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Sample records for phosphatases tc-ptp shp1

  1. Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas' disease

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2013-06-05

    Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed.

  2. Rapid TCR-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity

    PubMed Central

    Liu, Yin; Kruhlak, Michael J.; Hao, Jian-Jiang; Shaw, Stephen

    2007-01-01

    Since the tyrosine phosphatase SHP-1 plays a major role in regulating T-cell signaling, we investigated regulation thereof by Ser/Thr phosphorylation. We found that TCR stimulation induced fast (≤1min) and transient phosphorylation of SHP-1 S591 in both Jurkat and human peripheral blood T-cells (PBT). Phosphorylation of S591 in T-cells could be mediated artificially by a constitutive active PKC-theta construct, but the dose dependence of inhibition by PKC inhibitors indicated that PKCs were not the relevant basophilic kinase in the physiologic response. S591 phosphorylation inhibited phosphatase function since a S591D mutant had lower activity than the S591A mutant. Additional evidence that S591 phosphorylation alters SHP-1 function was provided by studies of Jurkat cells stably expressing SHP-1 wildtype or mutants. In those cells, S591D mutation reduced the capacity of transfected SHP-1 to inhibit TCR-induced phosphorylation of PLC-γ1. Interestingly, SHP-1 Y536 phosphorylation (previously shown to augment phosphatase activity) was also induced in PBT by TCR signal but at a much later time compared to S591 (~30 min). S591 phosphorylation also altered cellular distribution of SHP-1 because: 1) SHP-1 in lipid rafts and a sheared membrane fraction was hypo-phosphorylated; 2) In stably transfected Jurkat cell lines, S591D mutant protein had reduced presence in both lipid raft and the sheared membrane fraction; 3) S591 phosphorylation prevented nuclear localization of a C-terminal GFP tagged SHP-1 construct. Our studies also shed light on an additional mechanism regulating SHP-1 nuclear localization, namely conformational autoinhibition. These findings highlight elegant regulation of SHP-1 by sequential phosporylation of serine then tyrosine. PMID:17575265

  3. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    PubMed

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  4. An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

    PubMed

    Peng, Liu; Lei, Zhang; Xiao-na, Xie; Deli, Wang; Jing, Sun; Yong-sen, Wang; Zhi, Wang; Shu, Xing; Jun-feng, Ma; Wan-nan, Li; Xue-qi, Fu

    2015-03-01

    Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway.

  5. An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

    PubMed

    Peng, Liu; Lei, Zhang; Xiao-na, Xie; Deli, Wang; Jing, Sun; Yong-sen, Wang; Zhi, Wang; Shu, Xing; Jun-feng, Ma; Wan-nan, Li; Xue-qi, Fu

    2015-03-01

    Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway. PMID:25730798

  6. Antagonism or Synergism: Role of Tyrosine Phosphatases SHP-1 and SHP-2 in Growth Factor Signaling

    PubMed Central

    Wang, Ning; Li, Zhe; Ding, Ronghua; Frank, Gerald D.; Senbonmatsu, Takaaki; Landon, Erwin J.; Inagami, Tadashi; Zhao, Zhizhuang Joe

    2008-01-01

    SHP-1 and SHP-2 are two SH2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer while SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with siRNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 since it was abrogated by knockdown of SHP-2 and SHP-2, but not SHP-1, binds directly to tyrosine phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for hitherto unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases. PMID:16762922

  7. The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress.

    PubMed

    Jang, Min Kyoung; Kim, Sae-Hoon; Lee, Ki-Young; Kim, Tae-Bum; Moon, Keun Ae; Park, Chan Sun; Bae, Yun Jeong; Zhu, Zhou; Moon, Hee-Bom; Cho, You Sook

    2010-02-26

    Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H(2)O(2)), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H(2)O(2), compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress. PMID:20117097

  8. The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress.

    PubMed

    Jang, Min Kyoung; Kim, Sae-Hoon; Lee, Ki-Young; Kim, Tae-Bum; Moon, Keun Ae; Park, Chan Sun; Bae, Yun Jeong; Zhu, Zhou; Moon, Hee-Bom; Cho, You Sook

    2010-02-26

    Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H(2)O(2)), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H(2)O(2), compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.

  9. Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1.

    PubMed

    Nandan, Devki; Reiner, Neil E

    2005-03-01

    Protozoan parasites of the genus leishmania are obligate intracellular parasites of monocytes and macrophages. These pathogens have evolved to invade the mammalian immune system and typically survive for long periods of time. Leishmania have developed a variety of remarkable strategies to prevent their elimination by both innate and acquired immune effector mechanisms. One particular strategy of interest involves manipulation of host cell regulatory pathways so as to prevent macrophage activation required for efficient microbicidal activity. These interference mechanisms are the main focus of this review. Several lines of evidence have been developed to show that the Src homology-2 domain containing tyrosine phosphatase-1 (SHP-1) becomes activated in leishmania-infected cells and that this contributes to disease pathogenesis. Recent studies aimed at understanding the mechanism responsible for the change in activation state of SHP-1 led to the identification of leishmania EF-1alpha as an SHP-1 binding protein and SHP-1 activator. This was a surprising finding given that this ubiquitous and highly conserved protein plays an essential role in protein translation in both prokaryotic and eukaryotic cells. The role of leishmania EF-1alpha as an SHP-1 activator and its contribution to pathogenesis are reviewed with particular attention to the properties that distinguish it from host EF-1alpha. PMID:15721837

  10. Interferon-β treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1

    PubMed Central

    Christophi, George P.; Panos, Michael; Hudson, Chad A.; Tsikkou, Chriso; Mihai, Cornelia; Mejico, Luis J.; Jubelt, Burk; Massa, Paul T.

    2009-01-01

    Interferon-β is a current treatment for multiple sclerosis (MS). Interferon-β is thought to exert its therapeutic effects on MS by down-modulating the immune response by multiple potential pathways. Here, we document that treatment of MS patients with interferon β-1a (Rebif) results in a significant increase in the levels and function of the protein tyrosine phosphatase SHP-1 in PBMCs. SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and CNS demyelination as evidenced in mice deficient in SHP-1. In order to examine the functional significance of SHP-1 induction in MS PBMCs, we analyzed the activity of proinflammatory signaling molecules STAT1, STAT6, and NF-κB, which are known SHP-1 targets. Interferon-β treatment in vivo resulted in decreased NF-κB and STAT6 activation and increased STAT1 activation. Further analysis in vitro showed that cultured PBMCs of MS patients and normal subjects had a significant SHP-1 induction following interferon-β treatment that correlated with decreased NF-κB and STAT6 activation. Most importantly, experimental depletion of SHP-1 in cultured PBMCs abolished the anti-inflammatory effects of interferon-β treatment, indicating that SHP-1 is a predominant mediator of interferon-β activity. In conclusion, interferon-β treatment upregulates SHP-1 expression resulting in decreased transcription factor activation and inflammatory gene expression important in MS pathogenesis. PMID:19559654

  11. Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

    PubMed Central

    HUDSON, CHAD A.; CHRISTOPHI, GEORGE P.; CAO, LING; GRUBER, ROSS C.

    2007-01-01

    The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes. PMID:18250891

  12. Host SHP1 phosphatase antagonizes Helicobacter pylori CagA and can be downregulated by Epstein-Barr virus.

    PubMed

    Saju, Priya; Murata-Kamiya, Naoko; Hayashi, Takeru; Senda, Yoshie; Nagase, Lisa; Noda, Saori; Matsusaka, Keisuke; Funata, Sayaka; Kunita, Akiko; Urabe, Masayuki; Seto, Yasuyuki; Fukayama, Masashi; Kaneda, Atsushi; Hatakeyama, Masanori

    2016-01-01

    Most if not all gastric cancers are associated with chronic infection of the stomach mucosa with Helicobacter pylori cagA-positive strains(1-4). Approximately 10% of gastric cancers also harbour Epstein-Barr virus (EBV) in the cancer cells(5,6). Following delivery into gastric epithelial cells via type IV secretion(7,8), the cagA-encoded CagA protein undergoes tyrosine phosphorylation on the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs initially by Src family kinases (SFKs) and then by c-Abl(9,10). Tyrosine-phosphorylated CagA binds to the pro-oncogenic protein tyrosine phosphatase SHP2 and thereby deregulates the phosphatase activity(11,12), which has been considered to play an important role in gastric carcinogenesis(13). Here we show that the SHP2 homologue SHP1 interacts with CagA independently of the EPIYA motif. The interaction potentiates the phosphatase activity of SHP1 that dampens the oncogenic action of CagA by dephosphorylating the CagA EPIYA motifs. In vitro infection of gastric epithelial cells with EBV induces SHP1 promoter hypermethylation, which strengthens phosphorylation-dependent CagA action via epigenetic downregulation of SHP1 expression. Clinical specimens of EBV-positive gastric cancers also exhibit SHP1 hypermethylation with reduced SHP1 expression. The results reveal that SHP1 is the long-sought phosphatase that can antagonize CagA. Augmented H. pylori CagA activity, via SHP1 inhibition, might also contribute to the development of EBV-positive gastric cancer. PMID:27572445

  13. Dissociation of SHP-1 from spinophilin during platelet activation exposes an inhibitory binding site for protein phosphatase-1 (PP1).

    PubMed

    Ma, Peisong; Foote, Darci C; Sinnamon, Andrew J; Brass, Lawrence F

    2015-01-01

    We have recently shown that a critical regulatory node in the platelet signaling network lies immediately downstream of platelet receptors for thrombin and TxA2. This node is comprised of a scaffold protein (spinophilin, SPL), a protein tyrosine phosphatase (SHP-1), and either of the two members of the Regulators of G protein Signaling family predominantly expressed in platelets (RGS10 or RGS18). The SPL/RGS/SHP-1 complex is present in resting platelets, dissociating when thrombin or TxA2, but not ADP or collagen, activate SHP-1 and release RGS10 and RGS18 to dampen signaling. Here we demonstrate an additional regulatory role for spinophilin, showing that dissociation of SHP-1 from spinophilin is followed by an increase in the binding of spinophilin to PP1, a serine/threonine phosphatase whose binding site maps to a region close to the SHP-1 binding site. The increase in PP1 binding to spinophilin is limited to platelet agonists that cause dissociation of the complex and is selective for the α and γ isoforms of PP1. Studies in cell culture show that SHP-1 and PP1 can compete for binding to spinophilin and that binding inhibits PP1 activity since over-expression of wild type spinophilin, but not spinophilin with a disabled PP1 binding site, causes an increase in the phosphorylation of myosin light chain, a well-characterized PP1 substrate. Collectively, these results indicate that in addition to regulating RGS protein availability in resting platelets, spinophilin can serve as a time-dependent, agonist- and isoform-selective regulator of PP1, inhibiting its activity when decay of the SPL/RGS/SHP-1 complex releases SHP-1 from spinophilin, exposing a binding site for PP1.

  14. Dissociation of SHP-1 from Spinophilin during Platelet Activation Exposes an Inhibitory Binding Site for Protein Phosphatase-1 (PP1)

    PubMed Central

    Ma, Peisong; Foote, Darci C.; Sinnamon, Andrew J.; Brass, Lawrence F.

