration for maximum complexation was 85.2 mM for MDP, 72.8 mM for HEDP and 45.8 mM for EDTMP. The best amount of SnCl 2 .2H 2 O was 1.5 mg/mL for 188 Re-HEDP and 1 mg/mL for both 188 Re-MDP and 188 Re-EDTMP. In these conditions the three complexes showed a complexation rate above 95%. Reasonable radiochemical stability for 24 hours was achieved by 188 Re-EDTMP when employing ascorbic acid. All products showed a great uptake by the kidneys. 188 Re-EDTMP had the greatest uptake by femur (3.1 ± 0.2% ID/g) followed by 188 Re-MDP (1.2 ± 0.1% ID/g) and 188 Re-HEDP (1.0 ± 0.1% ID/g), 4 hours post injection. 188 Re-EDTMP displayed a femur bone/muscle ratio of 28.5, 188 Re-MDP 4.9 and 188 Re-HEDP 4.9. In conclusion 188 Re-EDTMP demonstrated the best potential as a radiopharmaceutical for bone cancer pain relief, encouraging further dosimetric studies and clinical trials. (orig.)

47 CFR 1.88 - Predesignation pleading procedure.

Science.gov (United States)

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Predesignation pleading procedure. 1.88 Section... Rules of Practice and Procedure Miscellaneous Proceedings § 1.88 Predesignation pleading procedure. In... staff, in its discretion, may in writing, advise such licensee of the general nature of the...

Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data.

Directory of Open Access Journals (Sweden)

Katrina M Waters

Full Text Available To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

Network Analysis of Epidermal Growth Factor Signaling using Integrated Genomic, Proteomic and Phosphorylation Data

Energy Technology Data Exchange (ETDEWEB)

Waters, Katrina M.; Liu, Tao; Quesenberry, Ryan D.; Willse, Alan R.; Bandyopadhyay, Somnath; Kathmann, Loel E.; Weber, Thomas J.; Smith, Richard D.; Wiley, H. S.; Thrall, Brian D.

2012-03-29

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

Studies of gel metal-oxide composite samples as filling materials for W-188/Re-188 generator column

Czech Academy of Sciences Publication Activity Database

Iller, E.; Polkowska-Motrenko, H.; Lada, W.; Wawszczak, D.; Sypula, M.; Doner, K.; Konior, M.; Milczarek, J.; Zoladek, J.; Ráliš, Jan

2009-01-01

Roč. 281, č. 1 (2009), s. 83-86 ISSN 0236-5731. [9th International Conference on Nuclear Analytical Methods in the Life Sciences. Lisbon, 07.09.2008-12.09.2008] Institutional research plan: CEZ:AV0Z10480505 Keywords : W-188/Re-188 generator * W-Zr gels * W-Zr composites * Sol-gel process Subject RIV: CH - Nuclear ; Quantum Chemistry Impact factor: 0.631, year: 2009

Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

DEFF Research Database (Denmark)

Xiang, B; Yu, G H; Guo, J

2001-01-01

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

Identification of ischemia-regulated phosphorylation sites in connexin43: A possible target for the antiarrhythmic peptide analogue rotigaptide (ZP123)

DEFF Research Database (Denmark)

Axelsen, Lene Nygaard; Stahlhut, Martin; Mohammed, Shabaz

2006-01-01

Previous studies suggest that dephosphorylation of connexin43 (Cx43) is related to uncoupling of gap junction communication, which plays an important role in the genesis of ischemia-induced ventricular tachycardia. We studied changes in Cx43 phosphorylation during global ischemia in the absence...... and presence of the antiarrhythmic peptide analogue rotigaptide (formerly known as ZP123). Phosphorylation analysis was performed on Cx43 purified from isolated perfused rat hearts using matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography electrospray ionization tandem mass...... of ischemia, the critical time interval where gap junction uncoupling occurs, Ser297 and Ser368 also became fully dephosphorylated. During the same time period, all untreated hearts developed asystole. Treatment with rotigaptide significantly increased the time to ischemia-induced asystole and suppressed...

46 CFR 154.188 - Membrane tank: Inner hull steel.

Science.gov (United States)

2010-10-01

... 46 Shipping 5 2010-10-01 2010-10-01 false Membrane tank: Inner hull steel. 154.188 Section 154.188... STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Design, Construction and Equipment Hull Structure § 154.188 Membrane tank: Inner hull steel. For a vessel with membrane tanks, the inner hull...

Paxillin: a crossroad in pathological cell migration

Directory of Open Access Journals (Sweden)

Ana María López-Colomé

2017-02-01

Full Text Available Abstract Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis.

Study on the preparation and stability of 188Re biomolecules via EHDP

International Nuclear Information System (INIS)

Ferro-Flores, G.; Garcia-Salinas, L.; Paredes-Gutierrez, L.; Hashimoto, K.; Melendez-Alafort, L.; Murphy, C.A.

2001-01-01

A direct labelling technique via ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand was developed for the preparation of several biomolecules: 188 Re-monoclonal antibody ior cea1 against carcinoembryonic antigen ( 188 Re-MoAb), biotinylated 188 Re-MoAb ( 188 Re-MoAb-biotin), 188 Re-polyclonal IgG ( 188 Re-IgG), 188 Re-peptide (somatostatine analogue peptide b-(2-naphtyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-amide), 188 Re-MoAb fragments ( 188 Re-F(ab') 2 ) and biotinylated 188 Re-F(ab') 2 ( 188 Re-F(ab') 2 -biotin). The reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were optimized during the radiolabelling of each biomolecule. Before the labelling procedure, disulphide bridge groups of the biomolecules were reduced with 2-mercaptoethanol (2-ME). To obtain 188 Re labelled antibodies and peptides in high radiochemical yields (>90%) via EHDP, it was necessary to use acidic conditions and a high concentration of stannous chloride to allow the redox reaction Re +7 →Re +5 :Re +4 . The labelling of MoAb and F(ab') 2 with 188 Re via EHDP was also evaluated employing a pretargeted technique by avidin-biotin strategy in normal mice, demonstrating that the 188 Re-labelled biotinylated antibodies are stable complexes in vivo. The 188 Re-peptide complex prepared by this method, was stable for 24 h and no radiolytic degradation was observed. (author)

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

Science.gov (United States)

Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

2016-09-01

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Active inhibitor-1 maintains protein hyper-phosphorylation in aging hearts and halts remodeling in failing hearts.

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Pritchard, Tracy J; Kawase, Yoshiaki; Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J; Kranias, Evangelia G

2013-01-01

Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.

The Synthesis And Characterization Of Wolfram Phthalocyanine For The Target Material Of High Specific Activity Radioisotope Wolfram - 188 (188W)

International Nuclear Information System (INIS)

Setiawan, Duyeh

2000-01-01

The application of 188 Re radioisotope separation on aluminia column through elution solution has increased significantly since the last two decades. The 188 Re radioisotope has been done fram 188 Re beta-decay through a neutron capture radiation on wolfram -186 target. In trhe column separation, high specific activity of 188 W radioisotope is required to get sufficient activity in small quality 188 W radioisotope has been carried out in this research. Wolfram-phthalocyanine compound was prepared by refluxing a mixture of wolfram trioxyde, (WO 3 ) and phthalonitrile, (C 8 H 4 N 2 ) at 250 o C for two hours. The synthesis of wolfram phthalocyanine is 70% purity yield, the product are green crystals, have a 193,0-193,8 o C melting points, and has a molecular formula C 3 2H 1 6 N8 WO 2 . The infra red spectrum of wolfram-phthalocyanine was the absorption band at 964,3 cm - 1 was due to the vibration of W=O bond of the wolfram dioxy-phthalocyanine. The x-ray diffraction of the wolfram dioxy-phthalocyanine was similar with molybdenum dioxy-phthalocyanine compound. This fact showed that the product was wolfram dioxy-phthalocyanine

Correlative SEM SERS for quantitative analysis of dimer nanoparticles.

Science.gov (United States)

Timmermans, F J; Lenferink, A T M; van Wolferen, H A G M; Otto, C

2016-11-14

A Raman microscope integrated with a scanning electron microscope was used to investigate plasmonic structures by correlative SEM-SERS analysis. The integrated Raman-SEM microscope combines high-resolution electron microscopy information with SERS signal enhancement from selected nanostructures with adsorbed Raman reporter molecules. Correlative analysis is performed for dimers of two gold nanospheres. Dimers were selected on the basis of SEM images from multi aggregate samples. The effect of the orientation of the dimer with respect to the polarization state of the laser light and the effect of the particle gap size on the Raman signal intensity is observed. Additionally, calculations are performed to simulate the electric near field enhancement. These simulations are based on the morphologies observed by electron microscopy. In this way the experiments are compared with the enhancement factor calculated with near field simulations and are subsequently used to quantify the SERS enhancement factor. Large differences between experimentally observed and calculated enhancement factors are regularly detected, a phenomenon caused by nanoscale differences between the real and 'simplified' simulated structures. Quantitative SERS experiments reveal the structure induced enhancement factor, ranging from ∼200 to ∼20 000, averaged over the full nanostructure surface. The results demonstrate correlative Raman-SEM microscopy for the quantitative analysis of plasmonic particles and structures, thus enabling a new analytical method in the field of SERS and plasmonics.

Production of carrier-free 188Re in the past ten years in Taiwan

International Nuclear Information System (INIS)

Bor-Tsung Hsieh; Institute of Nuclear Energy Research, Taiwan; Wan-Yu Lin; Tsai-Yueh Luo; Kai-Yuan Cheng

2007-01-01

Twenty clinical scale alumina-based 188 W/ 188 Re generators and carrier-free 188 Re has been produced at the Institute of Nuclear Energy Research (INER-Taiwan) for over ten years. 2845.6 GBq (76.9 Ci) of 188 Re-perrhenate solution has been eluted from generators during the past ten years. We have used the harvesting 188 Re solution for labeling radiopharmaceuticals, such as 188 Re-HEDP, 188 Re-MDP, 188 Re-microsphere, 188 Relipiodol, and 188 Re-sulfur colloid, etc. The average eluting yield of 188 Re is 78.6±5.8% that was investigated at 1115 harvesting times from 20 generators. Each generator can be used more than six months but the Millipore needs to be changed every two months for smooth harvesting and high yield of 188 Re solution. (author)

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

Directory of Open Access Journals (Sweden)

Richard M Sharpe

Full Text Available High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Crosstalk between phosphorylation and O-GlcNAcylation: friend or foe.

Science.gov (United States)

van der Laarse, Saar A M; Leney, Aneika C; Heck, Albert J R

2018-05-02

A wide variety of protein post-translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co-occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O-GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O-GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O-GlcNAc transferase), on the erasers (i.e., phosphatases and O-GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O-GlcNAcylation crosstalk. © 2018 Federation of European Biochemical Societies.

Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

Science.gov (United States)

Battistone, M A; Da Ros, V G; Salicioni, A M; Navarrete, F A; Krapf, D; Visconti, P E; Cuasnicú, P S

2013-09-01

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

Science.gov (United States)

Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

2014-12-05

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

Science.gov (United States)

Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

2018-05-01

Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

40 CFR 159.188 - Failure of performance information.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Failure of performance information. 159.188 Section 159.188 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... submitted concerning substantiation of any incident of a pest having developed resistance to any pesticide...

Phosphorylation of zona occludens-2 by protein kinase C epsilon regulates its nuclear exportation.

Science.gov (United States)

Chamorro, David; Alarcón, Lourdes; Ponce, Arturo; Tapia, Rocio; González-Aguilar, Héctor; Robles-Flores, Martha; Mejía-Castillo, Teresa; Segovia, José; Bandala, Yamir; Juaristi, Eusebio; González-Mariscal, Lorenza

2009-09-01

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Directory of Open Access Journals (Sweden)

Thomas R Caulfield

2014-11-01

Full Text Available Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin

PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation ▿

Science.gov (United States)

Meyer, Rosana D.; Srinivasan, Srimathi; Singh, Amrik J.; Mahoney, John E.; Gharahassanlou, Kobra Rezazadeh; Rahimi, Nader

2011-01-01

The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2. PMID:21402774

S4S8-RPA phosphorylation as an indicator of cancer progression in oral squamous cell carcinomas.