    2015-01-01

    We have recently shown that a critical regulatory node in the platelet signaling network lies immediately downstream of platelet receptors for thrombin and TxA2. This node is comprised of a scaffold protein (spinophilin, SPL), a protein tyrosine phosphatase (SHP-1), and either of the two members of the Regulators of G protein Signaling family predominantly expressed in platelets (RGS10 or RGS18). The SPL/RGS/SHP-1 complex is present in resting platelets, dissociating when thrombin or TxA2, but not ADP or collagen, activate SHP-1 and release RGS10 and RGS18 to dampen signaling. Here we demonstrate an additional regulatory role for spinophilin, showing that dissociation of SHP-1 from spinophilin is followed by an increase in the binding of spinophilin to PP1, a serine/threonine phosphatase whose binding site maps to a region close to the SHP-1 binding site. The increase in PP1 binding to spinophilin is limited to platelet agonists that cause dissociation of the complex and is selective for the α and γ isoforms of PP1. Studies in cell culture show that SHP-1 and PP1 can compete for binding to spinophilin and that binding inhibits PP1 activity since over-expression of wild type spinophilin, but not spinophilin with a disabled PP1 binding site, causes an increase in the phosphorylation of myosin light chain, a well-characterized PP1 substrate. Collectively, these results indicate that in addition to regulating RGS protein availability in resting platelets, spinophilin can serve as a time-dependent, agonist- and isoform-selective regulator of PP1, inhibiting its activity when decay of the SPL/RGS/SHP-1 complex releases SHP-1 from spinophilin, exposing a binding site for PP1. PMID:25785436

  15. The role of the Syk/Shp-1 kinase-phosphatase equilibrium in B cell development and signaling.

    PubMed

    Alsadeq, Ameera; Hobeika, Elias; Medgyesi, David; Kläsener, Kathrin; Reth, Michael

    2014-07-01

    Signal transduction from the BCR is regulated by the equilibrium between kinases (e.g., spleen tyrosine kinase [Syk]) and phosphatases (e.g., Shp-1). Previous studies showed that Syk-deficient B cells have a developmental block at the pro/pre-B cell stage, whereas a B cell-specific Shp-1 deficiency promoted B-1a cell development and led to autoimmunity. We generated B cell-specific Shp-1 and Syk double-knockout (DKO) mice and compared them to the single-knockout mice deficient for either Syk or Shp-1. Unlike Syk-deficient mice, the DKO mice can generate mature B cells, albeit at >20-fold reduced B cell numbers. The DKO B-2 cells are all Syk-negative, whereas the peritoneal B1 cells of the DKO mice still express Syk, indicating that they require this kinase for their proper development. The DKO B-2 cells cannot be stimulated via the BCR, whereas they are efficiently activated via TLR or CD40. We also found that in DKO pre-B cells, the kinase Zap70 is associated with the pre-BCR, suggesting that Zap70 is important to promote B cell maturation in the absence of Syk and SHP-1. Together, our data show that a properly balanced kinase/phosphatase equilibrium is crucial for normal B cell development and function.

  16. Lack of Phosphotyrosine Phosphatase SHP-1 Expression in Malignant T-Cell Lymphoma Cells Results from Methylation of the SHP-1 Promoter

    PubMed Central

    Zhang, Qian; Raghunath, Puthiyaveettil N.; Vonderheid, Eric; Ødum, Niels; Wasik, Mariusz A.

    2000-01-01

    SHP-1 is an important negative regulator of signaling by several receptors including receptors for interleukin-2 (IL-2R) and other cytokines. SHP-1 acts by dephosphorylating the receptors and receptor-associated kinases such as IL-2R-associated Jak3 kinase. We found that SHP-1 protein was not detectable or greatly diminished in most (six of seven) T cell lines derived from various types of T cell lymphomas and all (eight of eight) cutaneous T-cell lymphoma tissues with a transformed, large-cell morphology. All T-cell lymphoma lines tested (eight of eight) expressed diminished amounts or no detectable SHP-1 mRNA. These T cell lines did not, however, carry any mutations in the SHP-1 gene-coding, splice-junction, and promoter regions. Importantly, SHP-1 DNA promoter region in the T cell lines was resistant to digestion with three different methylation-sensitive restriction enzymes. This resistance was reversed by treatment of the cells with a demethylating agent, 5-deoxyazacytidine. The treatment resulted also in the expression of SHP-1 mRNA and, less frequently, SHP-1 protein. The expression of SHP-1 protein was associated with dephosphorylation of the Jak3 kinase. These results show that lack of SHP-1 expression is frequent in malignant T cells and results from methylation of the SHP-1 gene promoter. Furthermore, they indicate that SHP-1 loss may play a role in the pathogenesis of T cell lymphomas by permitting persistence of signals generated by IL-2R and, possibly, other receptor complexes. PMID:11021818

  17. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    PubMed

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  18. Gambogic Acid Inhibits STAT3 Phosphorylation Through Activation of Protein Tyrosine Phosphatase SHP-1: Potential Role in Proliferation and Apoptosis

    PubMed Central

    Prasad, Sahdeo; Pandey, Manoj K.; Yadav, Vivek R.; Aggarwal, Bharat B.

    2011-01-01

    The transcription factor, signal transducer and activator of transcription 3 (STAT3), is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Gamboge hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase (JAK) 1, and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced down-regulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by small interfering RNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis. PMID:21490133

  19. Morin inhibits STAT3 tyrosine 705 phosphorylation in tumor cells through activation of protein tyrosine phosphatase SHP1.

    PubMed

    Gupta, Subash C; Phromnoi, Kanokkarn; Aggarwal, Bharat B

    2013-04-01

    The major goal of cancer drug discovery is to find an agent that is safe and affordable, yet effective against cancer. Here we show that morin (3,5,7,2',4'-pentahydroxyflavone) has potential against cancer cells through suppression of the signal transducer and activator of transcription 3 (STAT3) pathway, which is closely linked to the transformation, survival, proliferation, and metastasis of cancer. We found that morin completely suppressed inducible and constitutively activated STAT3 and blocked the nuclear translocation of STAT3 and its DNA binding in multiple myeloma and head and neck squamous carcinoma cells. Morin inhibited activated Src, JAK-1, and JAK-2, all of which are linked to STAT3 activation, while up-regulating a protein inhibitor of activated STAT3, PIAS3. Pervanadate reversed the effects of morin on STAT3 phosphorylation, indicating the role of a protein tyrosine phosphatase. Furthermore, morin induced SHP1 expression at both the mRNA and protein levels, and silencing of SHP1 abrogated the effect of morin on STAT3 phosphorylation, indicating that morin mediates its effects on STAT3 through SHP1. Suppression of STAT3 correlated with the down-regulation of various gene products linked to tumor survival, proliferation, and angiogenesis and led to sensitization of tumor cells to thalidomide and bortezomib. Comparing the activities of morin with those of four structurally related flavonols demonstrated the importance of hydroxyl groups in the B ring in inhibiting STAT3 activation. These findings suggest that morin suppresses the STAT3 pathway, leading to the down-regulation of STAT3-dependent gene expression and chemosensitization of tumor cells.

  20. Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of B and T lymphocyte attenuator required for association with protein tyrosine phosphatases SHP-1 and SHP-2.

    PubMed

    Gavrieli, Maya; Watanabe, Norihiko; Loftin, Susan K; Murphy, Theresa L; Murphy, Kenneth M

    2003-12-26

    B and T lymphocytes express receptors providing positive and negative co-stimulatory signals. We recently identified a novel co-stimulatory molecule, B and T lymphocyte attenuator (BTLA), which exerts inhibitory effects on B and T lymphocytes. The cytoplasmic domain of murine and human BTLA share three conserved tyrosine-based signaling motifs, a Grb-2 recognition consensus, and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Phosphorylation of the cytoplasmic domain of BTLA induced the association with the protein tyrosine phosphatases SHP-1 and SHP-2. Association of SHP-1 and SHP-2 to other receptors can involve recruitment to either a single receptor ITIM or to two receptor ITIMs. Here, we analyzed the requirements of BTLA interaction with SHP-1 and SHP-2 in a series of murine and human BTLA mutants. For human BTLA, mutations of either Y257 or Y282, but not Y226, abrogated association with both SHP-1 and SHP-2. For murine BTLA, mutation of either Y274 or Y299, but not Y245, also abrogated association with both SHP-1 and SHP-2. These results indicate that for both murine and human BTLA, association with SHP-1 or SHP-2 requires both of conserved ITIM motifs and does not involve the conserved Grb-2 consensus. Thus, similar to the bisphosphoryl tyrosine-based activation motif (BTAM) by which the Grb-2 associated binder (Gab1), PDGF receptor, and PECAM-1 recruit SHP-2, BTLA also relies on dual ITIMs for its association with the phosphatases SHP-1 and SHP-2. PMID:14652006

  1. Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

    PubMed

    Krautwald, S; Büscher, D; Kummer, V; Buder, S; Baccarini, M

    1996-11-01

    Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways. PMID:8887625

  2. Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway.

    PubMed Central

    Krautwald, S; Büscher, D; Kummer, V; Buder, S; Baccarini, M

    1996-01-01

    Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways. PMID:8887625

  3. A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

    PubMed

    Wu, Ning; Zhong, Ming-Chao; Roncagalli, Romain; Pérez-Quintero, Luis-Alberto; Guo, Huaijian; Zhang, Zhanguang; Lenoir, Christelle; Dong, Zhongjun; Latour, Sylvain; Veillette, André

    2016-04-01

    Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

  4. Boswellic Acid Blocks STAT3 Signaling, Proliferation, and Survival of Multiple Myeloma via the Protein Tyrosine Phosphatase SHP-1

    PubMed Central

    Kunnumakkara, Ajaikumar B.; Nair, Asha S.; Sung, Bokyung; Pandey, Manoj K.; Aggarwal, Bharat B.

    2009-01-01

    Activation of signal transducers and activators of transcription (STAT)-3 factors has been linked with survival, proliferation, chemoresistance and angiogenesis of tumor cells, including human multiple myeloma (MM). Thus agents that can suppress STAT3 activation have potential as cancer therapeutics. In our search for such agents, we identified acetyl-11-keto-β-boswellic acid (AKBA), originally isolated from Boswellia serrata. Our results show that AKBA inhibited constitutive STAT3 activation in human MM cells. AKBA suppressed IL-6-induced STAT3 activation, and the inhibition was reversible. The phosphorylation of both Jak 2 and Src, constituents of the STAT3 pathway, was inhibited by AKBA. Interestingly, treatment of cells with pervanadate suppressed AKBA’s effect to inhibit the phosphorylation of STAT3, thus suggesting the involvement of a protein tyrosine phosphatase. We found that AKBA induced Src homology region 2 domain-containing phosphatase 1 (SHP-1), which may account for its role in dephosphorylation of STAT3. Moreover, deletion of SHP-1 gene by SiRNA abolished the ability of AKBA to inhibit STAT3 activation. The inhibition of STAT3 activation by AKBA led to the suppression of gene products involved in proliferation (cyclin D1), survival (Bcl-2, Bcl-xL and Mcl-1), and angiogenesis (VEGF). This affect correlated with the inhibition of proliferation and apoptosis in MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the AKBA induced apoptosis. Overall, our results suggest that AKBA is a novel inhibitor of STAT3 activation and has potential in the treatment of cancer. PMID:19147543

  5. SHP1-ing thymic selection.

    PubMed

    Gascoigne, Nicholas R J; Brzostek, Joanna; Mehta, Monika; Acuto, Oreste

    2016-09-01

    Thymocyte development and maintenance of peripheral T-cell numbers and functions are critically dependent on T-cell receptor (TCR) signal strength. SHP1 (Src homology region 2 domain-containing phosphatase-1), a tyrosine phosphatase, acts as a negative regulator of TCR signal strength. Moreover, germline SHP1 knockout mice have shown impaired thymic development. However, this has been recently questioned by an analysis of SHP1 conditional knockout mice, which reported normal thymic development of SHP1 deficient thymocytes. Using this SHP1 conditional knockout mice, in this issue of the European Journal of Immunology, Martinez et al. [Eur. J. Immunol. 2016. 46: 2103-2110] show that SHP1 indeed does have a role in the negative regulation of TCR signal strength in positively selected thymocytes, and in the final maturation of single positive thymocytes. They report that thymocyte development in such mice shows loss of mature, post-selection cells. This is due to increased TCR signal transduction in thymocytes immediately post positive-selection, and increased cell death in response to weak TCR ligands. Thus, SHP1-deficiency shows strong similarities to deficiency in the T-cell specific SHP1-associated protein Themis. PMID:27600672

  6. Ginkgetin Blocks Constitutive STAT3 Activation and Induces Apoptosis through Induction of SHP-1 and PTEN Tyrosine Phosphatases.