Science.gov (United States)

Rector, Jeff; Kapil, Sasha; Treude, Kelly J; Kumm, Phyllis; Glanzer, Jason G; Byrne, Brendan M; Liu, Shengqin; Smith, Lynette M; DiMaio, Dominick J; Giannini, Peter; Smith, Russell B; Oakley, Greg G

2017-02-07

Oral cancers are easily accessible compared to many other cancers. Nevertheless, oral cancer is often diagnosed late, resulting in a poor prognosis. Most oral cancers are squamous cell carcinomas that predominantly develop from cell hyperplasias and dysplasias. DNA damage is induced in these tissues directly or indirectly in response to oncogene-induced deregulation of cellular proliferation. Consequently, a DNA Damage response (DDR) and a cell cycle checkpoint is activated. As dysplasia transitions to cancer, proteins involved in DNA damage and checkpoint signaling are mutated or silenced decreasing cell death while increasing genomic instability and allowing continued tumor progression. Hyperphosphorylation of Replication Protein A (RPA), including phosphorylation of Ser4 and Ser8 of RPA2, is a well-known indicator of DNA damage and checkpoint activation. In this study, we utilize S4S8-RPA phosphorylation as a marker for cancer development and progression in oral squamous cell carcinomas (OSCC). S4S8-RPA phosphorylation was observed to be low in normal cells, high in dysplasias, moderate in early grade tumors, and low in late stage tumors, essentially supporting the model of the DDR as an early barrier to tumorigenesis in certain types of cancers. In contrast, overall RPA expression was not correlative to DDR activation or tumor progression. Utilizing S4S8-RPA phosphorylation to indicate competent DDR activation in the future may have clinical significance in OSCC treatment decisions, by predicting the susceptibility of cancer cells to first-line platinum-based therapies for locally advanced, metastatic and recurrent OSCC.

Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

Science.gov (United States)

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

2008-08-01

We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

Phosphorylation of chloroform soluble compounds in plasma membranes of human epidermoid carcinoma A431 cells

International Nuclear Information System (INIS)

Brautigan, D.L.; Randazzo, P.; Shriner, C.; Fain, J.N.

1985-01-01

This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. The authors found a novel chloroform-soluble product radiolabeled with [gamma- 32 P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32 P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid

19 CFR 122.188 - Issuance of temporary Customs access seal.

Science.gov (United States)

2010-04-01

... 19 Customs Duties 1 2010-04-01 2010-04-01 false Issuance of temporary Customs access seal. 122.188 Section 122.188 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY AIR COMMERCE REGULATIONS Access to Customs Security Areas § 122.188 Issuance of...

Dosimetric evaluation of anti-CD20 labelled with 188Re

International Nuclear Information System (INIS)

Barrio, Graciela; Osso Junior, Joao A.

2011-01-01

Radioimmunotherapy has the potential to deliver lethal radiation energy directly to malignant cells via targeting of radioisotope-conjugated monoclonal antibodies (MAbs) to specific antigens. B-cell lymphoma is a particularly good candidate for radioimmunotherapy because the disease is inherently radiosensitive, malignant cells in the blood, bone marrow, spleen and lymphonodes are accessible, and MAbs have been developed to B-cell surface antigens that do not shed or modulate. Rituximab (RTX), the human IgG1-type chimeric form of the parent murine antibody ibritumomab, is specifically targeted against CD20, a surface antigen expressed by pre-B and mature human B lymphocytes. The use of rhenium-188 from a 188 W/ 188 Re generator system represents an attractive alternative radionuclide for therapy. 188 Re is produced from beta decay of the 188 W parent. In addition to the emission of high-energy electrons (Eβ= 2118 keV), 188 Re also decays with emission of a gamma photon with an energy of 155 keV in 15% abundance. Besides the therapeutic usefulness of 188 Re, the emission of gamma photon is an added advantage since the biodistribution of 188 Re-labeled antibodies can be evaluated in vivo with a gamma camera. Also, rhenium has chemical properties similar to technetium. Thus, both can be conjugated to antibodies using similar chemistry methods. The objective of this work is to prove the usefulness of this radiopharmaceutical based on dosimetric studies, that are also required by the Brazilian Regulatory Agency (ANVISA). (author)

188Re labeling and biodistribution of magnetic nanoparticles for the tumor targeting

International Nuclear Information System (INIS)

Li Guiping; Zhang Hui; Wang Yongxian; Zhang Chunfu

2006-01-01

Objective: To prepare 188 Re labeled monoclonal antibody (Herceptin)-coated magnetic nanoparticles for tumor targeting and to study its biodistribution in mice. Methods: Herceptin and histidine were covalently linked to the amine group upon silica-coated magnetic nanoparticles modified by N-[3-(trimethyoxysilyl)prowl]-ethylenediamine using glutaraldehyde method. The Herceptin-coated magnetic nanoparticles and Herceptin were radiolabeled with 188 Re by a direct labelling method, whereas the histidine-coated magnetic nanoparticles was radiolabeled with 188 Re using fac-[ 188 Re(CO) 3 (H 2 0) 3 ] + as a precursor. The labelling efficiency and immunoreactivity as well as labelling stability were determined. Also, the biodistribution of 188 Re-magnetic and 188 Re-Herceptin-magnetic nanoparticles were observed in mice. Results: Herceptin-coated magnetic nanoparticles was characterized by transmission electron microscope (TEM) with diameter about 60 nm, while histidine-coated magnetic nanoparticles about 30 nm. The labeling efficiency for 188 Re-Herceptin, 188 Re-magnetic nanoparticles and 188 Re-Herceptin-magnetic nanoparticles were all > 90% and had a better stability in vitro. The immunoreactivity of Herceptin linked to magnetic nanoparticles was still high. The biodistribution in mice was shown that 188 Re-magnetic nanoparticles and 188 Re-Herceptin- magnetic nanoparticles had higher radioactivity levels in blood. Magnetic nanoparticles with diameter of 30 or 60 nm had a long half-life in blood stream and were accumulated in liver. Conclusion: The efficiency and stability of labelling Herceptin-coated magnetic nanoparticles and labelling magnetic nanoparticles with 188 Re are suitable for in vivo study in tumor-beating nude mice models. (authors)

Synthesis and in Vitro evaluation of ''1''8''8Re-biotinyl-hydrazino-etda

International Nuclear Information System (INIS)

Pervez, S; Mushtaq, A.; Jehangir, M.

2002-01-01

Pretargeting strategies have overcome many drawbacks associated with the use of directly labelled MoAbs in the diagnosis / treatment of various solid tumors. In particular the avidin-biotin system has received much attention due to extremely high affinity between avidin and biotin. An EDTA derivative of biotin has been synthesized (yield-35%). In order or label biotin derivative (biotinyl-hydrazino-EDTA) , stannous ion was used to reduce ''1''8''8ReO 4 (VII) to lower oxidation state and weak chelating agent glucoheptonate as stabilizer and trans chelating agent. Thin layer chromatography and high performance liquid chromatography techniques were employed to monitor the radiolabeling efficiency. The radiolabeling yield of ''1''8''8Re-EDTA B1 was >95%. The radiolabeled product was found to bind to avidin (70-80%), thereby demonstrating retention of its biological integrity

46 CFR 188.05-5 - Specific application noted in text.

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2010-10-01

... which the text pertains, and in many cases limits the application of the text to vessels contracted for... 46 Shipping 7 2010-10-01 2010-10-01 false Specific application noted in text. 188.05-5 Section 188... GENERAL PROVISIONS Application § 188.05-5 Specific application noted in text. (a) At the beginning of the...

Systematic inference of functional phosphorylation events in yeast metabolism.

Science.gov (United States)

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

Science.gov (United States)

Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

2014-01-01

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

Phosphorylated cAMP response element-binding protein as a molecular marker of memory processing in rat hippocampus: effect of novelty

OpenAIRE

Viola, Haydée Ana María; Furman, Melina; Izquierdo, Luciana Adriana; Alonso, Mariana; Barros, Daniela Martí; Souza, Márcia Maria de; Izquierdo, Ivan Antônio; Medina, Jorge Horacio

2000-01-01

From mollusks to mammals the activation of cAMP response element-binding protein (CREB) appears to be an important step in the formation of long-term memory (LTM). Here we show that a 5 min exposure to a novel environment (open field) 1 hr after acquisition of a one-trial inhibitory avoidance training hinders both the formation of LTM for the avoidance task and the increase in the phosphorylation state of hippocampal Ser 133 CREB [phosphorylated CREB (pCREB)] associated with the avoidance tra...

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

DEFF Research Database (Denmark)

Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

2012-01-01

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the a...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE51[OPEN

Science.gov (United States)

Zhou, Xiaona; Hao, Hongmei; Zhang, Yuguo; Bai, Yili; Zhu, Wenbo; Qin, Yunxia; Yuan, Feifei; Zhao, Feiyi; Wang, Mengyao; Hu, Jingjiang; Xu, Hong; Guo, Aiguang; Zhao, Huixian; Zhao, Yang; Cao, Cuiling; Yang, Yongqing; Schumaker, Karen S.; Guo, Yan; Xie, Chang Gen

2015-01-01

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-RELATED PROTEIN KINASE3-type protein kinase, SOS2-LIKE PROTEIN KINASE5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, ABSCISIC ACID-INSENSITIVE5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-LIKE CALCIUM BINDING PROTEINs) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana). PMID:25858916

Surface-enhanced Raman scattering (SERS) for detection of phenylketonuria for newborn screening

Science.gov (United States)

Javanmard, M.; Davis, R. W.

2014-02-01

Diagnosis of Phenylketonuria (PKU) in newborns is important because it can potentially help prevent mental retardation since it is treatable by dietary means. PKU results in phenylketonurics having phenylalanine levels as high as 2 mM whereas the normal upper limit in healthy newborns is 120 uM. To this end, we are developing a microfluidic platform integrated with a SERS substrate for detection of high levels of phenylalanine. We have successfully demonstrated SERS detection of phenylalanine using various SERS substrates fabricated using nanosphere lithography, which exhibit high levels of field enhancement. We show detection of SERS at clinically relevant levels.

Checkpoint kinase 1-induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis.

Science.gov (United States)

Li, Zhe; Li, Xueyan; Nai, Shanshan; Geng, Qizhi; Liao, Ji; Xu, Xingzhi; Li, Jing

2017-12-01

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O- linked β- N -acetylglucosamine ( O -GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an si OGT- based approach. Lastly, expression of plasmids resistant to si OGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Study of different absorbent materials for the preparation of generator systems of {sup 99}Mo - {sup 99m}Tc and {sup 188}W-{sup 188}Re

Energy Technology Data Exchange (ETDEWEB)

Lopes, Paula Regina Corain; Osso Junior, Joao Alberto, E-mail: paulinhacorain@usp.b, E-mail: jaosso@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2009-07-01

Amongst some currently radioisotopes, {sup 99m}Tc and {sup 188}Re present adequate decay properties for use in Nuclear Medicine. In the current times the most diagnosis examinations are performed with {sup 99m}Tc and {sup 188}Re is one radioisotope of potential use in therapy techniques. The objective of this work consists of determining the capacity of some adsorbent materials for retention of molybdenum and tungsten, aiming the optimization of generator systems of {sup 99}Mo-{sup 99m}Tc and {sup 188}W-{sup 188}Re with suitable characteristics for application in Nuclear Medicine. Known amounts, in mass, of molybdenum and tungsten were percolated through different chromatographic columns containing different commercial adsorbent materials such as: PZC (poly-zirconium compound), acid alumina and calcinated alumina used in the routine preparation of {sup 99}Mo-{sup 99m}Tc generators at IPEN. The tungsten ({sup 188}W), as well the PZC used in this project, supplied by Russia and Japan, respectively, through the International Atomic Energy Agency (IAEA) and used without any previous preparation. Also trace amounts of {sup 99}Mo and {sup 188}W were added to the initial solutions and the generators were assembled. The {sup 99}Mo-{sup 99m}Tc generators were then eluted with known volumes of 0.9% NaCl solution every 24 hours whereas the {sup 188}W-{sup 188}Re generators were eluted every 48 hours. The eluted samples were analyzed by Gamma Spectroscopy and later submitted to quality control evaluation. The results showed that the PZC presents superior retention capacity for Mo of 97.50mg Mo/gPZC, higher than in acid and calcinated alumina, however the elution efficiency at lower pHs is not so high. With regards to the experiments carried out with {sup 188}W and alumina, it was verified that the elution efficiency of {sup 188}Re was not reproducible and the retention capacity of W was 90.23mgW/gAl{sub 2}O{sub 3} at pH 7. (author)

Study of different adsorbent materials for the preparation of generator systems of 99Mo - 99mTc and 188W-188Re

International Nuclear Information System (INIS)