    PubMed

    Baek, Seung Ho; Lee, Jae Hwi; Ko, Jeong-Hyeon; Lee, Hanwool; Nam, Dongwoo; Lee, Seok Geun; Yang, Woong Mo; Um, Jae-Young; Lee, Junhee; Kim, Sung-Hoon; Shim, Bum Sang; Ahn, Kwang Seok

    2016-04-01

    Ginkgetin, a biflavone from Ginkgo biloba leaves, is known to exhibit antiinflammatory, antifungal, neuroprotective, and antitumor activities, but its precise mechanism of action has not been fully elucidated. Because the aberrant activation of STAT3 has been linked with regulation of inflammation, proliferation, invasion, and metastasis of tumors, we hypothesized that ginkgetin modulates the activation of STAT3 in tumor cells. We found that ginkgetin clearly suppressed constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK1 and c-Src kinases and nuclear translocation of STAT3 on both A549 and FaDu cells. Treatment with sodium pervanadate reversed the ginkgetin-induced down-modulation of STAT3, thereby indicating a critical role for a PTP. We also found that ginkgetin strongly induced the expression of the SHP-1 and PTEN proteins and its mRNAs. Further, deletion of SHP-1 and PTEN genes by siRNA suppressed the induction of SHP-1 and PTEN, and reversed the inhibition of STAT3 activation. Ginkgetin induced apoptosis as characterized by an increased accumulation of cells in subG1 phase, positive Annexin V binding, loss of mitochondrial membrane potential, down-regulation of STAT3-regulated gene products, and cleavage of PARP. Overall, ginkgetin abrogates STAT3 signaling pathway through induction of SHP-1 and PTEN proteins, thus attenuating STAT3 phosphorylation and tumorigenesis. PMID:27059688

  7. Tumoral Prostate Shows Different Expression Pattern of Somatostatin Receptor 2 (SSTR2) and Phosphotyrosine Phosphatase SHP-1 (PTPN6) According to Tumor Progression

    PubMed Central

    Cariaga-Martinez, Ariel Ernesto; Lorenzati, María Angelica; Riera, Mario Alejandro; Cubilla, Marisa Angelica; De La Rossa, Andrés; Giorgio, Ernesto Martín; Tiscornia, María Mercedes; Gimenez, Esteban Mariano; Rojas, María Eugenia; Chaneton, Bárbara Julieta; Rodríguez, Dora Isabel; Zapata, Pedro Darío

    2009-01-01

    Prostate proliferation is dependent of androgens and many peptide hormones. Recent reports suggest that SSTR2 and SHP-1 were two fundamental components on antiproliferative effect of somatostatin. Many studies on SHP-1 revealed that the expression of this protein was diminished or abolished in several of the cancer cell lines and tissues examined. However, it is necessary to confront the cell lines data with real situation in cancer cases. Our studies have shown that epithelial expressions of both proteins, SHP-1 and SSTR2, in normal and benign hyperplasia are localized in the luminal side of duct and acinar cells. Also, SSTR2 is expressed in stromal cells. In malignant prostate tissue, SHP-1 was diminished in 28/45 cases or absent in 12/45 cases, whereas SSTR2 epithelial was diminished in 38/45 cases or lost in only 2/45 cases. The intensity of immunostained was highly negative correlated with Gleason grade for two proteins. PMID:19365586

  8. Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer12

    PubMed Central

    Fan, Li-Ching; Teng, Hao-Wei; Shiau, Chung-Wai; Tai, Wei-Tien; Hung, Man-Hsin; Yang, Shung-Haur; Jiang, Jeng-Kai; Chen, Kuen-Feng

    2015-01-01

    STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC). Our previous data demonstrated that regorafenib (Stivarga) is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1) that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3Tyr705 level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43–induced apoptosis and decreased p-STAT3Tyr705 level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3Tyr705 level. These data suggest that SC-43–induced apoptosis mediated through the loss of p-STAT3Tyr705 was dependent on SHP-1 function. Importantly, SC-43–enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3Tyr705 signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3Tyr705expression in CRC patients (P = .038). Patients with strong SHP-1 and weak p-STAT3Tyr705 expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3Tyr705 expression (P = .029). In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC. PMID:26476076

  9. CD5-mediated inhibition of TCR signaling proceeds normally in the absence of SHP-1

    PubMed Central

    DONG, BAOXIA; SOMANI, ALLY-KHAN; LOVE, PAUL E.; ZHENG, XUAN; CHEN, XIEQUN; ZHANG, JINYI

    2016-01-01

    The CD5 transmembrane glycoprotein functions as a co-receptor in the signaling pathway linking T-cell antigen receptor (TCR) engagement to activation and differentiation. Although CD5 effects on TCR signaling have been shown to be primarily inhibitory, the underlying mechanisms remain unclear. In view of recent data revealing the ability of CD5 to associate with the SHP-1 tyrosine phosphatase, a protein that also downregulates TCR signaling, we examined the role of SHP-1 in modulating CD5 function using thymocytes from SHP-1-deficient viable motheaten (mev) mice. The results revealed the association of SHP-1 with CD5 to be markedly increased following TCR stimulation and indicated that this interaction was enhanced by and was dependent on CD5 tyrosine phosphorylation. However, there was no difference of the tyrosine phosphorylation status of CD5 between resting and TCR-stimulated cells in SHP-1-deficient compared to wild-type thymocytes. Lack of SHP-1 activity did not affect the levels of CD5 surface expression, CD5 co-immunoprecipitable tyrosine phosphatase activity and intracellular calcium increase following co-crosslinking of the TCR and CD5. Similarly, an analysis of T-cell thymocyte populations in mev mice expressing an H-Y transgene as well as a construct mediating T-cell restricted CD5 overexpression, revealed that the reduction in the positive selection conferred by CD5 overexpression was unaffected by SHP-1 deficiency. CD5 is not a SHP-1 substrate and SHP-1 is not required for and possibly not involved in the CD5-mediated modulation of TCR signaling. PMID:27221212

  10. SHP-1 deficiency and increased inflammatory gene expression in PBMCs of multiple sclerosis patients

    PubMed Central

    Christophi, George P; Hudson, Chad A; Gruber, Ross C; Christophi, Christoforos P; Mihai, Cornelia; Mejico, Luis J; Jubelt, Burk; Massa, Paul T

    2010-01-01

    Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination. PMID:18209728

  11. Regulation of acidification and apoptosis by SHP-1 and Bcl-2.

    PubMed

    Thangaraju, M; Sharma, K; Leber, B; Andrews, D W; Shen, S H; Srikant, C B

    1999-10-01

    Recruitment of the SH2 domain containing cytoplasmic protein-tyrosine phosphatase SHP-1 to the membrane by somatostatin (SST) is an early event in its antiproliferative signaling that induces intracellular acidification-dependent apoptosis in breast cancer cells. Fas ligation also induces acidification-dependent apoptosis in a manner requiring the presence of SHP-1 at the membrane. Moreover, we have recently reported that SHP-1 is required not only for acidification, but also for apoptotic events that follow acidification (Thangaraju, M., Sharma, K., Liu, D., Shen, S. H., and Srikant, C. B. (1999) Cancer Res. 59, 1649-1654). Here we show that ectopically expressed SHP-1 was predominantly membrane-associated and amplified the cytotoxic signaling initiated upon SST receptor activation and Fas ligation. The catalytically inactive mutant of SHP-1 (SHP-1C455S) abolished the ability of the SST agonists to signal apoptosis by preventing the recruitment of wild type SHP-1 to the membrane. Overexpression of the anti-apoptotic protein Bcl-2 in MCF-7 cells inhibited SST-induced apoptosis upstream of acidification by inhibiting p53-dependent induction of Bax as well as by raising the resting pH(i) and attenuating SST-induced decrease in pH(i). By contrast, Bcl-2 failed to prevent apoptosis triggered by direct acidification. These data demonstrate that (i) membrane-associated SHP-1 is required for receptor-mediated cytotoxic signaling that causes intracellular acidification and apoptosis, and (ii) Bcl-2 acts distal to SHP-1 and p53 to prevent SST-induced acidification but cannot inhibit the apoptotic events that ensue intracellular acidification.

  12. Shp-1 dephosphorylates TRPV1 in dorsal root ganglion neurons and alleviates CFA-induced inflammatory pain in rats.

    PubMed

    Xiao, Xing; Zhao, Xiao-Tao; Xu, Ling-Chi; Yue, Lu-Peng; Liu, Feng-Yu; Cai, Jie; Liao, Fei-Fei; Kong, Jin-Ge; Xing, Guo-Gang; Yi, Ming; Wan, You

    2015-04-01

    Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed in nociceptive neurons of rat dorsal root ganglions (DRGs) and mediate inflammatory pain. Nonspecific inhibition of protein-tyrosine phosphatases (PTPs) increases the tyrosine phosphorylation of TRPV1 and sensitizes TRPV1. However, less is known about tyrosine phosphorylation's implication in inflammatory pain, compared with that of serine/threonine phosphorylation. Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1) is a key phosphatase dephosphorylating TRPV1. In this study, we reported that Shp-1 colocalized with and bound to TRPV1 in nociceptive DRG neurons. Shp-1 inhibitors, including sodium stibogluconate and PTP inhibitor III, sensitized TRPV1 in cultured DRG neurons. In naive rats, intrathecal injection of Shp-1 inhibitors increased both TRPV1 and tyrosine-phosphorylated TRPV1 in DRGs and induced thermal hyperalgesia, which was abolished by pretreatment with TRPV1 antagonists capsazepine, BCTC, or AMG9810. Complete Freund's adjuvant (CFA)-induced inflammatory pain in rats significantly increased the expression of Shp-1, TRPV1, and tyrosine-phosphorylated TRPV1, as well as the colocalization of Shp-1 and TRPV1 in DRGs. Intrathecal injection of sodium stibogluconate aggravated CFA-induced inflammatory pain, whereas Shp-1 overexpression in DRG neurons alleviated it. These results suggested that Shp-1 dephosphorylated and inhibited TRPV1 in DRG neurons, contributing to maintain thermal nociceptive thresholds in normal rats, and as a compensatory mechanism, Shp-1 increased in DRGs of rats with CFA-induced inflammatory pain, which was involved in protecting against excessive thermal hyperalgesia.

  13. Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

    PubMed

    Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

    2013-06-01

    N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications.

  14. SHP-1: the next checkpoint target for cancer immunotherapy?

    PubMed

    Watson, H Angharad; Wehenkel, Sophie; Matthews, James; Ager, Ann

    2016-04-15

    The immense power of the immune system is harnessed in healthy individuals by a range of negative regulatory signals and checkpoints. Manipulating these checkpoints through inhibition has resulted in striking immune-mediated clearance of otherwise untreatable tumours and metastases; unfortunately, not all patients respond to treatment with the currently available inhibitors of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Combinatorial studies using both anti-CTLA-4 and anti-PD-1 demonstrate synergistic effects of targeting multiple checkpoints, paving the way for other immune checkpoints to be targeted. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T-cells. This review will discuss the potential value of SHP-1 inhibition in future tumour immunotherapy.

  15. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    SciTech Connect

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  16. Dephosphorylation of the adaptor LAT and phospholipase C-γ by SHP-1 inhibits natural killer cell cytotoxicity.

    PubMed

    Matalon, Omri; Fried, Sophia; Ben-Shmuel, Aviad; Pauker, Maor H; Joseph, Noah; Keizer, Danielle; Piterburg, Marina; Barda-Saad, Mira

    2016-01-01

    Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.

  17. A THEMIS:SHP1 complex promotes T-cell survival.

    PubMed

    Paster, Wolfgang; Bruger, Annika M; Katsch, Kristin; Grégoire, Claude; Roncagalli, Romain; Fu, Guo; Gascoigne, Nicholas R J; Nika, Konstantina; Cohnen, Andre; Feller, Stephan M; Simister, Philip C; Molder, Kelly C; Cordoba, Shaun-Paul; Dushek, Omer; Malissen, Bernard; Acuto, Oreste

    2015-02-01

    THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation. PMID:25535246

  18. A THEMIS:SHP1 complex promotes T-cell survival

    PubMed Central

    Paster, Wolfgang; Bruger, Annika M; Katsch, Kristin; Grégoire, Claude; Roncagalli, Romain; Fu, Guo; Gascoigne, Nicholas RJ; Nika, Konstantina; Cohnen, Andre; Feller, Stephan M; Simister, Philip C; Molder, Kelly C; Cordoba, Shaun-Paul; Dushek, Omer; Malissen, Bernard; Acuto, Oreste

    2015-01-01

    THEMIS is critical for conventional T-cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr-phosphorylation-independent fashion. Rather, SHP1 and THEMIS engage with the N-SH3 and C-SH3 domains of GRB2, respectively, a configuration that allows GRB2-SH2 to recruit the complex onto LAT. Consistent with THEMIS-mediated recruitment of SHP to the TCR signalosome, THEMIS knock-down increased TCR-induced CD3-ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock-down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK-mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T-cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T-cell development and differentiation. PMID:25535246

  19. Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1

    PubMed Central

    Ishikawa, Eri; Kosako, Hidetaka; Yasuda, Tomoharu; Ohmuraya, Masaki; Araki, Kimi; Kurosaki, Tomohiro; Saito, Takashi; Yamasaki, Sho

    2016-01-01

    Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4+CD8+ double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1S557A) impairs generation of CD4+ thymocytes. These results suggest that the PKD–SHP-1 axis positively regulates TCR signalling to promote CD4+ T cell development. PMID:27670070

  20. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  1. Purity of transferred CD8+ T cells is crucial for safety and efficacy of combinatorial tumor immunotherapy in the absence of SHP-1

    PubMed Central

    Watson, H Angharad; Dolton, Garry; Ohme, Julia; Ladell, Kristin; Vigar, Miriam; Wehenkel, Sophie; Hindley, James; Mohammed, Rebar N; Miners, Kelly; Luckwell, Rhys A; Price, David A; Matthews, R James; Ager, Ann

    2016-01-01

    Adoptive transfer of tumor-specific cytotoxic T cells is a promising advance in cancer therapy. Similarly, checkpoint inhibition has shown striking clinical results in some patients. Here we combine adoptive cell transfer with ablation of the checkpoint protein Src homology 2-domain-containing phosphatase 1 (SHP-1, Ptpn6). Naturally occurring motheaten mice lack SHP-1 and do not survive weaning due to extensive immunopathology. To circumvent this limitation, we created a novel SHP-1null mouse that is viable up to 12 weeks of age by knocking out IL1r1. Using this model, we demonstrate that the absence of SHP-1 augments the ability of adoptively transferred CD8+ T cells to control tumor growth. This therapeutic effect was only observed in situations where T-cell numbers were limited, analogous to clinical settings. However, adoptive transfer of non-CD8+ SHP-1null hematopoietic cells resulted in lethal motheaten-like pathology, indicating that systemic inhibition of SHP-1 could have serious adverse effects. Despite this caveat, our findings support the development of SHP-1 inhibition strategies in human T cells to complement adoptive transfer therapies in the clinic. PMID:27430370

  2. Leishmania infantum-chagasi activates SHP-1 and reduces NFAT5/TonEBP activity in the mouse kidney inner medulla.

    PubMed

    Zhou, Xiaoming; Wang, Hong; Koles, Nancy L; Zhang, Aihong; Aronson, Naomi E

    2014-09-01

    Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a transcription factor, NFAT5/TonEBP, because it activates expression of osmoprotective genes betaine/glycine transporter 1 (BGT1) and sodium/myo-inositol transporter (SMIT), and water channel aquaporin-2, all of which are imperative for concentrating urine. Leishmania parasites evade macrophage immune defenses by activating protein tyrosine phosphatases, among which SHP-1 is critical. We previously demonstrated that SHP-1 inhibits tonicity-dependent activation of NFAT5/TonEBP in HEK293 cells through screening a genome-wide small interfering (si) RNA library against phosphatases (Zhou X, Gallazzini M, Burg MB, Ferraris JD. Proc Natl Acad Sci USA 107: 7072-7077, 2010). We sought to examine whether Leishmania can activate SHP-1 and inhibit NFAT5/TonEBP activity in the renal inner medulla in a murine model of visceral leishmaniasis by injection of female BALB/c mice with a single intravenous dose of 5 × 10(5) L. chagasi metacyclic promastigotes. We found that SHP-1 is expressed in the kidney inner medulla. L. chagasi activates SHP-1 with an increase in stimulatory phosphorylation of SHP-1-Y536 in the region. L. chagasi reduces expression of NFAT5/TonEBP mRNA and protein as well as expression of its targeted genes: BGT1, SMIT, and aquaporin-2. The culture supernatant from L. chagasi metacyclic promastigotes increases SHP-1 protein abundance and potently inhibits NFAT5 transcriptional activity in mIMCD3 cells. However, L. chagasi in our animal model has no significant effect on urinary concentration. We conclude that L. chagasi, most likely through its secreted virulence factors, activates SHP-1 and reduces NFAT5/TonEBP gene expression, which leads to reduced NFAT5/TonEBP transcriptional activity in the kidney inner medulla. PMID:24990897

  3. Lyn- and PLC-beta3-dependent regulation of SHP-1 phosphorylation controls Stat5 activity and myelomonocytic leukemia-like disease.

    PubMed

    Xiao, Wenbin; Ando, Tomoaki; Wang, Huan-You; Kawakami, Yuko; Kawakami, Toshiaki

    2010-12-23

    Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus, PLC-β3-deficient mice develop myeloproliferative neoplasm, like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent, fatal myelodysplastic/myeloproliferative neoplasm, similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease, phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore, we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities.

  4. SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis

    PubMed Central

    Watson, Neva B.; Schneider, Karin M.; Massa, Paul T.

    2015-01-01

    Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the central nervous system causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator SHP-1, which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler’s murine encephalomyelitis virus infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification with loss of ambulation in wild type mice. Surprisingly, although similar extensive myofiber infection and inflammation is observed in SHP-1-deficient (SHP-1−/−) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1−/− muscle, and an increased infiltration of immature monocytes/macrophages correlated with absence of clinical disease in SHP-1−/− mice, while mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. PMID:25681345

  5. Continuous inhibitory signaling by both SHP-1 and SHIP-1 pathways is required to maintain unresponsiveness of anergic B cells

    PubMed Central

    Getahun, Andrew; Beavers, Nicole A.; Larson, Sandy R.; Shlomchik, Mark J.

    2016-01-01

    Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn–SHIP-1–PTEN–SHP-1 axis function in B cells from systemic lupus erythematosus patients. PMID:27114609

  6. Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity

    PubMed Central

    Prestwood, Tyler R.; Spitzer, Matthew H.; Linde, Ian L.; Chabon, Jonathan; Reticker-Flynn, Nathan E.; Bhattacharya, Nupur; Zhang, Hong; Zhang, Xiangyue; Basto, Pamela A.; Burt, Bryan M.; Alonso, Michael N.; Engleman, Edgar G.

    2016-01-01

    BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity. PMID:27812544

  7. Interdependent regulation of intracellular acidification and SHP-1 in apoptosis.

    PubMed

    Thangaraju, M; Sharma, K; Liu, D; Shen, S H; Srikant, C B

    1999-04-01

    The G protein-coupled receptor agonist somatostatin (SST)-induces apoptosis in MCF-7 human breast cancer cells. This is associated with induction of wild-type p53, Bax, and an acidic endonuclease. We have shown recently that its cytotoxic signaling is mediated via membrane-associated SHP-1 and is dependent on decrease in intracellular pH (pHi) to 6.5. Here we investigated the relationship between intracellular acidification and SHP-1 in cytotoxic signaling. Clamping of pHi at 7.25 by the proton-ionophore nigericin abolished SST-signaled apoptosis without affecting its ability to regulate SHP-1, p53, and Bax. Apoptosis could be induced by nigericin clamping of pHi to 6.5. Such acidification-induced apoptosis was not observed at pHi <6.0 or >6.7. pHi-dependent apoptosis was associated with the translocation of SHP-1 to the membrane, enhanced in cells overexpressing SHP-1, and was abolished by its inactive mutant SHP-1C455S. Acidification caused by inhibition of Na+/H+ exchanger and H+ ATPase (pHi = 6.55 and 6.65, respectively) also triggered apoptosis. The effect of concurrent inhibition of Na+/H+ exchanger and H(+)-ATPase on pHi and apoptosis was comparable with that of SST. Acidification-induced, SHP-1-dependent apoptosis occurred in breast cancer cell lines in which SST was cytotoxic (MCF-7 and T47D) or not (MDA-MB-231). We conclude that: (a) SST-induced SHP-1-dependent acidification occurs subsequent to or independent of the induction of p53 and Bax; (b) SST-induced intracellular acidification may arise due to inhibition of Na+/H+ exchanger and H(+)-ATPase; and (c) SHP-1 is necessary not only for agonist-induced acidification but also for the execution of acidification-dependent apoptosis. We suggest that combined targeting of SHP-1 and intracellular acidification may lead to a novel strategy of anticancer therapy bypassing the need for receptor-mediated signaling.

  8. Disrupting VEGF-A paracrine and autocrine loops by targeting SHP-1 suppresses triple negative breast cancer metastasis

    PubMed Central

    Su, Jung-Chen; Mar, Ai-Chung; Wu, Szu-Hsien; Tai, Wei-Tien; Chu, Pei-Yi; Wu, Chia-Yun; Tseng, Ling-Ming; Lee, Te-Chang; Chen, Kuen-Feng; Liu, Chun-Yu; Chiu, Hao-Chieh; Shiau, Chung-Wai

    2016-01-01

    Patients with triple-negative breast cancer (TNBC) had an increased likelihood of distant recurrence and death, as compared with those with non-TNBC subtype. Regorafenib is a multi-receptor tyrosine kinase (RTK) inhibitor targeting oncogenesis and has been approved for metastatic colorectal cancer and advanced gastrointestinal stromal tumor. Recent studies suggest regorafenib acts as a SHP-1 phosphatase agonist. Here, we investigated the potential of regorafenib to suppress metastasis of TNBC cells through targeting SHP-1/p-STAT3/VEGF-A axis. We found a significant correlation between cancer cell migration and SHP-1/p-STAT3/VEGF-A expression in human TNBC cells. Clinically, high VEGF-A expression is associated with worse disease-free and distant metastasis-free survival. Regorafenib induced significant anti-migratory effects, in association with downregulation of p-STAT3 and VEGF-A. To exclude the role of RTK inhibition in regorafenib-induced anti-metastasis, we synthesized a regorafenib derivative, SC-78, that had minimal effect on VEGFR2 and PDGFR kinase inhibition, while having more potent effects on SHP-1 activation. SC-78 demonstrated superior in vitro and in vivo anti-migration to regorafenib. Furthermore, VEGF-A dependent autocrine/paracrine loops were disrupted by regorafenib and SC-78. This study implies that SHP-1/p-STAT3/VEGF-A axis is a potential therapeutic target for metastatic TNBC, and the more potent SC-78 may be a promising lead for suppressing metastasis of TNBC. PMID:27364975

  9. Phosphonate derivatives of tetraazamacrocycles as new inhibitors of protein tyrosine phosphatases.

    PubMed

    Kobzar, Oleksandr L; Shevchuk, Michael V; Lyashenko, Alesya N; Tanchuk, Vsevolod Yu; Romanenko, Vadim D; Kobelev, Sergei M; Averin, Alexei D; Beletskaya, Irina P; Vovk, Andriy I; Kukhar, Valery P

    2015-07-21

    α,α-Difluoro-β-ketophosphonated derivatives of tetraazamacrocycles were synthesized and found to be potential inhibitors of protein tyrosine phosphatases. N-Substituted conjugates of cyclam and cyclen with bioisosteric phosphonate groups displayed good activities toward T-cell protein tyrosine phosphatase with IC50 values in the micromolar to nanomolar range and showed selectivity over PTP1B, CD45, SHP2, and PTPβ. Kinetic studies indicated that the inhibitors can occupy the region of the active site of TC-PTP. This study demonstrates a new approach which employs tetraazamacrocycles as a molecular platform for designing inhibitors of protein tyrosine phosphatases. PMID:26058329

  10. A combination of sorafenib and SC-43 is a synergistic SHP-1 agonist duo to advance hepatocellular carcinoma therapy.

    PubMed

    Chao, Tzu-I; Tai, Wei-Tien; Hung, Man-Hsin; Tsai, Ming-Hsien; Chen, Min-Hsuan; Chang, Mao-Ju; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-02-28

    Sorafenib is the first and currently the only standard treatment for advanced hepatocellular carcinoma (HCC). We previously developed a sorafenib derivative SC-43, which exhibits much more enhanced anti-HCC activity than sorafenib and also promotes apoptosis in sorafenib-resistant HCC cells. Herein, a novel "sorafenib plus" combination therapy was developed by coupling sorafenib treatment with SC-43. Both sorafenib and SC-43 are proven Src homology region 2 domain containing phosphatase 1 (SHP-1) agonists. The combined actions of sorafenib and SC-43 enhanced SHP-1 activity, which was associated with diminished STAT3-related signals and stronger expression of apoptotic genes above that of either drug alone, culminating in increased cell death. Decreased p-STAT3 signaling and tumor size, as well as increased SHP-1 activity were observed in mice receiving the combination therapy in a subcutaneous HCC model. More reduced orthotopic HCC tumor size and prolonged survival were also observed in mice in the combination treatment arm compared to mice in either of the monotherapy arms. These results in the preclinical setting pave the way for further clinical studies to treat unresectable HCC. PMID:26679051

  11. Calpain-dependent cleavage of SHP-1 and SHP-2 is involved in the dephosphorylation of Jurkat T cells induced by Entamoeba histolytica.