Lopes, Paula Regina Corain

2009-01-01

Amongst some existing radioisotopes, 99m Tc and 188 Re present adequate physical chemical properties for use in Nuclear Medicine in the areas of diagnosis and therapy, respectively. Moreover, these radioisotopes can be distributed to medical centers in the form of generator systems of 99 Mo- 99m Tc and 188 W- 188 Re, allowing autonomy and practicity in use. The objective of this work consists of determining the capacity of some adsorbent materials for retention of molibdenium and tungsten, aiming the optimization of generator systems of 99 Mo- 99 mTc and 188 W- 188 Re with suitable characteristics for application in Nuclear Medicine. Known amounts, in mass, of molybdenum (Mo) and tungsten (W) were added to the loading solutions previously prepared, with values of pH adjusted between 1 and 7, and these were then percolated through different devices containing in its interior alumina, resin or poly-zirconium compound also known as PZC. The elutions were carried out within an interval of time of approximately 24 hours for the generator systems of 99 Mo - 99 mTc and 48 hours for the generator systems of 188 W- 188 Re due to the difference between the half-lives of radionuclides involved in the reactions. The eluted samples from both generator systems containing 99m Tc or 188 Re were submitted to quality control tests aiming to evaluate the radionuclidic, radiochemical and chemical purity, but no significant contamination for 99 Mo, 188 W, technetium or rhenium in the colloidal states and zirconium were detected in the eluted solutions and in the solutions extracted with saline solution for any value of pH studied. The commercial alumina cartridges known as Acid Sep Pak were more efficient in retaining the molybdenum in the loading solutions when compared with the others commercial retention devices employed. However, when this comparison extends to the chromatographic alumina columns, the use of acid alumina as adsorbent is more efficient when compared to the Acid Sep

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Science.gov (United States)

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

Study on the pharmacal of 186,188Re-SZ39

International Nuclear Information System (INIS)

Zu Jianhua; Wu Yuanfang; Dong Mo; Li Huiyuan; ZhangYulong

2000-01-01

The stability, immunocompetence and cytotoxicity in tumor as well as tumor inhibiting rate of 186,188 Re-SZ39 are investigated. The results indicate that 186,188 Re-SZ39 is stable in vivo by imaging and the volume of tumor of nude mice reduced obviously. So 186,188 Re-SZ39 has a strong cytotoxicity effect against glioma cells and is of good prospect as immuno-radiotherapeutic agent

Optimization of labelling procedure of 188rE-DMSA(v)

International Nuclear Information System (INIS)

Dantas, Danielle M.; Brambilla, Tania P.; Reis, Nicoli F.; Osso Junior, Joao A.

2011-01-01

Radionuclide therapy (RNT) is emerging as an important tool of nuclear medicine. Apart from the well established 131 I, several other promising radionuclides have been identified, among them 188 Re, 90 Y and 177 Lu. 188 Re has received a lot of attention in the past decade, due to its favourable nuclear characteristics [t 1/2 16.9 h, E b eta m ax 2.12 MeV and E g amma 155 keV (15%) suitable for imaging, including the fact that it is carrier-free and can be obtained cost-effectively through the generator 188 W- 188 Re. Biodistribution studies of 188 Re-DMSA(V) have shown that its general pharmacokinetic properties are similar to that of 99m Tc-DMSA(V), so this agent could be used for targeted radiotherapy of medullary thyroid carcinoma, bone metastases, soft tissue and others tumors. The aim of this work is to evaluate two labeling procedures for the preparation of 188 Re-DMSA(V). The first method was prepared using a commercial kit of DMSA(III) for labeling with 99m Tc at high temperature (100 deg C). The second method was prepared in a vial containing 2.5 mg of DMSA, 1.00 mg of SnCl 2 .2H 2 O and 10 mg of sodium oxalate, 10 mg of cyclodextrin, in a total volume of 2.0 mL. The pH was adjusted to 3 with 37% HCl. After labeling the solution was stirred and incubated for 30 min at room temperature. The radiochemical purity was determined using TLC-SG developed with two different solvent systems: Acetone and glycine. Preliminary results for both methods of labeling 188 Re-DMSA(V) showed that the labeling yield was >95%. Further experiments are also necessary to optimize the labeling methodology of 188 Re-DMSA(V).author)

Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

Science.gov (United States)

Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

2011-01-01

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

Synthesis and characterization of hydroxyapatite (HAp) for binder tungstate in 188W/188Re generator system

International Nuclear Information System (INIS)

Eni Hartati; Yati B Yuliyati; Duyeh Setiawan

2014-01-01

In this study, the synthesis of hydroxyapatite has been carried out through a process of precipitation of calcium hydroxide (Ca(OH) 2 ) with phosphoric acid (H 3 PO 4 ) based on acid base reaction process that produce crystalline solid and then it will be used for binder tungstate in 188 W/ 188 Re generator system. The purpose of this study were to characterize the results of synthesized hydroxyapatite and to determine the optimum conditions on generator 188 W/ 188 Re performance such as activation of the heating hydroxyapatite, pH of Na 2 WO 4 , reaction temperature, distribution coefficient and the adsorption capacity (adsorption coefficient) of hydroxyapatite. The characterization of synthesized hydroxyapatite was done using FTIR, XRD and SEM-EDAX, while the determination of the optimum condition of each parameters based on distribution coefficient and adsorption capacity hydroxyapatite using spectrophotometric method. The FTIR results of hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ) showed the appearance of peaks in the O-H stretching wave number (υ) and 3434.9 cm -1 and 563.8 cm -1 , while the group PO 4 -3 is shown by the P-O bond in (υ) 1033.8 cm -1 . The XRD results indicate that hydroxyapatite had three peaks at 2θ region is 31,875° , 32,205° and 33,080°. Characterization by SEM showed columnar morphology with a particle size of 200 nm. The optimum conditions of each parameter obtained at hydroxyapatite heating temperature 100 °C, Na 2 WO 4 solution pH 3, and the reaction temperature of 60 °C, which gave result on distribution coefficients of 193.74 mL/g and adsorption capacity (adsorption coefficient) of 5.20 mg W/g HAp. (author)

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

International Nuclear Information System (INIS)

Schulz, P.; Moscarello, M.A.; Cruz, T.F.

1988-01-01

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [ 32 P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein

A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

DEFF Research Database (Denmark)

Dang, Pham My-Chan; Stensballe, Allan; Boussetta, Tarek

2006-01-01

mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM......Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem...

SERS-Based Prognosis of Kidney Transplant Outcome

Science.gov (United States)

Chi, Jingmao

the unique SERS spectral features that indicate kidney AR. By integrating SERS analysis with statistical and chemical analysis and with the promising outcomes, this research has made a significant contribution in exploring the frontier of SERS analysis in biomedical sensing and diagnosis.

Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

Science.gov (United States)

Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

2015-01-01

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

Phosphorylation of MgrA and its effect on expression of the NorA and NorB efflux pumps of Staphylococcus aureus.

Science.gov (United States)

Truong-Bolduc, Que Chi; Hooper, David C

2010-05-01

MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrA(S110A-S113A) bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

Lithium in old open clusters - NGC 188

International Nuclear Information System (INIS)

Hobbs, L.M.; Pilachowski, C.

1988-01-01

Echelle spectra which include the Li I line at 6707 A are reported for seven main-sequence stars and one subgiant in NGC 188. The Li I line is detected in five of the six dwarfs which are highly probable cluster members. The derived atmospheric Li/H ratios exceed the solar value by factors ranging approximately from 10 to 40, although these apparently closely solarlike stars are about twice as old as the sun. The variation of the lithium abundance with stellar mass along the main sequences of the Pleiades, the Hyades, NGC 752, and NGC 188 are compared. The resulting evolutionary pattern indicates that the lithium fraction in the Galactic gas has shown no appreciable change from Li/H of roughly 10 to the -9th since the birth of NGC 188 about 10 Gyr ago, except that the abundance could have been higher by an uncertain but possibly appreciable factor at the beginning of that epoch. 51 references

Development of an optical fiber SERS microprobe for minimally invasive sensing applications

Science.gov (United States)

Mamun, Md Abdullah Al; Juodkazis, Saulius; Mahadevan-Jansen, Anita; Stoddart, Paul R.

2018-02-01

Numerous potential biomedical sensing applications of surface-enhanced Raman scattering (SERS) have been reported, but its practical use has been limited by the lack of a robust sensing platform. Optical fiber SERS probes show great promise, but are limited by the prominent silica Raman background, which requires the use of bulky optics for filtering the signal collection and excitation delivery paths. In the present study, a SERS microprobe has been designed and developed to eliminate the bottlenecks outlined above. For efficient excitation and delivery of the SERS signal, both hollow core photonic crystal fiber and double clad fiber have been investigated. While the hollow core fiber was still found to have excessive silica background, the double clad fiber allows efficient signal collection via the multi-mode inner cladding. A micro filtering mechanism has been designed, which can be integrated into the tip of the optical fiber SERS probe, providing filtering to suppress silica Raman background and thus avoiding the need for bulky optics. The design also assists in the efficient collection of SERS signal from the sample by rejecting Rayleigh scattered light from the sample. Optical fiber cleaving using ultra-short laser pulses was tested for improved control of the fiber tip geometry. With this miniaturized and integrated filtering mechanism, it is expected that the developed probe will promote the use of SERS for minimally invasive biomedical monitoring and sensing applications in future. The probe could potentially be placed inside a small gauge hypodermic needle and would be compatible with handheld portable spectrometers.

Lifetime measurements in shape transition nucleus 188Pt

Science.gov (United States)

Rohilla, Aman; Gupta, C. K.; Singh, R. P.; Muralithar, S.; Chakraborty, S.; Sharma, H. P.; Kumar, A.; Govil, I. M.; Biswas, D. C.; Chamoli, S. K.

2017-04-01

Nuclear level lifetimes of high spin states in yrast and non-yrast bands of 188Pt nucleus have been measured using recoil distance plunger setup present at IUAC, Delhi. In the experiment nuclear states of interest were populated via 174Yb(18O,4 n)188Pt reaction at a beam energy of 79MeV provided by 15 UD Pelletron accelerator. The extracted B(E2\\downarrow) values show an initial rise up to 4+ state and then a nearly constant behavior with spin along yrast band, indicating change of nuclear structure in 188Pt at low spins. The good agreement between experimental and TPSM model B(E2\\downarrow) values up to 4^+ state suggests an increase in axial deformation of the nucleus. The average absolute β2 = 0.20 (3) obtained from measured B(E2\\downarrow) values matches well the values predicted by CHFB and IBM calculations for oblate ( β2 ˜ -0.19) and prolate (β2 ˜ 0.22) shapes. As the lifetime measurements do not yield the sign of β2, no definite conclusion can be drawn on the prolate or oblate collectivity of 188Pt on the basis of present measurements.

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

Science.gov (United States)

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

A central role for CK1 in catalysing phosphorylation of the P53 transactivation domain at serine 20 after HHV-6B viral infection

DEFF Research Database (Denmark)

Maclaine, NJ; Øster, Bodil; Bundgaard, Bettina

2008-01-01

The tumour suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type-I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at serine 20 (Ser20) whose modification stabilises the binding of the transc...... was not blocked by D4476. These data highlight a central role for CK1 as the Ser20-site kinase for p53 in DNA virus-infected cells, but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser20....

46 CFR 188.27-1 - Lifesaving appliances and arrangements.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Lifesaving appliances and arrangements. 188.27-1 Section... VESSELS GENERAL PROVISIONS Lifesaving Appliances and Arrangements § 188.27-1 Lifesaving appliances and arrangements. All lifesaving appliances and arrangements shall be in accordance with the requirements for...

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

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Cho, Seong-Jun; Kang, Hana [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Kim, Min Young [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Pyo, Suhkneung [College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do (Korea, Republic of); Yang, Kwang Hee, E-mail: kwangheey@khnp.co.kr [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of)

2016-04-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a {sup 137}Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

Science.gov (United States)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

International Nuclear Information System (INIS)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-01-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a "1"3"7Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

AMPA receptor phosphorylation and recognition memory: learning-related, time-dependent changes in the chick brain following filial imprinting.

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Solomonia, Revaz O; Meparishvili, Maia; Mikautadze, Ekaterine; Kunelauri, Nana; Apkhazava, David; McCabe, Brian J

2013-04-01

There is strong evidence that a restricted part of the chick forebrain, the intermediate medial mesopallium (IMM), stores information acquired through the learning process of visual imprinting. We have previously demonstrated that at 1 h but not 24 h after imprinting training, a learning-specific increase in the amount of membrane Thr286-autophosphorylated α-calcium/calmodulin-dependent protein kinase II (αCaMKII), and in the proportion of total αCaMKII that is phosphorylated, occurs in the IMM but not in a control brain region, the posterior pole of the nidopallium (PPN). αCaMKII directly phosphorylates Ser831 in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. In the present study we have inquired whether the learning-related increase in αCaMKII autophosphorylation is followed by changes in the Ser831 phosphorylation of GluA1 (P-GluA1) and in the total amount of this subunit (T-GluA1). Trained chicks together with untrained control chicks were killed either 1 or 24 h after training. Tissue was removed from the IMM together with tissue from the PPN as a control. Amounts of P-GluA1 and T-GluA1 were measured. In the left IMM of the 1 h group the P-GluA1/T-GluA1 ratio increased in a learning-specific way. No learning-related changes were observed in other brain regions at 1 h or in any region 24 h after training. The results indicate that a time- and regionally-dependent, learning-specific increase in GluA1 phosphorylation occurs early in recognition memory formation.