    PubMed

    Kim, K A; Lee, Y A; Shin, M H

    2010-03-01

    Host cell death induced by Entamoeba histolytica is an important mechanism for both host defence and microbial immune evasion during human amoebiasis. However, the signalling pathways underlying cell death induced by E. histolytica are not fully understood. This study investigated the involvement of the protein tyrosine phosphatases (PTPs) SHP-1 and SHP-2 in the dephosphorylation associated with E. histolytica-induced host cell death. Incubation with E. histolytica resulted in a marked decrease in protein tyrosine phosphorylation levels and degradation of SHP-1 or SHP-2 in Jurkat cells. Pre-treatment of cells with a calpain inhibitor, calpeptin, impeded the amoeba-induced dephosporylation and cleavage of SHP-1 or SHP-2. Additionally, inhibition of PTPs with phenylarsine oxide (PAO) attenuated Entamoeba-induced dephosphorylation and DNA fragmentation in Jurkat T cells. These results suggest that calpain-dependent cleavage of SHP-1 and SHP-2 may contribute to protein tyrosine dephosphorylation in Jurkat T cell death induced by E. histolytica. PMID:20398180

  12. Insights into monocyte-driven osteoclastogenesis and its link with hematopoiesis: regulatory roles of PECAM-1 (CD31) and SHP-1.

    PubMed

    Wu, Yue; Madri, Joseph

    2010-01-01

    Osteoclasts are derived from hematopoietic cells of monocyte-macrophage lineage. Osteoclastogenesis is orchestrated by the migration of monocytic osteoclast progenitor cells in close proximity to bone surfaces destined for resorption. Although the overall roles of monocyte migratory behavior in osteoclastogenesis remain enigmatic, impaired monocyte migration can lead to either decreased or increased osteoclastogenesis, which appears contingent upon the roles of migration in either fusion events required for osteoclast formation or terminal differentiation of osteoclasts. The cell adhesion molecule PECAM-1 (platelet endothelial cell adhesion molecule 1), in concert with the tyrosine phosphatase SHP-1 (Src homology 2-containing protein tyrosine phosphatase 1) and tyrosine kinase Syk-1 (spleen tyrosine kinase 1), functions as a negative regulator of osteoclastogenesis. Both PECAM-1 (CD31) and SHP-1 knockout mice exhibit not only increased osteoclastogenesis but also abnormal hematopoiesis, which is suggestive of the intricate interplay between hematopoiesis and osteoclastogenesis. Interestingly, the most pronounced effect of PECAM-1 deficiency on hematopoiesis is reflected by excessive megakaryocytopoiesis. Emerging data have suggested the role of megakaryocytes in bone remodeling. Megakaryocytopoiesis-osteoclastogenesis interactions are discussed herein, reconciling the discrepancies shown by different studies in this area. PECAM-1 and non-receptor tyrosine phosphatase polymorphisms have been revealed in a spectrum of diseases. The complex regulatory roles of PECAM-1 and SHP-1 in vivo suggest the potential utilization of polymorphisms of these genes for diagnostic purposes. PMID:21083524

  13. Small molecule 1'-acetoxychavicol acetate suppresses breast tumor metastasis by regulating the SHP-1/STAT3/MMPs signaling pathway.

    PubMed

    Wang, Jieqiong; Zhang, Li; Chen, Guoliang; Zhang, Jing; Li, Zhenxi; Lu, Weiqiang; Liu, Mingyao; Pang, Xiufeng

    2014-11-01

    Signal transducer and activator of transcription 3 (STAT3) is implicated breast cancer metastasis and represents a potential target for developing new anti-tumor metastasis drugs. The purpose of this study is to investigate whether the natural agent 1'-acetoxychavicol acetate (ACA), derived from the rhizomes and seeds of Languas galanga, could suppress breast cancer metastasis by targeting STAT3 signaling pathway. ACA was examined for its effects on breast cancer migration/invasion and metastasis using Transwell assays in vitro and breast cancer skeletal metastasis mouse model in vivo (n = 10 mice per group). The inhibitory effect of ACA on cellular STAT3 signaling pathway was investigated by series of biochemistry analysis. The chavicol preferentially suppressed cancer cell migration and invasion, and this activity was superior to its cytotoxic effects. ACA suppressed both constitutive and interleukin-6-inducible STAT3 activation and diminished the accumulation of STAT3 in the nucleus and its DNA-binding activity. More importantly, ACA treatment led to significant up-regulation of Src homology region 2 domain-containing phosphatase 1 (SHP-1), and the ACA-induced depression of cancer cell migration and STAT3 signaling could be apparently reversed by blockade of SHP-1. Matrix metalloproteinase (MMP)-2 and -9, gene products of STAT3 that regulate cell invasion, were specifically suppressed by ACA. In tumor metastasis model, ACA potently inhibited the human breast cancer cell-induced osteolysis, and had little apparent in vivo toxicity at the test concentrations. ACA is a novel drug candidate for the inhibition of tumor metastasis through interference with the SHP-1/STAT3/MMPs signaling pathway.

  14. A derivative of epigallocatechin-3-gallate induces apoptosis via SHP-1-mediated suppression of BCR-ABL and STAT3 signalling in chronic myelogenous leukaemia

    PubMed Central

    Jung, Ji Hoon; Yun, Miyong; Choo, Eun-Jeong; Kim, Sun-Hee; Jeong, Myoung-Seok; Jung, Deok-Beom; Lee, Hyemin; Kim, Eun-Ok; Kato, Nobuo; Kim, Bonglee; Srivastava, Sanjay K; Kaihatsu, Kunihiro; Kim, Sung-Hoon

    2015-01-01

    Background and Purpose Epigallocatechin-3-gallate (EGCG) is a component of green tea known to have chemo-preventative effects on several cancers. However, EGCG has limited clinical application, which necessitates the development of a more effective EGCG prodrug as an anticancer agent. Experimental Approach Derivatives of EGCG were evaluated for their stability and anti-tumour activity in human chronic myeloid leukaemia (CML) K562 and KBM5 cells. Key Results EGCG-mono-palmitate (EGCG-MP) showed most prolonged stability compared with other EGCG derivatives. EGCG-MP exerted greater cytotoxicity and apoptosis in K562 and KBM5 cells than the other EGCG derivatives. EGCG-MP induced Src-homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) leading decreased oncogenic protein BCR-ABL and STAT3 phosphorylation in CML cells, compared with treatment with EGCG. Furthermore, EGCG-MP reduced phosphorylation of STAT3 and survival genes in K562 cells, compared with EGCG. Conversely, depletion of SHP-1 or application of the tyrosine phosphatase inhibitor pervanadate blocked the ability of EGCG-MP to suppress phosphorylation of BCR-ABL and STAT3, and the expression of survival genes downstream of STAT3. In addition, EGCG-MP treatment more effectively suppressed tumour growth in BALB/c athymic nude mice compared with untreated controls or EGCG treatment. Immunohistochemistry revealed increased caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group. Conclusions and Implications EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling in vitro and in vivo more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment. PMID:25825203

  15. Age-related impairment of GM-CSF-induced signalling in neutrophils: role of SHP-1 and SOCS proteins.

    PubMed

    Tortorella, Cosimo; Simone, Olivia; Piazzolla, Giuseppina; Stella, Isabella; Antonaci, Salvatore

    2007-08-01

    Functional activities of mature human neutrophils are strongly influenced by the pro-inflammatory cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). Accordingly, a defective response to GM-CSF might have dramatic consequences for neutrophil functions and the host defence against infections. Such an event is most likely to occur in senescence. A number of studies have, in fact, reported an impairment of the GM-CSF capacity to prime and/or to activate respiratory burst, as well as to delay apoptotic events, in neutrophils from elderly individuals. In the last 2 decades many efforts have been made to explore at molecular levels the mechanism underlying these defects. Recent studies let us depict a scenario in which an increased activity of inhibitory molecules, such as Src homology domain-containing protein tyrosine phosphatase-1 (SHP-1) and suppressors of cytokine signalling (SOCS), is responsible for the age-related failure of GM-CSF to stimulate neutrophil functions via inhibition of Lyn-, phosphoinositide 3-kinase (PI3-K)/extracellular signal-regulated kinase (ERK)- and signal transducers and activators of transcription (STAT)-dependent pathways. The control of SHP-1 and/or SOCS activity might therefore be an important therapeutic target for the restoration of normal immune responses during senescence.

  16. Schistosoma mansoni Soluble Egg Antigens Induce Expression of the Negative Regulators SOCS1 and SHP1 in Human Dendritic Cells via Interaction with the Mannose Receptor

    PubMed Central

    Klaver, Elsenoor J.; Kuijk, Loes M.; Lindhorst, Thisbe K.; Cummings, Richard D.; van Die, Irma

    2015-01-01

    Schistosomiasis is a common debilitating human parasitic disease in (sub)tropical areas, however, schistosome infections can also protect against a variety of inflammatory diseases. This has raised broad interest in the mechanisms by which Schistosoma modulate the immune system into an anti-inflammatory and regulatory state. Human dendritic cells (DCs) show many phenotypic changes upon contact with Schistosoma mansoni soluble egg antigens (SEA). We here show that oxidation of SEA glycans, but not heat-denaturation, abrogates the capacity of SEA to suppress both LPS-induced cytokine secretion and DC proliferation, indicating an important role of SEA glycans in these processes. Remarkably, interaction of SEA glycans with DCs results in a strongly increased expression of Suppressor Of Cytokine Signalling1 (SOCS1) and SH2-containing protein tyrosine Phosphatase-1 (SHP1), important negative regulators of TLR4 signalling. In addition, SEA induces the secretion of transforming growth factor β (TGF-β), and the surface expression of the costimulatory molecules Programmed Death Ligand-1 (PD-L1) and OX40 ligand (OX40L), which are known phenotypic markers for the capacity of DCs to polarize naïve T cells into Th2/Treg cell subsets. Inhibition of mannose receptor (MR)-mediated internalization of SEA into DCs by blocking with allyl α-D-mannoside or anti-MR antibodies, significantly reduced SOCS1 and SHP1 expression. In conclusion, we demonstrate that SEA glycans are essential for induction of enhanced SOCS1 and SHP1 levels in DCs via the MR. Our data provide novel mechanistic evidence for the potential of S. mansoni SEA glycans to modulate human DCs, which may contribute to the capacity of SEA to down-regulate inflammatory responses. PMID:25897665

  17. Schistosoma mansoni Soluble Egg Antigens Induce Expression of the Negative Regulators SOCS1 and SHP1 in Human Dendritic Cells via Interaction with the Mannose Receptor.

    PubMed

    Klaver, Elsenoor J; Kuijk, Loes M; Lindhorst, Thisbe K; Cummings, Richard D; van Die, Irma

    2015-01-01

    Schistosomiasis is a common debilitating human parasitic disease in (sub)tropical areas, however, schistosome infections can also protect against a variety of inflammatory diseases. This has raised broad interest in the mechanisms by which Schistosoma modulate the immune system into an anti-inflammatory and regulatory state. Human dendritic cells (DCs) show many phenotypic changes upon contact with Schistosoma mansoni soluble egg antigens (SEA). We here show that oxidation of SEA glycans, but not heat-denaturation, abrogates the capacity of SEA to suppress both LPS-induced cytokine secretion and DC proliferation, indicating an important role of SEA glycans in these processes. Remarkably, interaction of SEA glycans with DCs results in a strongly increased expression of Suppressor Of Cytokine Signalling1 (SOCS1) and SH2-containing protein tyrosine Phosphatase-1 (SHP1), important negative regulators of TLR4 signalling. In addition, SEA induces the secretion of transforming growth factor β (TGF-β), and the surface expression of the costimulatory molecules Programmed Death Ligand-1 (PD-L1) and OX40 ligand (OX40L), which are known phenotypic markers for the capacity of DCs to polarize naïve T cells into Th2/Treg cell subsets. Inhibition of mannose receptor (MR)-mediated internalization of SEA into DCs by blocking with allyl α-D-mannoside or anti-MR antibodies, significantly reduced SOCS1 and SHP1 expression. In conclusion, we demonstrate that SEA glycans are essential for induction of enhanced SOCS1 and SHP1 levels in DCs via the MR. Our data provide novel mechanistic evidence for the potential of S. mansoni SEA glycans to modulate human DCs, which may contribute to the capacity of SEA to down-regulate inflammatory responses. PMID:25897665

  18. Jak2 inhibition deactivates Lyn kinase through the SET–PP2A–SHP1 pathway, causing apoptosis in drug-resistant cells from chronic myelogenous leukemia patients

    PubMed Central

    Samanta, AK; Chakraborty, SN; Wang, Y; Kantarjian, H; Sun, X; Hood, J; Perrotti, D; Arlinghaus, RB

    2010-01-01

    Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr–Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr–Abl+ cells inhibition of Jak2, a downstream target of Bcr–Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr–Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET–PP2A–Shp1 pathway. PMID:19234487

  19. A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation.

    PubMed Central

    Wright, J H; Drueckes, P; Bartoe, J; Zhao, Z; Shen, S H; Krebs, E G

    1997-01-01

    SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth. Images PMID:9285826

  20. A PKC-SHP1 signaling axis desensitizes Fcγ receptor signaling by reducing the tyrosine phosphorylation of CBL and regulates FcγR mediated phagocytosis

    PubMed Central

    2014-01-01

    Background Fcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes. Results Using the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S). Conclusions Our results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings

  1. Inhibition of STAT3 signaling and induction of SHP1 mediate antiangiogenic and antitumor activities of ergosterol peroxide in U266 multiple myeloma cells

    PubMed Central

    2012-01-01

    Background Ergosterol peroxide (EP) derived from edible mushroom has been shown to exert anti-tumor activity in several cancer cells. In the present study, anti-angiogenic activity of EP was investigated with the underlying molecular mechanisms in human multiple myeloma U266 cells. Results Despite weak cytotoxicity against U266 cells, EP suppressed phosphorylation, DNA binding activity and nuclear translocalization of signal transducer and activator of transcription 3 (STAT3) in U266 cells at nontoxic concentrations. Also, EP inhibited phosphorylation of the upstream kinases Janus kinase 2 (JAK2) and Src in a time-dependent manner. Furthermore, EP increased the expression of protein tyrosine phosphatase SHP-1 at protein and mRNA levels, and conversely silencing of the SHP-1 gene clearly blocked EP-mediated STAT3 inactivation. In addition, EP significantly decreased vascular endothelial growth factor (VEGF), one of STAT3 target genes at cellular and protein levels as well as disrupted in vitro tube formation assay. Moreover, EP significantly suppressed the growth of U266 cells inoculated in female BALB/c athymic nude mice and immunohistochemistry revealed that EP effectively reduced the expression of STAT3 and CD34 in tumor sections compared to untreated control. Conclusion These findings suggest that EP can exert antitumor activity in multiple myeloma U266 cells partly with antiangiogenic activity targeting JAK2/STAT3 signaling pathway as a potent cancer preventive agent for treatment of multiple myeloma cells. PMID:22260501

  2. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma

    PubMed Central

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-01-01

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC. PMID:26959741

  3. CEACAM1 negatively regulates IL-1β production in LPS activated neutrophils by recruiting SHP-1 to a SYK-TLR4-CEACAM1 complex.

    PubMed

    Lu, Rongze; Pan, Hao; Shively, John E

    2012-01-01

    LPS-activated neutrophils secrete IL-1β by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1β in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1β secretion in neutrophils. The absence of this regulation in Ceacam1⁻/⁻ neutrophils led to hyper production of IL-1β in response to LPS. The hyper production of IL-1β was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1⁻/⁻ neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1β production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1β production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation.

  4. SHP-1 promoter 2 methylation in cerebrospinal fluid for diagnosis of leptomeningeal epithelial-derived malignancy (carcinomatous meningitis).

    PubMed

    Vinayanuwattikun, Chanida; Mingmalairak, Siyamol; Jittapiromsak, Nutchawan; Thaipisuttikul, Iyavut; Sriuranpong, Virote; Mutirangura, Apiwat; Shuangshoti, Shanop

    2016-09-01

    Current diagnostic methods for leptomeningeal metastasis (LM) from epithelial-derived malignancy (EDM) have limited sensitivity. Here, we explored SHP-1 promoter 2 methylation (SHP1P2)-an epithelial-specific methylation marker previously proven as risk stratification and potential diagnostic marker in non-small cell lung cancer-for EDM with LM. We prospectively recruited 136 patients who were diagnosed EDM with LM (n = 25), EDM without LM (n = 14), non-EDM with LM (n = 8), and benign meningeal diseases (n = 89). The primary cancer sites for EDM with LM were lung (n = 17), breast (n = 5), and colon (n = 3). We performed quantitative analyses of cell-free (cfSHP1P2) and whole fraction (wSHP1P2) from cerebrospinal fluid (CSF); results were correlated with the clinicopathological data, including CSF cytology. Median cfSHP1P2 and wSHP1P2 were 3.08 [range: 0-163.5] and 9.35 [0.69-91.63] ng/ml, respectively, in EDM with LM; 0 [0-0.08] and 0.23 [0-7.84] ng/ml in EDM without LM; and were undetectable in most cases of benign meningeal diseases and non-EDM with LM. The cut-off values of 0.22 ng/ml for methylated cfSHP1P2 and 0.59 ng/ml for wSHP1P2 were the best to discriminate EDM with LM from EDM without LM (sensitivity: 79-100 %; specificity: 83-100 %), as well as from other benign conditions (sensitivity: 85-100 % specificity: 78-100 %). CSF cytology yielded 76 % sensitivity for diagnosing EDM with LM. Further validation of CSF SHP1P2 methylation detection as a role of adjunctive tool for LM from EDM should be interested based on our study.

  5. SHP-1 promoter 2 methylation in cerebrospinal fluid for diagnosis of leptomeningeal epithelial-derived malignancy (carcinomatous meningitis).

    PubMed

    Vinayanuwattikun, Chanida; Mingmalairak, Siyamol; Jittapiromsak, Nutchawan; Thaipisuttikul, Iyavut; Sriuranpong, Virote; Mutirangura, Apiwat; Shuangshoti, Shanop

    2016-09-01

    Current diagnostic methods for leptomeningeal metastasis (LM) from epithelial-derived malignancy (EDM) have limited sensitivity. Here, we explored SHP-1 promoter 2 methylation (SHP1P2)-an epithelial-specific methylation marker previously proven as risk stratification and potential diagnostic marker in non-small cell lung cancer-for EDM with LM. We prospectively recruited 136 patients who were diagnosed EDM with LM (n = 25), EDM without LM (n = 14), non-EDM with LM (n = 8), and benign meningeal diseases (n = 89). The primary cancer sites for EDM with LM were lung (n = 17), breast (n = 5), and colon (n = 3). We performed quantitative analyses of cell-free (cfSHP1P2) and whole fraction (wSHP1P2) from cerebrospinal fluid (CSF); results were correlated with the clinicopathological data, including CSF cytology. Median cfSHP1P2 and wSHP1P2 were 3.08 [range: 0-163.5] and 9.35 [0.69-91.63] ng/ml, respectively, in EDM with LM; 0 [0-0.08] and 0.23 [0-7.84] ng/ml in EDM without LM; and were undetectable in most cases of benign meningeal diseases and non-EDM with LM. The cut-off values of 0.22 ng/ml for methylated cfSHP1P2 and 0.59 ng/ml for wSHP1P2 were the best to discriminate EDM with LM from EDM without LM (sensitivity: 79-100 %; specificity: 83-100 %), as well as from other benign conditions (sensitivity: 85-100 % specificity: 78-100 %). CSF cytology yielded 76 % sensitivity for diagnosing EDM with LM. Further validation of CSF SHP1P2 methylation detection as a role of adjunctive tool for LM from EDM should be interested based on our study. PMID:27401153

  6. SC-2001 Overcomes STAT3-mediated Sorafenib Resistance through RFX-1/SHP-1 Activation in Hepatocellular Carcinoma☆☆☆

    PubMed Central

    Su, Jung-Chen; Tseng, Ping-Hui; Wu, Szu-Hsien; Hsu, Cheng-Yi; Tai, Wei-Tien; Li, Yong-Shi; Chen, I-Ting; Liu, Chun-Yu; Chen, Kuen-Feng; Shiau, Chung-Wai

    2014-01-01

    Hepatocellular carcinoma is the fifth most common solid cancer worldwide. Sorafenib, a small multikinase inhibitor, is the only approved therapy for advanced HCC. The clinical benefit of sorafenib is offset by the acquisition of sorafenib resistance. Understanding of the molecular mechanism of STAT3 overexpression in sorafenib resistance is critical if the clinical benefits of this drug are to be improved. In this study, we explored our hypothesis that loss of RFX-1/SHP-1 and further increase of p-STAT3 as a result of sorafenib treatment induces sorafenib resistance as a cytoprotective response effect, thereby, limiting sorafenib sensitivity and efficiency. We found that knockdown of RFX-1 protected HCC cells against sorafenib-induced cell apoptosis and SHP-1 activity was required for the process. SC-2001, a molecule with similar structure to obatoclax, synergistically suppressed tumor growth when used in combination with sorafenib in vitro and overcame sorafenib resistance through up-regulating RFX-1 and SHP-1 resulting in tumor suppression and mediation of dephosphorylation of STAT3. In addition, sustained sorafenib treatment in HCC led to increased p-STAT3 which was a key mediator of sorafenib sensitivity. The combination of SC-2001 and sorafenib strongly inhibited tumor growth in both wild-type and sorafenib-resistant HCC cell bearing xenograft models. These results demonstrate that inactivation of RFX/SHP-1 induced by sustained sorafenib treatment confers sorafenib resistance to HCC through p-STAT3 up-regulation. These effects can be overcome by SC-2001 through RFX-1/SHP-1 dependent p-STAT3 suppression. In conclusion, the use of SC-2001 in combination with sorafenib may constitute a new strategy for HCC therapy. PMID:25047655

  7. SC-2001 overcomes STAT3-mediated sorafenib resistance through RFX-1/SHP-1 activation in hepatocellular carcinoma.

    PubMed

    Su, Jung-Chen; Tseng, Ping-Hui; Wu, Szu-Hsien; Hsu, Cheng-Yi; Tai, Wei-Tien; Li, Yong-Shi; Chen, I-Ting; Liu, Chun-Yu; Chen, Kuen-Feng; Shiau, Chung-Wai

    2014-07-01

    Hepatocellular carcinoma is the fifth most common solid cancer worldwide. Sorafenib, a small multikinase inhibitor, is the only approved therapy for advanced HCC. The clinical benefit of sorafenib is offset by the acquisition of sorafenib resistance. Understanding of the molecular mechanism of STAT3 overexpression in sorafenib resistance is critical if the clinical benefits of this drug are to be improved. In this study, we explored our hypothesis that loss of RFX-1/SHP-1 and further increase of p-STAT3 as a result of sorafenib treatment induces sorafenib resistance as a cytoprotective response effect, thereby, limiting sorafenib sensitivity and efficiency. We found that knockdown of RFX-1 protected HCC cells against sorafenib-induced cell apoptosis and SHP-1 activity was required for the process. SC-2001, a molecule with similar structure to obatoclax, synergistically suppressed tumor growth when used in combination with sorafenib in vitro and overcame sorafenib resistance through up-regulating RFX-1 and SHP-1 resulting in tumor suppression and mediation of dephosphorylation of STAT3. In addition, sustained sorafenib treatment in HCC led to increased p-STAT3 which was a key mediator of sorafenib sensitivity. The combination of SC-2001 and sorafenib strongly inhibited tumor growth in both wild-type and sorafenib-resistant HCC cell bearing xenograft models. These results demonstrate that inactivation of RFX/SHP-1 induced by sustained sorafenib treatment confers sorafenib resistance to HCC through p-STAT3 up-regulation. These effects can be overcome by SC-2001 through RFX-1/SHP-1 dependent p-STAT3 suppression. In conclusion, the use of SC-2001 in combination with sorafenib may constitute a new strategy for HCC therapy. PMID:25047655

  8. The MADS Box Genes ABS, SHP1, and SHP2 Are Essential for the Coordination of Cell Divisions in Ovule and Seed Coat Development and for Endosperm Formation in Arabidopsis thaliana

    PubMed Central

    Tekleyohans, Dawit G.; Wittkop, Benjamin; Snowdon, Rod J.