Dosimetric evaluation of anti-CD20 labelled with {sup 188}Re

Energy Technology Data Exchange (ETDEWEB)

Barrio, Graciela; Osso Junior, Joao A., E-mail: gracielabarrio@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2011-07-01

Radioimmunotherapy has the potential to deliver lethal radiation energy directly to malignant cells via targeting of radioisotope-conjugated monoclonal antibodies (MAbs) to specific antigens. B-cell lymphoma is a particularly good candidate for radioimmunotherapy because the disease is inherently radiosensitive, malignant cells in the blood, bone marrow, spleen and lymphonodes are accessible, and MAbs have been developed to B-cell surface antigens that do not shed or modulate. Rituximab (RTX), the human IgG1-type chimeric form of the parent murine antibody ibritumomab, is specifically targeted against CD20, a surface antigen expressed by pre-B and mature human B lymphocytes. The use of rhenium-188 from a {sup 188}W/{sup 188}Re generator system represents an attractive alternative radionuclide for therapy. {sup 188}Re is produced from beta decay of the {sup 188}W parent. In addition to the emission of high-energy electrons (E{beta}= 2118 keV), {sup 188}Re also decays with emission of a gamma photon with an energy of 155 keV in 15% abundance. Besides the therapeutic usefulness of {sup 188}Re, the emission of gamma photon is an added advantage since the biodistribution of {sup 188}Re-labeled antibodies can be evaluated in vivo with a gamma camera. Also, rhenium has chemical properties similar to technetium. Thus, both can be conjugated to antibodies using similar chemistry methods. The objective of this work is to prove the usefulness of this radiopharmaceutical based on dosimetric studies, that are also required by the Brazilian Regulatory Agency (ANVISA). (author)

Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

Science.gov (United States)

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-07-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

188Re-microspheres of albumin - the potential preparation for radiotherapy

International Nuclear Information System (INIS)

Dyomin, D.N.; Petriev, V.M.

2000-01-01

In this paper author describe preparation the albumin microspheres labelled with rhenium-188. We undertake an attempt to develop kits to the generator of rhenium-188 on the basis of albumin microspheres for radiotherapy of both oncological and non-oncological diseases. Microspheres, rhenium-188 with sizes 1 0-20 micron for treatment of rheumatoid arthritis (damage of large and intermediate joints), intraperitoneal administration and intrapleural administration at metastases covering a cavity. Microspheres, Re-188 with sizes 40-60 micron for treatment of disseminated kidney cancer (intraarterial, selectively), intratumoral administration to damaged nodules less than 2-3 cm. Microspheres, Re-188 with sizes 80-100 micron for large neoplasms and metastases of liver (intraarterial, selectively), intratumoral administration to damaged nodules with sizes over 3 cm. Preparation of albumin microspheres is carried out by thermal denaturation of protein in vegetable oil. Microspheres are obtained with the necessary range of sizes by ultrasonic fractionation. At our laboratory the method of preparation of albumin microspheres with any sizes of particles (from 5 -10 up to 800 -1000 microns) has been developed. (authors)

Comparative studies of antibody anti-CD20 labeled with 188Re

International Nuclear Information System (INIS)

Dias, Carla Roberta de Barros Rodrigues

2010-01-01

Nuclear Medicine is an unique and important modality in oncology and the development of new tumor-targeted radiopharmaceuticals for both diagnosis and therapy is an area of interest for researchers. Rituximab (RTX) is a quimeric monoclonal antibody (mAb) (IgG 1) that specifically binds to CD20 antigen with high affinity and has been successfully used for the treatment of Non-Hodgkin Lymphoma (NHL) of cell B. The CD20 antigen is expressed over more than 90% of cell B NHL. Technetium-99m ( 99m Tc) and rhenium-188 ( 188 Re) are an attractive radionuclide pair for clinical use due to their favorable decay properties for diagnosis ( 99m Tc: T 1/2 = 6 h, γ radiation = 140 keV) and therapy ( 188 Re: T 1/2 = 17 h, maximum β energy = 2.12 MeV) and to their availability in the form of 99 Mo/ 99 mTc and 188 W/ 188 Re generators. The radionuclides can be conjugated to mAb using similar chemical procedures. The aim of this work was to study the labeling of anti-CD20 mAb (RTX) with 188 Re using two techniques: the direct labeling method [ 188 Re(V)] and the labeling method via the carbonyl nucleus [ 188 Re(I)]. Besides the quality control, the radiolabeled mAb was submitted to in vivo, in vitro and ex vivo biological studies. For the direct labeling, RTX was reducing by incubation with 2-mercaptoethanol for generating sulphydryl groups (-SH) and further labeled with 188 Re(V), in a study of several parameters in order to reach an optimized formulation. The labeling via the carbonyl nucleus both 99 mTc and 188 Re were employed through 2 different procedures: (1) labeling of intact RTX with 99 mTc(I) and (2) reduced RTX (RTX red ) labeled with 99 mTc(I)/ 188 Re(I). Also a parameter study was performed to obtain an optimized formulation. The quality control method for evaluating the radiochemical purity showed a good labeling yield (93%) for the direct method. The labeling method via carbonyl group, the results showed that the - SH groups of RTX red are a possible way of labeling

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

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Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

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Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

2017-08-05

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

Science.gov (United States)

Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

2015-01-01

Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

OpenAIRE

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-01-01

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid subs...

Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

Science.gov (United States)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-07-12

Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

International Nuclear Information System (INIS)

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-01-01

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

GSK3-mediated MAF phosphorylation in multiple myeloma as a potential therapeutic target

International Nuclear Information System (INIS)

Herath, N I; Rocques, N; Garancher, A; Eychène, A; Pouponnot, C

2014-01-01

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the proliferation of mature antibody-secreting plasma B cells in the bone marrow. MM can arise from initiating translocations, of which the musculoaponeurotic fibrosarcoma (MAF) family is implicated in ∼5%. MMs bearing Maf translocations are of poor prognosis. These translocations are associated with elevated Maf expression, including c-MAF, MAFB and MAFA, and with t(14;16) and t(14;20) translocations, involving c-MAF and MAFB, respectively. c-MAF is also overexpressed in MM through MEK/ERK activation, bringing the number of MMs driven by the deregulation of a Maf gene close to 50%. Here we demonstrate that MAFB and c-MAF are phosphorylated by the Ser/Thr kinase GSK3 in human MM cell lines. We show that LiCl-induced GSK3 inhibition targets these phosphorylations and specifically decreases proliferation and colony formation of Maf-expressing MM cell lines. Interestingly, bortezomib induced stabilisation of Maf phosphorylation, an observation that could explain, at least partially, the low efficacy of bortezomib for patients carrying Maf translocations. Thus, GSK3 inhibition could represent a new therapeutic approach for these patients

Study of radiation synovectomy using 188Re-sulfide

International Nuclear Information System (INIS)

Chen Gang; Li Peiyong; Jiang Xufeng; Zhang Liying; Wang Xuefeng; Sun Zhenming; Zhang Huan

2002-01-01

Objective: To study the radiation synovectomy with 188 Re-sulfide. Methods: Thirty cases were divided into 2 groups, the group with hemophilia and the group with rheumatoid arthritis (RA). Patients with joint synovitis were injected different doses of 188 Re-sulfide, 222 - 444 MBq intra-articular. MRI was taken before and 3 - 6 months after the radiation synovectomy to evaluate the treatment efficacy, and the symptoms were also evaluated. Results: MRI study showed that after the treatment the synovium became thiner and the edema was reduced in the lesioned joint. The symptoms were improved with the pain relieved and duration of intra-articular hemorrhage reduced. Conclusions: Radiation synovectomy using 188 Re-sulfide has effects on synovitis. It can be used clinically to improve the symptoms of joint synovitis and reduce the duration of intra-articular hemorrhage

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17*

Science.gov (United States)

Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi

2016-01-01

Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. PMID:26499836

PhosphoRice: a meta-predictor of rice-specific phosphorylation sites

Directory of Open Access Journals (Sweden)

Que Shufu

2012-02-01

Full Text Available Abstract Background As a result of the growing body of protein phosphorylation sites data, the number of phosphoprotein databases is constantly increasing, and dozens of tools are available for predicting protein phosphorylation sites to achieve fast automatic results. However, none of the existing tools has been developed to predict protein phosphorylation sites in rice. Results In this paper, the phosphorylation site predictors, NetPhos 2.0, NetPhosK, Kinasephos, Scansite, Disphos and Predphosphos, were integrated to construct meta-predictors of rice-specific phosphorylation sites using several methods, including unweighted voting, unreduced weighted voting, reduced unweighted voting and weighted voting strategies. PhosphoRice, the meta-predictor produced by using weighted voting strategy with parameters selected by restricted grid search and conditional random search, performed the best at predicting phosphorylation sites in rice. Its Matthew's Correlation Coefficient (MCC and Accuracy (ACC reached to 0.474 and 73.8%, respectively. Compared to the best individual element predictor (Disphos_default, PhosphoRice archieved a significant increase in MCC of 0.071 (P Conclusions PhosphoRice is a powerful tool for predicting unidentified phosphorylation sites in rice. Compared to the existing methods, we found that our tool showed greater robustness in ACC and MCC. PhosphoRice is available to the public at http://bioinformatics.fafu.edu.cn/PhosphoRice.

The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia.

Directory of Open Access Journals (Sweden)

Katherine Mills-Lujan

Full Text Available Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV. The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1 mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2 Ser49 is phosphorylated in planta; and 3 plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.

Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2 in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA.

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Wimolpak Sriwai

release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser(188.

Labelling of Re-ABP with 188Re for bone pain palliation

International Nuclear Information System (INIS)

Arteaga de Murphy, Consuelo; Ferro-Flores, Guillermina; Pedraza-Lopez, Martha; Melendez-Alafort, Laura; Croft, B.Y.Barbara Y.; Ramirez, Flor de Maria; Padilla, Juan

2001-01-01

Etidronate and medronate have been labelled with technetium-99m ( 99m Tc-HEDP, 99m Tc-MDP) for bone scanning and, with rhenium-188 ( 188 Re-HEDP) to palliate the pain resulting from bone metastases. The objective of this study was to label alendronate, ABP, a new bisphosphonate, with SnF 2 -reduced- 188 Re. The reagents for the 5 mg ABP kit were SnF 2 , KReO 4 and gentisic acid at acid pH. The chemical, spectroscopic and microscopic characteristics, quality control, rat bone uptake of [ 188 Re]Re-ABP and similarities with 99m Tc-ABP are presented. We conclude that this is a promising new radiopharmaceutical for bone metastases pain palliation

hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

International Nuclear Information System (INIS)

Berglund, Fredrik M.; Clarke, Paul R.

2009-01-01

Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

Comparison of the phosphorylation events in membranes prepared from proliferating versus quiescent endothelial cells

International Nuclear Information System (INIS)

Kazlauskas, A.; DiColeto, P.E.

1986-01-01

Little is known of the intracellular events which regulate the proliferation of endothelial cells (EC). Triton-solubilized membranes from proliferating (sparse) and quiescent (confluent) EC were incubated at pH 6.5 in the presence of divalent cations and [ 32 P]ATP. Membrane proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The overall kinase activity per mg protein was slightly greater in membranes prepared from proliferating versus quiescent cells. They found four proteins labeled in sparse cells to a dramatically greater extent having the following approximate molecular masses: 180, 100, 97 and 55 kilodalton (kd). The first two phosphoproteins were phosphorylated on serine residues exclusively; the 97 kd phosphoprotein contained 39% phosphoserine (p-ser) and 61% phosphothreonine (p-thr); and the 55 kd phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr). The kinases acting on all four phosphoproteins were independent of Ca 2+ , cAMP, cGMP, or phorbol 12-myristate 13-acetate. The observed differences in phosphorylation events between sparse and confluent membranes occurred in membranes from two EC lines - pig aortic and bovine aortic - but were not apparent in membranes prepared from human foreskin fibroblasts or 3T3 cells. Sparse endothelial cells made quiescent by serum deprivation were found to resemble confluent cells in the kinase activity; therefore, the enhanced kinase activity in sparse membranes may be growth dependent

Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

Science.gov (United States)

Grant, Nicola J; Coates, Philip J; Woods, Yvonne L; Bray, Susan E; Morrice, Nicholas A; Hastie, C James; Lamont, Douglas J; Carey, Francis A; Sutherland, Calum

2015-11-10

Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

Science.gov (United States)

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

Experimental study of the biological properties of 188Re-Hepama-1 biologic superparamagnetic nanoparticles

International Nuclear Information System (INIS)

Feng Yanlin; Tan Jiaju; Sun Jing; Wen Guanghua; Wu Xiaolian; Liang Sheng; Xia Jiaoyun

2007-01-01

Objective: To investigate a new biologic-superparamagnetic nanoparticles's characteristics of immunological activity, biological distributing in vivo, targeting and inhibiting tumor effect. Methods: The experimental group 188 Re-Hepama-l-superparamagnetic nanoparticles, and control groups, including 188 ReO 4 - , 188 Re-Hepama-1, and 188 Re-superparamagnetic nanoparticles, were set up. The distributions were measured after injection 4 h and 24 h by caudal vein of Kuming mice. The magnetic targeting experiments in vivo were clone with and without magnetic field in liver after injection in New Zealand rabbit. The inhibiting tumor effect on hepatic cancer cell lines SMMC-7721 of the above four 188 Re labeled products were measured by mono nuclear cell direct cytotoxicity assay method. Results: After injection 4 h and 24 h by vein, the liver taking was highest in group 188 Re-Hepama-l-superparamagnetic nanoparticles. The radiative activity in liver in magnetism zoo was higher than in non magnetism zoo in 188 Re- Hepama-1-superparamagnetic nanoparticles after applying magnetic field in left lobe of liver, and the ratio of in magnetism zoo to non magnetism zoo was 1.87. And the half effective inhibition radioactive concentrations (IC 50 ) in 188 Re-Hepama-l-superparamagnetic nanoparticles was one forth of 188 ReO 4 - . Conclusion: 188 Re- Hepama-l-superparamagnetic nanoparticles showed its fine stability in intro, good immunological activity and significant liver target. (authors)

Poloxamer [corrected] 188 has a deleterious effect on dystrophic skeletal muscle function.

Directory of Open Access Journals (Sweden)

Rebecca L Terry

Full Text Available Duchenne muscular dystrophy (DMD is an X-linked, fatal muscle wasting disease for which there is currently no cure and limited palliative treatments. Poloxomer 188 (P188 is a tri-block copolymer that has been proposed as a potential treatment for cardiomyopathy in DMD patients. Despite the reported beneficial effects of P188 on dystrophic cardiac muscle function, the effects of P188 on dystrophic skeletal muscle function are relatively unknown. Mdx mice were injected intraperitoneally with 460 mg/kg or 30 mg/kg P188 dissolved in saline, or saline alone (control. The effect of single-dose and 2-week daily treatment was assessed using a muscle function test on the Tibialis Anterior (TA muscle in situ in anaesthetised mice. The test comprises a warm up, measurement of the force-frequency relationship and a series of eccentric contractions with a 10% stretch that have previously been shown to cause a drop in maximum force in mdx mice. After 2 weeks of P188 treatment at either 30 or 460 mg/kg/day the drop in maximum force produced following eccentric contractions was significantly greater than that seen in saline treated control mice (P = 0.0001. Two week P188 treatment at either dose did not significantly change the force-frequency relationship or maximum isometric specific force produced by the TA muscle. In conclusion P188 treatment increases susceptibility to contraction-induced injury following eccentric contractions in dystrophic skeletal muscle and hence its suitability as a potential therapeutic for DMD should be reconsidered.

Flux control through protein phosphorylation in yeast

DEFF Research Database (Denmark)

Chen, Yu; Nielsen, Jens

2016-01-01

Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

Science.gov (United States)

Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

International Nuclear Information System (INIS)

O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

2008-01-01

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by ∼3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 (angstrom) X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state

Radiolabeling of anti-CD20 with Re0-188: liquid kit

International Nuclear Information System (INIS)

Dias, Carla Roberta; Osso Junior, Joao Alberto

2007-01-01

Radioimmunotherapy uses the targeting features of monoclonal antibody to deliver radiation from an attached radionuclide. The radionuclide 188 Re is currently produced from the father nuclide 188 W through a transportable generator system. Because of its easy availability and suitable nuclear properties (E βMAX =2.1 MeV, t 1/2 =16.9 h, E γ =155 keV), this radionuclide is considered an attractive candidate for application as therapeutic agents and could be conveniently utilized for imaging and dosimetric purposes. The objective of this work is the optimization of radiolabeling of anti-CD20 with 188 Re using a liquid formulation. Anti-CD20 was reduced by incubation with 2-ME and purified over a PD-10 column. The number of resulting free SH was assayed with Ellman's reagent. Optimization of radiolabeling was achieved by varying parameters: antibody mass, reducing agent, reaction time and 188 Re volume in the liquid kit. Radiochemical purity of 188 Re-anti-CD20 was evaluated. An average of 12 SH groups per mol in the reductions was found. The best labeling efficiency (> 93%) was achieved in the following conditions: 1 mg anti-CD20; 82.8 mg sodium tartrate; 1 mg SnCl 2 ; 0.25 mg gentisic acid, 1 mL 188 Re and reaction time of 1 hour at room temperature. (author)

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

Directory of Open Access Journals (Sweden)

Ann De Vos

2015-04-01

Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

Intracerebroventricular administration of okadaic acid induces hippocampal glucose uptake dysfunction and tau phosphorylation.

Science.gov (United States)

Broetto, Núbia; Hansen, Fernanda; Brolese, Giovana; Batassini, Cristiane; Lirio, Franciane; Galland, Fabiana; Dos Santos, João Paulo Almeida; Dutra, Márcio Ferreira; Gonçalves, Carlos-Alberto

2016-06-01

Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization of labelling procedure of {sup 188}rE-DMSA(v)

Energy Technology Data Exchange (ETDEWEB)

Dantas, Danielle M.; Brambilla, Tania P.; Reis, Nicoli F.; Osso Junior, Joao A., E-mail: jaosso@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2011-07-01

Radionuclide therapy (RNT) is emerging as an important tool of nuclear medicine. Apart from the well established {sup 131}I, several other promising radionuclides have been identified, among them {sup 188}Re, {sup 90}Y and {sup 177}Lu. {sup 188}Re has received a lot of attention in the past decade, due to its favourable nuclear characteristics [t{sub 1/2} 16.9 h, E{sub b}eta{sub m}ax 2.12 MeV and E{sub g}amma 155 keV (15%) suitable for imaging, including the fact that it is carrier-free and can be obtained cost-effectively through the generator {sup 188}W-{sup 188}Re. Biodistribution studies of {sup 188}Re-DMSA(V) have shown that its general pharmacokinetic properties are similar to that of {sup 99m}Tc-DMSA(V), so this agent could be used for targeted radiotherapy of medullary thyroid carcinoma, bone metastases, soft tissue and others tumors. The aim of this work is to evaluate two labeling procedures for the preparation of {sup 188}Re-DMSA(V). The first method was prepared using a commercial kit of DMSA(III) for labeling with {sup 99m}Tc at high temperature (100 deg C). The second method was prepared in a vial containing 2.5 mg of DMSA, 1.00 mg of SnCl{sub 2}.2H{sub 2}O and 10 mg of sodium oxalate, 10 mg of cyclodextrin, in a total volume of 2.0 mL. The pH was adjusted to 3 with 37% HCl. After labeling the solution was stirred and incubated for 30 min at room temperature. The radiochemical purity was determined using TLC-SG developed with two different solvent systems: Acetone and glycine. Preliminary results for both methods of labeling {sup 188}Re-DMSA(V) showed that the labeling yield was >95%. Further experiments are also necessary to optimize the labeling methodology of {sup 188}Re-DMSA(V).author)

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17.

Science.gov (United States)

Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi; Wu, Shih-Hsiung

2016-01-01

Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S(88)VS(90)K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

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Zhuo Zhan

Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

Structural characterization by NMR of a double phosphorylated chimeric peptide vaccine for treatment of Alzheimer's disease.

Science.gov (United States)

Ramírez-Gualito, Karla; Richter, Monique; Matzapetakis, Manolis; Singer, David; Berger, Stefan

2013-04-26

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer's disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau₂₂₉₋₂₃₇[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B₂₄₁₋₂₅₅ originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

Ebselen inhibits iron-induced tau phosphorylation by attenuating DMT1 up-regulation and cellular iron uptake.

Science.gov (United States)

Xie, Ling; Zheng, Wei; Xin, Na; Xie, Jing-Wei; Wang, Tao; Wang, Zhan-You

2012-08-01

Dysregulation of iron homeostasis is involved in the pathological process of Alzheimer's disease (AD). We have recently reported that divalent metal transporter 1 (DMT1) is upregulated in an AD transgenic mouse brain, and that silencing of DMT1, which reduces cellular iron influx, results in inhibition of amyloidogenesis in vitro, suggesting a potential target of DMT1 for AD therapy. In the present study, we tested the hypothesis that inhibition of DMT1 with ebselen, a DMT1 transport inhibitor, could affect tau phosphorylation. Human neuroblastoma SH-SY5Y cells were pre-treated with ebselen and then treated with ferrous sulfate (dissolved in ascorbic acid), and the effects of ebselen on tau phosphorylation and the relative signaling pathways were examined. Our results showed that ebselen decreased iron influx, reduced iron-induced ROS production, inhibited the activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3β, and ultimately attenuated the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. The present study indicates that the neuroprotective effect of ebselen on AD is not only related to its antioxidant activity as reported previously, but is also associated with a reduction in tau phosphorylation by inhibition of DMT1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development of SERS active fibre sensors

International Nuclear Information System (INIS)

Polwart, Ewan

2002-01-01

Surface-enhanced Raman scattering (SERS) is sensitive and selective and when coupled with fibre-optics could potentially produce an effective chemical sensing system. This thesis concerns the development of a single-fibre-based sensor, with an integral SERS-active substrate. A number of different methods for the manufacture of SERS-active surfaces on glass substrates were investigated and compared. The immobilisation of metal nanoparticles on glass functionalised with (3-aminopropyl)trimethoxysilane emerged as a suitable approach for the production of sensors. Substrates prepared by this approach were characterised using UV-visible spectroscopy, electron microscopy and Raman mapping. It was found that exposure of substrates to laser radiation led to a decrease in the signal recorded from adsorbed analytes. This speed of the decrease was shown to depend on the analyte, and the exciting wavelength and power. SERS-active fibre sensors were produced by immobilisation of silver nanoparticles at the distal end of a (3-aminopropyl)trimethoxysilane-derivatised optical fibre. These sensors were used to obtain spectra with good signal to noise ratios from 4-(benzotriazol-5-ylazo)-3,5-dimethoxyphenylamine and crystal violet. Sensing of dyes in effluent was also investigated. The development of sensors for the measurement of pH, by treating the SERS-active fibre tip with pH sensitive dyes is also described. Spectral changes were observed with these sensors as a response to the pH. Partial least squares regression was used to produce linear calibration models for the pH range 5-11 from which it was possible to predict the pH with an accuracy of ∼0.2 pH units. Some of the limitations of these sensors were explored. The feasibility of using these sensors for measurement of oxygen and thiols, was investigated. The measurement of oxygen using methylene blue as a transducer was demonstrated. Two transduction methodologies--reactions with iron porphyrins and pyrrole-2,5-diones

Country Report: Rep. of Korea. {sup 188}re-Tricabonyl Labeling Strategy: Preparation of 1{sup 188}Re(I) Tricarbonyl Precursor [{sup 188}Re(OH{sub 2}){sub 3}(CO){sub 3}]+ for the Labeling of Biomolecules

Energy Technology Data Exchange (ETDEWEB)

Park, Sang Hyun [Radiation Research Division for Biotechnology, Korea Atomic Energy Research Institute (Korea, Republic of)

2010-07-01

The {sup 99m}Tc(I) and {sup 188}Re(I) tricarbonyl precursors [M(OH{sub 2}){sub 3}(CO){sub 3}]{sup +} have been shown to be excellent starting materials for the synthesis of {sup 99m}Tc(I) and {sup 188}Re(I) tricarbonyl complexes as well as radiolabeling of target-specific biomolecules.{sup 1-8} Recently, a user-friendly kit formulation (IsoLink{sup TM}) was developed using potassium boranocarbonate, K{sub 2}[BH{sub 3}CO{sub 2}], for preparation of the {sup 99m}Tc precursor complex. This solid reagent serves both as a source of carbon monoxide and a reducing agent for technetium. It was also used by Schibli and coworkers for the preparation of the corresponding {sup 188}Re precursor complex (“Schibili’s kit”).{sup 9} This approach involved the reduction of [{sup 188}Re]perrhenate eluate (1 ml) in neutral solution with 3 mg K{sub 2}[BH{sub 3}CO{sub 2}] and 5 mg BH3≅NH3 by incubation at 60 °C for 15 min. The amounts of reducing agents and acid (concentrated phosphoric acid) were carefully balanced not only to avoid fast hydrolysis of the boranes, but also to maintain a sufficiently low pH to stabilize reduced rhenium intermediates. The preparations resulted in yields >85 % of the desired precursor complex. In addition, perrhenate (7±3 %), colloidal {sup 188}ReO{sub 2} (<5 %), and a byproduct of unknown composition were also detected. To increase the yields even further, we evaluated the recently described borohydride exchange resin (BER) as an additional reducing agent and an anion scavengers.