    2016-01-01

    Seed formation is a pivotal process in plant reproduction and dispersal. It begins with megagametophyte development in the ovule, followed by fertilization and subsequently coordinated development of embryo, endosperm, and maternal seed coat. Two closely related MADS-box genes, SHATTERPROOF 1 and 2 (SHP1 and SHP2) are involved in specifying ovule integument identity in Arabidopsis thaliana. The MADS box gene ARABIDOPSIS BSISTER (ABS or TT16) is required, together with SEEDSTICK (STK) for the formation of endothelium, part of the seed coat and innermost tissue layer formed by the maternal plant. Little is known about the genetic interaction of SHP1 and SHP2 with ABS and the coordination of endosperm and seed coat development. In this work, mutant and expression analysis shed light on this aspect of concerted development. Triple tt16 shp1 shp2 mutants produce malformed seedlings, seed coat formation defects, fewer seeds, and mucilage reduction. While shp1 shp2 mutants fail to coordinate the timely development of ovules, tt16 mutants show less peripheral endosperm after fertilization. Failure in coordinated division of the innermost integument layer in early ovule stages leads to inner seed coat defects in tt16 and tt16 shp1 shp2 triple mutant seeds. An antagonistic action of ABS and SHP1/SHP2 is observed in inner seed coat layer formation. Expression analysis also indicates that ABS represses SHP1, SHP2, and FRUITFUL expression. Our work shows that the evolutionary conserved Bsister genes are required not only for endothelium but also for endosperm development and genetically interact with SHP1 and SHP2 in a partially antagonistic manner. PMID:27776173

  9. Specific dephosphorylation of Janus Kinase 2 by protein tyrosine phosphatases.

    PubMed

    Li, Jianzhuo; Liu, Xidong; Chu, Huiying; Fu, Xueqi; Li, Tianbao; Hu, Lianghai; Xing, Shu; Li, Guohui; Gu, Jingkai; Zhao, Zhizhuang Joe

    2015-01-01

    Many protein kinases are activated through phosphorylation of an activation loop thereby turning on downstream signaling pathways. Activation of JAK2, a nonreceptor tyrosine kinase with an important role in growth factor and cytokine signaling, requires phosphorylation of the 1007 and 1008 tyrosyl residues. Dephosphorylation of these two sites by phosphatases presumably inactivates the enzyme, but the underlying mechanism is not known. In this study, we employed MALDI-TOF/TOF and triple quadrupole mass spectrometers to analyze qualitatively and quantitatively the dephosphorylation process by using synthetic peptides derived from the tandem autophosphorylation sites (Y1007 and Y1008) of human JAK2. We found that tyrosine phosphatases catalyzed the dephosphorylation reaction sequentially, but different enzymes exhibited different selectivity. Protein tyrosine phosphatase 1B caused rapid dephosphorylation of Y1008 followed by Y1007, while SHP1 and SHP2 selectively dephosphorylated Y1008 only, and yet HePTP randomly removed a single phosphate from either Y1007 or Y1008, leaving behind mono-phosphorylated peptides. The specificity of dephosphorylation was further confirmed by molecular modeling. The data reveal multiple modes of JAK2 regulation by tyrosine phosphatases, reflecting a complex, and intricate interplay between protein phosphorylation and dephosphorylation.

  10. ALP (Alkaline Phosphatase) Test

    MedlinePlus

    ... known as: ALK PHOS; Alkp Formal name: Alkaline Phosphatase Related tests: AST ; ALT ; GGT ; Bilirubin ; Liver Panel ; Bone Markers ; Alkaline Phosphatase Isoenzymes; Bone Specific ALP All content on Lab ...

  11. Apoptosis Induced by Tanshinone IIA and Cryptotanshinone Is Mediated by Distinct JAK/STAT3/5 and SHP1/2 Signaling in Chronic Myeloid Leukemia K562 Cells

    PubMed Central

    Jung, Ji Hoon; Kwon, Tae-Rin; Jeong, Soo-Jin; Kim, Eun-Ok; Sohn, Eun Jung; Yun, Miyong; Kim, Sung-Hoon

    2013-01-01

    Though tanshinone IIA and cryptotanshinone possess a variety of biological effects such as anti-inflammatory, antioxidative, antimetabolic, and anticancer effects, the precise molecular targets or pathways responsible for anticancer activities of tanshinone IIA and cryptotanshinone in chronic myeloid leukemia (CML) still remain unclear. In the present study, we investigated the effect of tanshinone IIA and cryptotanshinone on the Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling during apoptotic process. We found that both tanshinone IIA and cryptotanshinone induced apoptosis by activation of caspase-9/3 and Sub-G1 accumulation in K562 cells. However, they have the distinct JAK/STAT pathway, in which tanshinone IIA inhibits JAK2/STAT5 signaling, whereas cryptotanshinone targets the JAK2/STAT3. In addition, tanshinone IIA enhanced the expression of both SHP-1 and -2, while cryptotanshinone regulated the expression of only SHP-1. Both tanshinone IIA and cryptotanshinone attenuated the expression of bcl-xL, survivin, and cyclin D1. Furthermore, tanshinone IIA augmented synergy with imatinib, a CML chemotherapeutic drug, better than cryptotanshinone in K562 cells. Overall, our findings suggest that the anticancer activity of tanshinone IIA and cryptotanshinone is mediated by the distinct the JAK/STAT3/5 and SHP1/2 signaling, and tanshinone IIA has the potential for combination therapy with imatinib in K562 CML cells. PMID:23878608

  12. Inhibition of dual-specificity phosphatase 26 by ethyl-3,4-dephostatin: Ethyl-3,4-dephostatin as a multiphosphatase inhibitor.

    PubMed

    Seo, Huiyun; Cho, Sayeon

    2016-04-01

    Protein tyrosine phosphatases (PTPs) regulate protein function by dephosphorylating phosphorylated proteins in many signaling cascades and some of them have been targets for drug development against many human diseases. There have been many reports that some chemical inhibitors could regulate particular phosphatases. However, there was no extensive study on specificity of inhibitors towardss phosphatases. We investigated the effects of ethyl-3,4-dephostatin, a potent inhibitor of five PTPs including PTP-1B and Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), on thirteen other PTPs using in vitro phosphatase assays. Of them, dual-specificity protein phosphatase 26 (DUSP26), which inhibits mitogen-activated protein kinase (MAPK) and p53 tumor suppressor and is known to be overexpressed in anaplastic thyroid carcinoma, was inhibited by ethyl-3,4-dephostatin in a concentration-dependent manner. Kinetic studies with ethyl-3,4-dephostatin and DUSP26 revealed competitive inhibition, suggesting that ethyl-3,4-dephostatin binds to the catalytic site of DUSP26 like other substrate PTPs. Moreover, ethyl-3,4-dephostatin protects DUSP26-mediated dephosphorylation of p38, a member of the MAPK family, and p53. Taken together, these results suggest that ethyl-3,4-dephostatin functions as a multiphosphatase inhibitor and is useful as a therapeutic agent for cancers overexpressing DUSP26. PMID:27209699

  13. Identification of New Substrates of the Protein-tyrosine Phosphatase PTP1B by Bayesian Integration of Proteome Evidence*

    PubMed Central

    Ferrari, Emanuela; Tinti, Michele; Costa, Stefano; Corallino, Salvatore; Nardozza, Aurelio Pio; Chatraryamontri, Andrew; Ceol, Arnaud; Cesareni, Gianni; Castagnoli, Luisa

    2011-01-01

    There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and “closeness” in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation. PMID:21123182

  14. Programmed cell death receptor ligand 1 modulates the regulatory T cells' capacity to repress shock/sepsis-induced indirect acute lung injury by recruiting phosphatase SRC homology region 2 domain-containing phosphatase 1.

    PubMed

    Tang, Lunxian; Bai, Jianwen; Chung, Chun-Shiang; Lomas-Neira, Joanne; Chen, Yaping; Huang, Xin; Ayala, Alfred

    2015-01-01

    We recently reported that adoptively transferred (AT) exogenous CD4+ CD25+ regulatory T cells (Tregs) to wild-type (WT) mice can directly act to repress shock/sepsis-induced experimental indirect acute lung injury (iALI), and this is mediated in part by programmed cell death receptor 1 (PD-1). In this study, we further determine whether recipient mouse lacking PD-L1, one of the primary ligands for PD-1, contributes to the manipulation of the Tregs' capacity to repress lung injury. To do this, Tregs isolated from the spleen of WT mice were AT into PD-L1 mice subjected to hemorrhagic shock and subsequent to cecal ligation and puncture to induce iALI. Samples were collected for analyses 24 h after cecal ligation and puncture. We found that in PD-L1-recipient mice, AT WT-Tregs lost the ability to reverse the development of iALI seen in WT recipient mice (i.e., no reduction of lung injury indices assessed by histology and vascular leakage, failure to decrease the lung neutrophil influx [myeloperoxidase activity], or the rise in lung apoptosis [caspase 3 activity]). Also, a significant increase in interleukin 1β (IL-1β) and keratinocyte-derived chemokine, but no changes in IL-6, IL-10, and IL-17A levels in lung tissues were seen in these mice compared with iALI mice without AT of Tregs. Furthermore, we noted that the lung tissue tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1), but not SHP-2, was activated with the AT of Tregs in PD-L1(-/-) iALI mice. Finally, through local depletion of CD4+ T cells or CD25+ (Tregs) in the lung, prior to inducing iALI, we found that SHP-1 activation was associated with the loss of Tregs' protective effects in vivo. Collectively, our data reveal that PD-L1 is a critical modulator of Tregs' ability to suppress iALI, and this appears to involve SHP-1 activation.

  15. TGF-β1 re-programs TLR4 signaling in L. donovani infection: enhancement of SHP-1 and ubiquitin-editing enzyme A20.

    PubMed

    Das, Sushmita; Pandey, Krishna; Kumar, Ashish; Sardar, Abul H; Purkait, Bidyut; Kumar, Manish; Kumar, Sudeep; Ravidas, Vidya N; Roy, Syamal; Singh, Dharmendra; Das, Pradeep

    2012-07-01

    Visceral leishmaniasis (VL), caused by Leishmania donovani, is a major health concern in India. It represents T-helper type 2 (Th2) bias of cytokines in active state and Th1 bias at cure. However, the role of the parasite in regulating Toll-like receptor (TLR)-mediated macrophage activation in VL patients remains elusive. In this report, we demonstrated that later stages of L. donovani infection rendered tolerance to macrophages, leading to incapability for the production of inflammatory cytokines like tumor necrosis factor (TNF)-α and interleukin (IL)-1β in response to TLR stimulation. Overexpression of transforming growth factor (TGF)-β(1), but not IL-10, resulted in suppressed lipopolysaccharide (LPS)-induced production of TNF-α and downregulation of TLR4 expression in L. donovani-infected macrophages. Recombinant human (rh)TGF-β(1) markedly enhanced tyrosine phosphatase (Src homology region 2 domain-containing phosphatase-1) activity, but inhibited IL-1 receptor-activated kinase (IRAK)-1 activation. Addition of neutralizing TGF-β(1) antibody reversed these effects, and thus suggesting the pivotal role of TGF-β(1) in promoting refractoriness for LPS in macrophages. Surprisingly, the use of a tyrosine phosphatase inhibitor (sodium orthovanadate, Na(3)VO(4)) promoted IRAK-1 activation, confirming the negative inhibitory role of tyrosine phosphatase in macrophage activation. Furthermore, rhTGF-β(1) induced tolerance in infected macrophages by reducing inhibitory protein (IκBα) degradation in a time-dependent manner. In addition, short interfering RNA studies proved that overexpression of A20 ubiquitin-editing protein complex induced inhibitory activity of TGF-β(1) on LPS-mediated nuclear factor-κB activation. Thus, these findings suggest that TGF-β(1) promotes overexpression of A20 through tyrosine phosphatase activity that ensures transient activation of inflammatory signaling pathways in macrophages in active L. donovani infection.