Integrated Lateral Flow Test Strip with Electrochemical Sensor for Quantification of Phosphorylated Cholinesterase: Biomarker of Exposure to Organophosphorus Agents

Energy Technology Data Exchange (ETDEWEB)

Du, Dan; Wang, Jun; Wang, Limin; Lu, Donglai; Lin, Yuehe

2012-02-08

An integrated lateral flow test strip with electrochemical sensor (LFTSES) device with rapid, selective and sensitive response for quantification of exposure to organophosphorus (OP) pesticides and nerve agents has been developed. The principle of this approach is based on parallel measurements of post-exposure and baseline acetylcholinesterase (AChE) enzyme activity, where reactivation of the phosphorylated AChE is exploited to enable measurement of total amount of AChE (including inhibited and active) which is used as a baseline for calculation of AChE inhibition. Quantitative measurement of phosphorylated adduct (OP-AChE) was realized by subtracting the active AChE from the total amount of AChE. The proposed LFTSES device integrates immunochromatographic test strip technology with electrochemical measurement using a disposable screen printed electrode which is located under the test zone. It shows linear response between AChE enzyme activity and enzyme concentration from 0.05 to 10 nM, with detection limit of 0.02 nM. Based on this reactivation approach, the LFTSES device has been successfully applied for in vitro red blood cells inhibition studies using chlorpyrifos oxon as a model OP agent. This approach not only eliminates the difficulty in screening of low-dose OP exposure because of individual variation of normal AChE values, but also avoids the problem in overlapping substrate specificity with cholinesterases and avoids potential interference from other electroactive species in biological samples. It is baseline free and thus provides a rapid, sensitive, selective and inexpensive tool for in-field and point-of-care assessment of exposures to OP pesticides and nerve agents.

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

Radiolabeling of anti-CD20 with Re0-188: liquid kit

Energy Technology Data Exchange (ETDEWEB)

Dias, Carla Roberta; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail: carlarobertab@yahoo.com.br; jaosso@ipen.br

2007-07-01

Radioimmunotherapy uses the targeting features of monoclonal antibody to deliver radiation from an attached radionuclide. The radionuclide {sup 188}Re is currently produced from the father nuclide {sup 188}W through a transportable generator system. Because of its easy availability and suitable nuclear properties (E{sub {beta}}{sub MAX} =2.1 MeV, t{sub 1/2}=16.9 h, E{sub {gamma}}=155 keV), this radionuclide is considered an attractive candidate for application as therapeutic agents and could be conveniently utilized for imaging and dosimetric purposes. The objective of this work is the optimization of radiolabeling of anti-CD20 with {sup 188}Re using a liquid formulation. Anti-CD20 was reduced by incubation with 2-ME and purified over a PD-10 column. The number of resulting free SH was assayed with Ellman's reagent. Optimization of radiolabeling was achieved by varying parameters: antibody mass, reducing agent, reaction time and {sup 188}Re volume in the liquid kit. Radiochemical purity of {sup 188}Re-anti-CD20 was evaluated. An average of 12 SH groups per mol in the reductions was found. The best labeling efficiency (> 93%) was achieved in the following conditions: 1 mg anti-CD20; 82.8 mg sodium tartrate; 1 mg SnCl{sub 2}; 0.25 mg gentisic acid, 1 mL {sup 188}Re and reaction time of 1 hour at room temperature. (author)

34 CFR 668.188 - Preventing evasion of the consequences of cohort default rates.

Science.gov (United States)

2010-07-01

... 34 Education 3 2010-07-01 2010-07-01 false Preventing evasion of the consequences of cohort default rates. 668.188 Section 668.188 Education Regulations of the Offices of the Department of Education... Two Year Cohort Default Rates § 668.188 Preventing evasion of the consequences of cohort default rates...

Review on SERS of Bacteria

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Pamela A. Mosier-Boss

2017-11-01

Full Text Available Surface enhanced Raman spectroscopy (SERS has been widely used for chemical detection. Moreover, the inherent richness of the spectral data has made SERS attractive for use in detecting biological materials, including bacteria. This review discusses methods that have been used to obtain SERS spectra of bacteria. The kinds of SERS substrates employed to obtain SERS spectra are discussed as well as how bacteria interact with silver and gold nanoparticles. The roll of capping agents on Ag/Au NPs in obtaining SERS spectra is examined as well as the interpretation of the spectral data.

Lifetime measurements in shape transition nucleus {sup 188}Pt

Energy Technology Data Exchange (ETDEWEB)

Rohilla, Aman; Gupta, C.K.; Chamoli, S.K. [University of Delhi, Department of Physics and Astrophysics, New Delhi (India); Singh, R.P.; Muralithar, S. [Inter University Accelerator Centre, New Delhi (India); Chakraborty, S.; Sharma, H.P. [Banaras Hindu University, Department of Physics, Varanasi (India); Kumar, A.; Govil, I.M. [Panjab University, Department of Physics, Chandigarh (India); Biswas, D.C. [Bhabha Atomic Research Center, Nuclear Physics Division, Trombay, Mumbai (India)

2017-04-15

Nuclear level lifetimes of high spin states in yrast and non-yrast bands of {sup 188}Pt nucleus have been measured using recoil distance plunger setup present at IUAC, Delhi. In the experiment nuclear states of interest were populated via {sup 174}Yb({sup 18}O,4n){sup 188}Pt reaction at a beam energy of 79MeV provided by 15 UD Pelletron accelerator. The extracted B(E2 ↓) values show an initial rise up to 4{sup +} state and then a nearly constant behavior with spin along yrast band, indicating change of nuclear structure in {sup 188}Pt at low spins. The good agreement between experimental and TPSM model B(E2 ↓) values up to 4{sup +} state suggests an increase in axial deformation of the nucleus. The average absolute β{sub 2} = 0.20 (3) obtained from measured B(E2 ↓) values matches well the values predicted by CHFB and IBM calculations for oblate (β{sub 2} ∝ -0.19) and prolate (β{sub 2} ∝ 0.22) shapes. As the lifetime measurements do not yield the sign of β{sub 2}, no definite conclusion can be drawn on the prolate or oblate collectivity of {sup 188}Pt on the basis of present measurements. (orig.)

Labeling of MDP with 188Re for bone tumour therapy

International Nuclear Information System (INIS)

Barbezan, Angelica B.; Osso Junior, Joao A.

2011-01-01

188 Re is one of the most attractive radioisotopes for a variety of therapeutic applications in nuclear medicine, due to its physical decay properties, such as β - emission of 2.12 MeV, γ emission of 155 keV and half life of 16.9 hours. Biphosphonates are potent inhibitors of osteoclastic bone resorption and are effective in several diseases that cause bone fragility and bone metastases. Because of these characteristics, labeled biphosphonates have been studied for bone pathologies, also acting as palliation of bone pain in case of metastasis.The aim of this study was to optimize the labeling of a phosphonate-MDP (Sodium Methylene Diphosphonate) with 188 Re for use in bone pain palliation. 188 Re was obtained by eluting a 188 W- 188 Re generator from POLATOM. The labeling was performed at room temperature using MDP, SnCl 2 as reducing agent and ascorbic acid. The variables studied were: Mass of ligand (3, 6 and 10 mg), reducing agent mass (5, 7, 10 and 11 mg), ascorbic acid mass (1, 3, 5 and 6 mg), pH (1 and 2) and time of reaction (15, 60, 120, 360 and 4320 minutes), that also reflected the stability of the radiopharmaceutical. The radiochemical control, that also measures the labeling efficiency was evaluated by paper chromatography using Whatman 3MM paper and the solvents acetone and 0.9%NaCl. The best formulation was the following: Mass of ligand MDP: 10 mg, mass of SnCl 2 : 5 mg, ascorbic acid mass: 3 mg, time of reaction: 30 minutes, pH: 1. Under optimum conditions, 188 Re MDP radiolabeling yield was 98,07% and the radiopharmaceutical was stable up to 72 h. (author)

Ralstonia eutropha's Poly(3-hydroxybutyrate)(PHB) polymerase PhaC1 and PHB depolymerase PhaZa1 are phosphorylated in vivo.

Science.gov (United States)

Jüngert, Janina R; Patterson, Cameron; Jendrossek, Dieter

2018-04-20

In this study, we screened PHB synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phase. Thr373 of PHB synthase PhaC1 was phosphorylated in the stationary growth phase but was not modified in the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in phosphorylated form both in the exponential and in the stationary growth phase. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modification of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for modulation of the activities of PHB synthase and PHB depolymerase. Importance Polyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and other stress conditions. The simultaneous presence of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation Cupriavidus necator ) has been previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of identified residues. Furthermore, we conducted in vitro and in vivo analysis of PHB synthase activity and PHB contents. Copyright © 2018 American Society for Microbiology.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Science.gov (United States)

Ying, Sheng; Hill, Allyson T; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

2017-06-01

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds ( Ricinus communis ) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca 2+ -dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: ( i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 μM, ( ii ) a Ca 2+ -dependent electrophoretic mobility shift, and ( iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ( Arabidopsis thaliana ) CPK4 and soybean ( Glycine max ) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. © 2017 American Society of Plant

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction

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Anna Simó

2018-06-01

Full Text Available Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1 synaptic activity at the neuromuscular junction, (2 nPKCε and cPKCβI isoforms activity, (3 muscle contraction per se, and (4 the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min with or without contraction (abolished by μ-conotoxin GIIIB. Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB. Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction.

Science.gov (United States)

Simó, Anna; Just-Borràs, Laia; Cilleros-Mañé, Víctor; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A; Tomàs, Josep

2018-01-01

Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se , and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity-induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity-induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

Rhenium 188 labelling of peptide conjugates

International Nuclear Information System (INIS)

Melendez-Alafort, Laura

2001-01-01

Many human tumours express high levels, of somatostatin receptors. In order to make possible a radiotherapeutic treatment of this kind for tumour a series of somatostatin analogues that can tightly chelate beta emitting isotopes have been developed in recent years. The work carried out for this thesis has been aimed towards development of a new therapeutic radiopharmaceutical for treatment of somatostatin receptor positive tumours. The first chapters describe work with technetium-99m to establish the labelling and analytical conditions for a somatostatin analogue, [Tyr 3 ]-octreotide (TOC), as a precursor to undertaking labelling studies with the beta emitter rhenium-188. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was conjugated to TOC and labelled with 99m using different coligands. Then the stability, receptor binding and biodistribution of each complex were assessed. 99m Tc-HYNIC-TOC using EDDA as coligand showed the best characteristics, and was superior for tumour imaging in humans than the commercially available 111 In-DTPA-octreotide. The conditions for labelling the HYNIC-TOC conjugate with 188 Re were then optimised using tricine as a co-ligand. A labelling yield of ∼80% was achieved. After purification however, the stability of the complex was low. The use of other coligand systems which had proved useful for 99m Tc labelling was explored, but yields were very poor. Other chelators such as diethylenetriamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and mercaptoacetyltriglycine (MAG 3 ) were studied as potential co-ligand agents to label the HYNIC-TOC conjugate with 188 Re but, again low yields of the labelled peptide complexes were achieved. A novel 188 Re-HYNIC complex was prepared in high yields using N-N-disubstituted dithiocarbamates as coligands. However to date, the specific activities achieved with this system are relatively low. The use of the [ 99m Tc(CO) 3 (H 2 O) 3 ] complex to label the HYNIC-TOC conjugate was investigated

Preparation of 188 Re-lanreotide as a potential tumor therapeutic agent

International Nuclear Information System (INIS)

Bai Hongsheng; Jin Xiaohai; Fan Hongqiang; Jia Bing; Wang Yuqing; Lu Weiwei

2001-01-01

Radiolabeled peptides hold unlimited potential in diagnostic applications and therapy of malignant tumor. Somatostatin analogue peptide (Lanreotide) is labeled directly with 188 Re via the mixture of citrate and tartrate. The influences of reaction conditions such as pH, temperature, amount of stannous chloride, Lanreotide quantity, reaction time on labeling yield are investigated in detail. At the same time, the stability in vitro, quality control and animal test are evaluated. The experimental results show that Lanreotide reacts with 188 Re for 40 min at pH 2 - 3 and 60 degree C, the labeling yield is at range of 88% - 94%. After purification of 188 Re-Lanreotide with Sep-Pak C 18 reverse phase extraction cartridge, the radiochemical purity (RP) is more than 95%. 188 Re-Lanreotide is eliminated rapidly from the blood and is excreted through liver, the uptake of lung and intestine is high

Treatment of liver cancer with Rhenium-188 Lipiodol: Colombian experience

International Nuclear Information System (INIS)

Bernal, P.; Osorio, M.; Mendoza, M.; Esguerra, R.; Ucros, G.; Gutierrez, C.; Velez, O.; Cerquera, A.M.; Padhy, A.K.

2002-01-01

Aim:Trans-arterial Radio-conjugate therapy plays an important role in the palliative treatment of inoperable liver cancer. It also helps in reduction of the tumor to an operable state from an inoperable one. As a part of an IAEA sponsored coordinated research project, a new radiopharmaceutical, Rhenium-188 Lipiodol has been developed. The aim of this study was to establish the safety of the radiopharmaceutical and to find out the efficacy of treatment. Materials and Methods: Eight patients suffering from various forms of liver cancer (Hepatocellular carcinoma-4, Metastases from carcinoma of colon-3 and Carcinoid- 1) were treated with Rhenium -188 Lipiodol. Seven out of the eight patients were classified as ECOG- 1 and one as ECOG- 3. Labelling of Rhenium-188 with Lipiodol was carried out according to a protocol developed under the CRP and standardized in our service. Rhenium-188 Lipiodol was administered through the trans-arterial route by either selective (75%) or ultra selective (25%) hepatic arteriography. Administered therapeutic doses ranged between 170 MBq and 4181 MBq. Dosimetric evaluations were made using the IAEA developed dosimetry spreadsheet. All patients were followed up (1-5 months, average = 2 months) after treatment by clinical examination, liver function tests, haematological examinations and CT scans of liver to determine the size of hepatic tumor. Results: Rhenium-188 Lipiodol treatment was well tolerated by all patients. No immediate systemic complications were noted in any of the patients within 72 hrs. following therapy. Only two patients had mild rise in temperature in the immediate post-therapy period, which subsided subsequently. One patient who was classified as Child B and ECOG 3, developed encephalopathy on the seventh day after treatment. He died of hepatic failure. Another one present depressive syndrome, didn't accept food and died Follow-up CT scans in all the surviving (6/8) patients revealed significant reduction of the tumours

Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic.

Science.gov (United States)

Mahoney, Brendan J; Zhang, Meiling; Zintsmaster, John S; Peng, Jeffrey W

2018-03-02

Pin1 is a two-domain human protein that catalyzes the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs in numerous cell-cycle regulatory proteins. These pS/T-P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved "latches" between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T-P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fabrication of Semiconductor ZnO Nanostructures for Versatile SERS Application

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Lili Yang

2017-11-01

Full Text Available Since the initial discovery of surface-enhanced Raman scattering (SERS in the 1970s, it has exhibited a huge potential application in many fields due to its outstanding advantages. Since the ultra-sensitive noble metallic nanostructures have increasingly exposed themselves as having some problems during application, semiconductors have been gradually exploited as one of the critical SERS substrate materials due to their distinctive advantages when compared with noble metals. ZnO is one of the most representative metallic oxide semiconductors with an abundant reserve, various and cost-effective fabrication techniques, as well as special physical and chemical properties. Thanks to the varied morphologies, size-dependent exciton, good chemical stability, a tunable band gap, carrier concentration, and stoichiometry, ZnO nanostructures have the potential to be exploited as SERS substrates. Moreover, other distinctive properties possessed by ZnO such as biocompatibility, photocatcalysis and self-cleaning, and gas- and chemo-sensitivity can be synergistically integrated and exerted with SERS activity to realize the multifunctional potential of ZnO substrates. In this review, we discuss the inevitable development trend of exploiting the potential semiconductor ZnO as a SERS substrate. After clarifying the root cause of the great disparity between the enhancement factor (EF of noble metals and that of ZnO nanostructures, two specific methods are put forward to improve the SERS activity of ZnO, namely: elemental doping and combination of ZnO with noble metals. Then, we introduce a distinctive advantage of ZnO as SERS substrate and illustrate the necessity of reporting a meaningful average EF. We also summarize some fabrication methods for ZnO nanostructures with varied dimensions (0–3 dimensions. Finally, we present an overview of ZnO nanostructures for the versatile SERS application.

Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

International Nuclear Information System (INIS)

Grant, Nicola J.; Coates, Philip J.; Woods, Yvonne L.; Bray, Susan E.; Morrice, Nicholas A.; Hastie, C. James; Lamont, Douglas J.; Carey, Francis A.; Sutherland, Calum

2015-01-01

Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

International Nuclear Information System (INIS)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

2010-01-01

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Energy Technology Data Exchange (ETDEWEB)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates); Galadari, Sehamuddin, E-mail: sehamuddin@uaeu.ac.ae [Cell Signaling Laboratory, Department of Biochemistry, Faculty of Medicine and Health Sciences, UAE University, P.O. Box 17666, Al Ain (United Arab Emirates)

2010-04-09

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Stefan Berger

2013-04-01

Full Text Available Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

MAG2GABA-biocytin synthesized with new intermediates for radiolabelling 99mTc and 188Re

International Nuclear Information System (INIS)

Ahn, S.H.; Choi, T.H.; Choi, C.W.; Yang, S.D.; Woo, K.S.; Chung, W.S.; Lim, S.M.

2001-01-01

188 Re from 188 W- 188 Re generator, is recently introduced in therapeutic nuclear medicine and made it possible to use whenever needed. We synthesized MAG 2 GABA-Biocytin (MGB), labelled with 188 Re for pretargeted radioimmunotherapy and evaluated biological behavior of 188 Re-MGB. N-hydroxysuccinimidyl ester of S-benzoyl mercaptoacetyldiglycine (NHS-MAG 2 ) was synthesized first, reacted with aminobutyric acid(GABA) to give MAG 2 GABA and then converted to NHS-MAG 2 GABA and conjugated to biocytin to give the MGB. To label MGB with 188 Re (50mA), ag, 200ml 1M tartrate pH7, 200 l stannous (10mg/mL) and 180MBq perrhenate were mixed and heated for 30min at 100 deg. C. The reactant was purified with C 18 Sep-Pak. HPLC analysis of the 188 Re-MGB performed on reverse phase C 18 column with a gradient. To see the stability, 188 Re-MGB was added to serum at 37 deg. C. Binding capacity of 188 Re-MGB to avidin or streptavidin was determined by size exclusion HPLC system. Biodistribution was studied in ICR normal mice(n=4/group), from 5min to 120min. In Raji cells tumour bearing nude mice(n=3), biotinylated Lym-1(40MEG) injection after 48 h, streptavidin(50MEG) was injected. 24 h later, 188 Re-MGB(0.5MEG) was injected, and biodistribution was observed 2 h later. 188 Re-MGB was obtained with labelling yield 98%. Stability in serum was maintained over 70% until 3 h. Binding capacity of 188 Re-MGB to streptavidin was greater than avidin. In normal mice, 188 Re-MGB was excreted via hepatobiliary pathway,%ID/g of GI tract was 52.1 at 120min. In Raji cells tumour bearing nude mice, liver and colon were higher than those of normal mouse. Tumour uptake at 120min was 0.05%ID/g. 188 Re-MGB was effectively labelled and retained binding activity with streptavidin. 188 Re-MGB may have a role in pretargeted radioimmunotherapy. (author)

Probing intruder configurations in $^{186, 188}$Pb using Coulomb excitation

CERN Multimedia

Columb excitation measurements to study the shape coexistence, mixing and quadrupole collectivity of the low-lying levels in neutron-deficient $^{188}$Pb nuclei are proposed with a view to extending similar studies to the $^{186}$Pb midshell nucleus. The HIE-ISOLDE beam of $^{186,188}$Pb nuclei will be delivered to MINIBALL+SPEDE set-up for simultaneous in-beam $\\gamma$-ray and conversion electron spectroscopy. The proposed experiment will allow the sign of the quadrupole deformation parameter to be extracted for the two lowest 2$^{+}$ states in $^{188}$Pb. Moreover, the advent of SPEDE will allow probing of the bandhead 0$^{+}$ states via direct measurements of E0 transitions. Beam development is requested to provide pure and instense $^{186}$Pb beam.

Protein kinase A-alpha directly phosphorylates FoxO1 in vascular endothelial cells to regulate expression of vascular cellular adhesion molecule-1 mRNA.

Science.gov (United States)

Lee, Ji-Won; Chen, Hui; Pullikotil, Philomena; Quon, Michael J

2011-02-25

FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase α (PKA-α). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-α. Using an immune complex kinase assay with [γ-(32)P]ATP, purified PKA-α directly phosphorylated wild-type FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-α in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-α and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-α decreased forskolin- or prostaglandin E(2)-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-α, catalytically inactive mutant PKA-α, or siRNA against PKA-α caused corresponding increases or decreases in transactivation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-α or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-α treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-α in vascular endothelial cells.

Potential application of SERS for arsenic speciation in biological matrices.

Science.gov (United States)

Yang, Mingwei; Matulis, Shannon; Boise, Lawrence H; McGoron, Anthony J; Cai, Yong

2017-08-01

Speciation of arsenic is usually carried out using chromatography-based methods coupled with spectroscopic determination; however, the inevitable procedures involving sample preparation and separation could potentially alter the integrity of the arsenic metabolites present in biological samples. Surface-enhanced Raman spectroscopy (SERS) could be a promising alternative for providing a reliable arsenic analysis under the influence of a cellular matrix. A method for arsenic speciation using SERS in cellular matrix was developed in this study and four arsenicals were selected, including arsenite (As III ), arsenate (As V ), monomethylarsonic acid (MMA V ) and dimethylarsinic acid (DMA V ). Silver nanoparticles in the form of colliodal suspension with different surface charges, i.e., coated with citrate (AgNPs-Citrate) and spermine (AgNPs-Spermine) were employed as SERS substrates. Adsorption of arsenicals on nanoparticles in colloidal suspensions and the cellular matrix and the pH, size, and zeta potential of the colloidal suspensions were investigated for a better understanding of the SERS signal response of arsenicals in the colloidal suspensions or under the influence of cellular matrix. Arsenicals showed substantially different SERS responses in the two colloidal suspensions, mainly because of the distinct difference in the interaction between the arsenicals and the nanoparticles. Arsenic speciation in cell lysate could be successfully carried out in AgNPs-Spermine suspension, while AgNPs-Citrate could not yield significant SERS signals under the experimental conditions. This study proved that AgNPs-Spermine colloidal suspension could be a promising SERS substrate for studying arsenic metabolism in a biological matrix, reducing the bias caused by traditional techniques that involve sample extraction and pretreatment.

IL6 induces TAM resistance via kinase-specific phosphorylation of ERα in OVCA cells.

Science.gov (United States)

Wang, Yue; Niu, Xiu Long; Guo, Xiao Qin; Yang, Jing; Li, Ling; Qu, Ye; Xiu Hu, Cun; Mao, Li Qun; Wang, Dan

2015-06-01

About 40-60% of ovarian cancer (OVCA) cases express ERα, but only a small proportion of patients respond clinically to anti-estrogen treatment with estrogen receptor (ER) antagonist tamoxifen (TAM). The mechanism of TAM resistance in the course of OVCA progression remains unclear. However, IL6 plays a critical role in the development and progression of OVCA. Our recent results indicated that IL6 secreted by OVCA cells may promote the resistance of these cells to TAM via ER isoforms and steroid hormone receptor coactivator-1. Here we demonstrate that both exogenous (a relatively short period of treatment with recombinant IL6) and endogenous IL6 (generated as a result of transfection with a plasmid encoding sense IL6) increases expression of pERα-Ser118 and pERα-Ser167 in non-IL6-expressing A2780 cells, while deleting endogenous IL6 expression in IL6-overexpressing CAOV-3 cells (by transfection with a plasmid encoding antisense IL6) reduces expression of pERα-Ser118 and pERα-Ser167, indicating that IL6-induced TAM resistance may also be associated with increased expression of pERα-Ser118 and pERα-Ser167 in OVCA cells. Results of further investigation indicate that IL6 phosphorylates ERα at Ser118 and Ser167 by triggering activation of MEK/ERK and phosphotidylinositol 3 kinase/Akt signaling, respectively, to activate the ER pathway and thereby induce OVCA cells resistance to TAM. These results indicate that IL6 secreted by OVCA cells may also contribute to the refractoriness of these cells to TAM via the crosstalk between ER and IL6-mediated intracellular signal transduction cascades. Overexpression of IL6 not only plays an important role in OVCA progression but also promotes TAM resistance. Our results indicate that TAM-IL6-targeted adjunctive therapy may lead to a more effective intervention than TAM alone. © 2015 Society for Endocrinology.

Phenobarbital Meets Phosphorylation of Nuclear Receptors.

Science.gov (United States)

Negishi, Masahiko

2017-05-01

Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

Therapeutic applications of Rhenium-188 in nuclear medicine and oncology - Current status and expected future perspectives

International Nuclear Information System (INIS)

Knapp, F. F. Jr.

2005-01-01

Full text: The increasing use of unsealed radioactive targeting agents for cancer treatment requires the routine availability of cost-effective radioisotopes. Rhenium-188 (Re-188; half-life 16.9 hours) is a high-energy beta-emitter (E max 2.12 MeV), readily available no- max carrier-added from the alumina-based tungsten-188 (half-life 69 days)/rhenium-188 generator system. Rhenium-188 also emits a 155 keV (15%) gamma photon, permitting gamma camera imaging for biodistribution and dosimetry evaluation. The versatile chemistry of rhenium allows attachment to a wide variety of targeting molecules for Re-188 applications in nuclear oncology for both palliative metastatic treatment and targeted tumor therapy - radionuclide synovectomy, and coronary restenosis therapy. The long parent half-life and consistent performance provide an indefinite generator shelf-life of several months with high Re-188 elution yields (75-85 %) and consistently low W-188 parent breakthrough ( -6 ). Simple post-elution concentration methods have been developed which provide very high specific volume solution of Re-188 for radiolabeling (> 700 mCi/mL saline/1 Ci generator). Over 60 physician-sponsored clinical trials are currently in progress worldwide with applications in nuclear medicine, nuclear oncology and interventional cardiology. A variety of Re-188-labeled therapeutic radiopharmaceuticals and devices are being developed for clinical trials currently in progress for treatment of both benign and metastatic oncological disorders. Palliation of metastatic bone pain with Re-188-HEDP - prepared from a simple 'kit' - has been demonstrated as a cost-effective alternative to similar agents. Recent studies have in fact demonstrated the enhancement of progression-free interval and survival time by repeated Re-188-HEDP injections to patients with metastatic disease from prostate cancer. The use of the Re-188-labeled antiNCA95 (CD66) antibody in conjunction with external beam irradiation is an

Biological behavior of 188Re-biotin chelate for multistep therapy with the avidin-biotin system

International Nuclear Information System (INIS)

Choi, T. H.; Ahn, S. H.; Choi, C. W.; Woo, K. S.; Jung, W. S.; Lim, S. J.; Lim, S. M.

1999-01-01

The purpose of this study was to test the three-step targeting of tumors in mice using biotinylated antibody, streptavidin and radiolabeled biotin for radioimmunotherapy (RAIT). Three-step pretargetting can potentially decreases harmful radiation to normal tissues in radioimmunotherapy. 188 Re from 188 W- 188 Re generator, is recently introduced in therapeutic nuclear medicine and made it possible to use whenever needed. We studied biotin-chelates MGB for use in the avidin/biotin pretargetting system. Chelates that hold radiometals with high stability under physiological conditions are essential to avoid excessive radiation damage to non-target cells. We synthesized MAG 2 GABA-Biocytin (MGB), labeled with 188 Re and evaluated biological behavior of 188 Re-MGB. biotinyl MAG 2 GABA bind the therapeutic radiometal 188 Re with excellent in vitro stability and have the required physiological properties for pretargetted therapy. In normal mice, 188 Re-MGB was excreted via hepatobiliary pathway, %ID/g of GI tract was 52.1 at 120min. In Raji cells tumor bearing nude mice, liver and colon were higher than those of normal mouse. Tumor uptake at 120min was 0.05%ID/g. 188 Re-MGB may have a role in pretargetted radioimmunotherapy

A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host

Directory of Open Access Journals (Sweden)

Xiaofeng Zhou

2018-01-01

Full Text Available Most pathogenic bacteria deliver virulence factors into host cytosol through type III secretion systems (T3SS to perturb host immune responses. The expression of T3SS is often repressed in rich medium but is specifically induced in the host environment. The molecular mechanisms underlying host-specific induction of T3SS expression is not completely understood. Here we demonstrate in Xanthomonas citri that host-induced phosphorylation of the ATP-dependent protease Lon stabilizes HrpG, the master regulator of T3SS, conferring bacterial virulence. Ser/Thr/Tyr phosphoproteome analysis revealed that phosphorylation of Lon at serine 654 occurs in the citrus host. In rich medium, Lon represses T3SS by degradation of HrpG via recognition of its N terminus. Genetic and biochemical data indicate that phosphorylation at serine 654 deactivates Lon proteolytic activity and attenuates HrpG proteolysis. Substitution of alanine for Lon serine 654 resulted in repression of T3SS gene expression in the citrus host through robust degradation of HrpG and reduced bacterial virulence. Our work reveals a novel mechanism for distinct regulation of bacterial T3SS in different environments. Additionally, our data provide new insight into the role of protein posttranslational modification in the regulation of bacterial virulence.

Oxidative phosphorylation revisited

DEFF Research Database (Denmark)

Nath, Sunil; Villadsen, John

2015-01-01

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

Interaction between lipid monolayers and poloxamer 188: An X-ray reflectivity and diffraction study

DEFF Research Database (Denmark)

Wu, G.H.; Majewski, J.; Ege, C.

2005-01-01

The mechanism by which poloxamer 188 (P188) seals a damaged cell membrane is examined using the lipid monolayer as a model system. X-ray reflectivity and grazing-incidence x-ray diffraction results show that at low nominal lipid density, P188, by physically occupying the available area and phase ...

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

Science.gov (United States)

Stenner, Frank; Liewen, Heike; Göttig, Stephan; Henschler, Reinhard; Markuly, Norbert; Kleber, Sascha; Faust, Michael; Mischo, Axel; Bauer, Stefan; Zweifel, Martin; Knuth, Alexander; Renner, Christoph; Wadle, Andreas

2013-01-01

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

Directory of Open Access Journals (Sweden)

Frank Stenner

Full Text Available RP1 (synonym: MAPRE2, EB2 is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

EFFICACY of P188 ON LAPINE MENISCUS PRESERVATION FOLLOWING BLUNT TRAUMA

Science.gov (United States)

Coatney, Garrett A.; Abraham, Adam C.; Fischenich, Kristine M.; Button, Keith D.; Haut, Roger C.; Haut Donahue, Tammy L.

2015-01-01

Traumatic injury to the knee leads to the development of posttraumatic osteoarthritis. The objective of this study was to characterize the effects of a single intra-articular injection of a non-ionic surfactant, Poloxamer 188 (P188), in preservation of meniscal tissue following trauma through maintenance of meniscal glycosaminoglycan (GAG) content and mechanical properties. Flemish Giant rabbits were subjected to a closed knee joint, traumatic compressive impact with the joint constrained to prevent anterior tibial translation. The contralateral limb served as an un-impacted control. Six animals (treated) received an injection of P188 in phosphate buffered saline (PBS) post trauma, and another six animals (sham) received a single injection of PBS to the impacted limb. Histological analyses for GAG was determined 6 weeks post trauma, and functional outcomes were assessed using stress relaxation micro-indentation. The impacted limbs of the sham group demonstrated a significant decrease in meniscal GAG coverage compared to non-impacted limbs (p < 0.05). GAG coverage of the impacted P188 treated limbs was not significantly different than contralateral non-impacted limbs in all regions except the medial anterior (p < 0.05). No significant changes were documented in mechanics for either the sham or treated groups compared to their respective control limbs. This suggests that a single intra-articular injection of P188 shows promise in prevention of trauma induced GAG loss. PMID:25846264

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

Science.gov (United States)

Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

2014-08-18

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

Science.gov (United States)

Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

2010-12-01

Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models

46 CFR 188.10-21 - Compressed gas.

Science.gov (United States)

2010-10-01

... PROVISIONS Definition of Terms Used in This Subchapter § 188.10-21 Compressed gas. This term includes any... by the Reid method covered by the American Society for Testing Materials Method of Test for Vapor...

Labeling of MDP with {sup 188}Re for bone tumour therapy

Energy Technology Data Exchange (ETDEWEB)

Barbezan, Angelica B.; Osso Junior, Joao A., E-mail: jaosso@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2011-07-01

{sup 188}Re is one of the most attractive radioisotopes for a variety of therapeutic applications in nuclear medicine, due to its physical decay properties, such as {beta}{sup -} emission of 2.12 MeV, {gamma} emission of 155 keV and half life of 16.9 hours. Biphosphonates are potent inhibitors of osteoclastic bone resorption and are effective in several diseases that cause bone fragility and bone metastases. Because of these characteristics, labeled biphosphonates have been studied for bone pathologies, also acting as palliation of bone pain in case of metastasis.The aim of this study was to optimize the labeling of a phosphonate-MDP (Sodium Methylene Diphosphonate) with {sup 188}Re for use in bone pain palliation. {sup 188}Re was obtained by eluting a {sup 188}W-{sup 188}Re generator from POLATOM. The labeling was performed at room temperature using MDP, SnCl{sub 2} as reducing agent and ascorbic acid. The variables studied were: Mass of ligand (3, 6 and 10 mg), reducing agent mass (5, 7, 10 and 11 mg), ascorbic acid mass (1, 3, 5 and 6 mg), pH (1 and 2) and time of reaction (15, 60, 120, 360 and 4320 minutes), that also reflected the stability of the radiopharmaceutical. The radiochemical control, that also measures the labeling efficiency was evaluated by paper chromatography using Whatman 3MM paper and the solvents acetone and 0.9%NaCl. The best formulation was the following: Mass of ligand MDP: 10 mg, mass of SnCl{sub 2}: 5 mg, ascorbic acid mass: 3 mg, time of reaction: 30 minutes, pH: 1. Under optimum conditions, {sup 188}Re MDP radiolabeling yield was 98,07% and the radiopharmaceutical was stable up to 72 h. (author)

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds1[OPEN

Science.gov (United States)

Hill, Allyson T.; Anderson, Erin M.; She, Yi-Min

2017-01-01

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. PMID:28363991

Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders*

Science.gov (United States)

Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G.; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

2015-01-01

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. PMID:26499801

Study of radiation synovectomy using 188Re-sulfide in hemophilic arthritis

International Nuclear Information System (INIS)

Li, P.Y.; Cheng, G.; Jiang, X.F.; Wang, X.F.; Shen, Z.M.; Zhang, Z.H.

2002-01-01

Purpose: Based on results of previous animal studies, the efficacy of 188 Re-sulfide on radiation synovectomy in hemophilia synovitis.was evaluated. Material and Methods: 188 Re-sulfide suspension was produced by dispersion method. 25 hemophilic patients with 30 synovitic joints including 22 knees and 8 ankles received the radiation synovectomy. The stage of synovitic joint was classified by joint score including the pain, stability and range of motion and MR score. The doses of 188 Re-sulfide injected into knee and ankle were determined as 12mCi and 6mCi respectively, according to the depth and curve and the results of our previous animal study. To exam the distribution of 188 Re-sulfide in vivo after the injection, a whole-body scan was taken 24 and 48 hours later to calculate the retention of 188 Re-sulfide in joint by percentage of join counts in whole body. The follow up was take place at 6-12 months after the synovectomy by joint score, MRI score, synovial structure, the times and interval of hemorrhage of the joints. Results: Few patients complained discomfort after the injection such as hurt of the superficial tissues around the injected point and swelling (2 patients,.8%).The symptoms in this two patients continued up to 3 days and gradually decreased in severity. All patients felt relief of the pain and swelling in joints. 90% joints including 20 knees and 7 ankles did not bleed any more during the 3-month term of follow up, 3 joints from 2 patients with intra-article bleeding had hemorrhage in one month after long distance walk. 16%(5/30) of joints including 4 knees and 1 ankles had recurrent hemorrhage in 12 months after the radiation synovectomy. However, their interval of intra-article bleeding was prolonged MRI showed the thick synovium became thin, villi reduced and the joint edema relieved. The retention of 188 Re-sulfide in administrated joint was more than 95% until 48 hours later. No any sign of radioactive distribution was found in bone marrow

Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach

International Nuclear Information System (INIS)