  16. [Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.

    PubMed Central

    Desmarais, S; Friesen, R W; Zamboni, R; Ramachandran, C

    1999-01-01

    Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs. PMID:9882618

  17. Regulation of synthase phosphatase and phosphorylase phosphatase in rat liver.

    PubMed

    Tan, A W; Nuttall, F Q

    1976-08-12

    Using substrates purified from liver, the apparent Km values of synthase phosphatase ([UDPglucose--glycogen glucosyltransferase-D]phosphohydrolase, EC 3.1.3.42) and phosphorylase phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17) were found to be 0.7 and 60 units/ml respectively. The maximal velocity of phosphorylase phosphatase was more than a 100 times that of synthase phosphatase. In adrenalectomized, fasted animals there was a complete loss of synthase phosphatase but only a slight decrease in phosphorylase phosphatase when activity was measured using endogenous substrates in a concentrated liver extract. When assayed under optimal conditions with purified substrates, both activities were present but had decreased to very low levels. Mixing experiments indicated that synthase D present in the extract of adrenalectomized fasted animals was altered such that it was no longer a substrate for synthase phosphatase from normal rats. Phosphorylase a substrate on the other hand was unaltered and readily converted. When glucose was given in vivo, no change in percent of synthase in the I form was seen in adrenalectomized rats but the percent of phosphorylase in the a form was reduced. Precipitation of protein from an extract of normal fed rats with ethanol produced a large activation of phosphorylase phosphatase activity with no corresponding increase in synthase phosphatase activity. Despite the low phosphorylase phosphatase present in extracts of adrenalectomized fasted animals, ethanol precipitation increased activity to the same high level as obtained in the normal fed rats. Synthase phosphatase and phosphorylase phosphatase activities were also decreased in normal fasted, diabetic fed and fasted, and adrenalectomized fed rats. Both enzymes recovered in the same manner temporally after oral glucose administration to adrenalectomized, fasted rats. These results suggest an integrated regulatory mechanism for the two phosphatase.

  18. Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases.

    PubMed

    Selner, Nicholas G; Luechapanichkul, Rinrada; Chen, Xianwen; Neel, Benjamin G; Zhang, Zhong-Yin; Knapp, Stefan; Bell, Charles E; Pei, Dehua

    2014-01-21

    The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic

  19. Ternary oxovanadium(IV) complexes of ONO-donor Schiff base and polypyridyl derivatives as protein tyrosine phosphatase inhibitors: synthesis, characterization, and biological activities.

    PubMed

    Yuan, Caixia; Lu, Liping; Gao, Xiaoli; Wu, Yanbo; Guo, Maolin; Li, Ying; Fu, Xueqi; Zhu, Miaoli

    2009-08-01

    A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic acid (SAA)], and a bidentate NN ligand [viz., 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2',3'-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry, UV-vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of both complexes, [V(IV)O(SAA)(bpy)].0.25bpy and [V(IV)O(SAA)(phen)].0.33H(2)O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO(3)N(3)). The oxidation state of V(IV) with d(1) configuration was confirmed by EPR spectroscopy. The speciation of VO-SAA-bpy in aqueous solution was investigated by potentiomtreic pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0-7.4, and one is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine phosphatase 1B (PTP1B) (IC(50) approximately 30-61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly, the [V(IV)O(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents. PMID:19290551

  20. Stimulation of the alveolar macrophage respiratory burst by ADP causes selective glutathionylation of protein tyrosine phosphatase 1B.

    PubMed

    Rinna, Alessandra; Torres, Martine; Forman, Henry Jay

    2006-07-01

    H(2)O(2) produced by stimulation of the macrophage NADPH oxidase is involved both in bacterial killing and as a second messenger in these cells. Protein tyrosine phosphatases (PTPs) are targets for H(2)O(2) signaling through oxidation of their catalytic cysteine, resulting in inhibition of their activity. Here, we show that, in the rat alveolar macrophage NR8383 cell line, H(2)O(2) produced through the ADP-stimulated respiratory burst induces the formation of a disulfide bond between PTP1B and GSH that was detectable with an antibody to glutathione-protein complexes and was reversed by DTT addition. PTP1B glutathionylation was dependent on H(2)O(2) as the presence of catalase at the time of ADP stimulation inhibited the formation of the conjugate. Interestingly, other PTPs, i.e., SHP-1 and SHP-2, did not undergo glutathionylation in response to ADP stimulation of the respiratory burst, although glutathionylation of these proteins could be shown by reaction with 25 mM glutathione disulfide in vitro. While previous studies have suggested the reversible oxidation of PTP1B during signaling or showed PTP1B glutathionylation in vitro, the present study directly demonstrates that physiological stimulation of H(2)O(2) production results in PTP1B glutathionylation in intact cells, which may affect downstream signaling.

  1. Structural Genomics of Protein Phosphatases

    SciTech Connect

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  2. Glucose-6-phosphatase deficiency

    PubMed Central

    2011-01-01

    Glucose-6-phosphatase deficiency (G6P deficiency), or glycogen storage disease type I (GSDI), is a group of inherited metabolic diseases, including types Ia and Ib, characterized by poor tolerance to fasting, growth retardation and hepatomegaly resulting from accumulation of glycogen and fat in the liver. Prevalence is unknown and annual incidence is around 1/100,000 births. GSDIa is the more frequent type, representing about 80% of GSDI patients. The disease commonly manifests, between the ages of 3 to 4 months by symptoms of hypoglycemia (tremors, seizures, cyanosis, apnea). Patients have poor tolerance to fasting, marked hepatomegaly, growth retardation (small stature and delayed puberty), generally improved by an appropriate diet, osteopenia and sometimes osteoporosis, full-cheeked round face, enlarged kydneys and platelet dysfunctions leading to frequent epistaxis. In addition, in GSDIb, neutropenia and neutrophil dysfunction are responsible for tendency towards infections, relapsing aphtous gingivostomatitis, and inflammatory bowel disease. Late complications are hepatic (adenomas with rare but possible transformation into hepatocarcinoma) and renal (glomerular hyperfiltration leading to proteinuria and sometimes to renal insufficiency). GSDI is caused by a dysfunction in the G6P system, a key step in the regulation of glycemia. The deficit concerns the catalytic subunit G6P-alpha (type Ia) which is restricted to expression in the liver, kidney and intestine, or the ubiquitously expressed G6P transporter (type Ib). Mutations in the genes G6PC (17q21) and SLC37A4 (11q23) respectively cause GSDIa and Ib. Many mutations have been identified in both genes,. Transmission is autosomal recessive. Diagnosis is based on clinical presentation, on abnormal basal values and absence of hyperglycemic response to glucagon. It can be confirmed by demonstrating a deficient activity of a G6P system component in a liver biopsy. To date, the diagnosis is most commonly confirmed

  3. The protein tyrosine phosphatase PTPN4/PTP-MEG1, an enzyme capable of dephosphorylating the TCR ITAMs and regulating NF-κB, is dispensable for T cell development and/or T cell effector functions

    PubMed Central

    Young, Jennifer A.; Becker, Amy M.; Medeiros, Jennifer J.; Shapiro, Virginia S.; Wang, Andrew; Farrar, J. David; Quill, Timothy A.; van Huijsduijnen, Rob Hooft; van Oers, Nicolai S.C.

    2008-01-01

    T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR ζ subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-κB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogeneswere equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations. PMID:18614237

  4. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  5. [Phosphoprotein phosphatase nonspecifically hydrolyzes CoA].

    PubMed

    Reziapkin, V I; Moiseenok, A G

    1988-01-01

    CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate). PMID:2849829

  6. Protein phosphatases in pancreatic islets

    PubMed Central

    Ortsäter, Henrik; Grankvist, Nina; Honkanen, Richard E.; Sjöholm1, Åke

    2014-01-01

    The prevalence of diabetes is increasing rapidly world-wide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. However, far less is known about the role and regulation of protein dephosphorylation by various protein phosphatases. Herein we review extant data implicating serine/threonine and tyrosine phosphatases in various aspects of healthy and diabetic islet biology, ranging from control of hormonal stimulus-secretion coupling to mitogenesis and apoptosis. PMID:24681827

  7. Phosphoinositide Phosphatases in Cell Biology and Disease

    PubMed Central

    Liu, Yang; Bankaitis, Vytas A.

    2010-01-01

    Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

  8. Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle.

    PubMed Central

    Cohen, P; Nimmo, G A; Antoniw, J F

    1977-01-01

    A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian

  9. TPIP: a novel phosphoinositide 3-phosphatase.

    PubMed Central

    Walker, S M; Downes, C P; Leslie, N R

    2001-01-01

    The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor is a phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] 3-phosphatase that plays a critical role in regulating many cellular processes by antagonizing the phosphoinositide 3-kinase signalling pathway. We have identified and characterized two human homologues of PTEN, which differ with respect to their subcellular localization and lipid phosphatase activities. The previously cloned, but uncharacterized, TPTE (transmembrane phosphatase with tensin homology) is localized to the plasma membrane, but lacks detectable phosphoinositide 3-phosphatase activity. TPIP (TPTE and PTEN homologous inositol lipid phosphatase) is a novel phosphatase that occurs in several differentially spliced forms of which two, TPIP alpha and TPIP beta, appear to be functionally distinct. TPIP alpha displays similar phosphoinositide 3-phosphatase activity compared with PTEN against PtdIns(3,4,5)P(3), PtdIns(3,5)P(2), PtdIns(3,4)P(2) and PtdIns(3)P, has N-terminal transmembrane domains and appears to be localized on the endoplasmic reticulum. This is unusual as most signalling-lipid-metabolizing enzymes are not integral membrane proteins. TPIP beta, however, lacks detectable phosphatase activity and is cytosolic. TPIP has a wider tissue distribution than the testis-specific TPTE, with specific splice variants being expressed in testis, brain and stomach. TPTE and TPIP do not appear to be functional orthologues of the Golgi-localized and more distantly related murine PTEN2. We suggest that TPIP alpha plays a role in regulating phosphoinositide signalling on the endoplasmic reticulum, and might also represent a tumour suppressor and functional homologue of PTEN in some tissues. PMID:11716755

  10. Ratiometric electrochemical detection of alkaline phosphatase.

    PubMed

    Goggins, Sean; Naz, Christophe; Marsh, Barrie J; Frost, Christopher G

    2015-01-11

    A novel ferrocene-derived substrate for the ratiometric electrochemical detection of alkaline phosphatase (ALP) was designed and synthesised. It was demonstrated to be an excellent electrochemical substrate for the ALP-labelled enzyme-linked immunosorbent assay (ELISA).

  11. Multiple Functions of the Eya Phosphotyrosine Phosphatase

    PubMed Central

    2015-01-01

    Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling. PMID:26667035

  12. Assessing the Biological Activity of the Glucan Phosphatase Laforin.

    PubMed

    Romá-Mateo, Carlos; Raththagala, Madushi; Gentry, Mathew S; Sanz, Pascual

    2016-01-01

    Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity. PMID:27514803

  13. Bacterial-like PPP protein phosphatases

    PubMed Central

    Kerk, David; Uhrig, R Glen; Moorhead, Greg B

    2013-01-01

    Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, “bacterial-like” enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the “eukaryotic-like” phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research. PMID:24675170

  14. Acid phosphatase/phosphotransferases from enteric bacteria.

    PubMed

    Mihara, Y; Utagawa, T; Yamada, H; Asano, Y

    2001-01-01

    We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.

  15. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    SciTech Connect

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

  16. Inhibition of sucrose phosphatase by sucrose

    PubMed Central

    Hawker, J. S.

    1967-01-01

    1. Partially purified sucrose phosphatase from immature stem tissue of sugarcane is inhibited by sucrose. The enzyme was also inhibited by maltose, melezitose and 6-kestose but not by eight other sugars, including glucose and fructose. 2. The relative effectiveness of sucrose, maltose and melezitose as inhibitors is different for sucrose phosphatase from different plants. 3. The inhibition of the sugar-cane enzyme by sucrose was shown to be partially competitive. The Ki for sucrose is about 10mm. 4. Melezitose is also a partially competitive inhibitor of the enzyme but the inhibition by maltose is probably mixed. 5. The possibility that sucrose controls both the rate of accumulation of sucrose in stems of sugar-cane and sucrose synthesis in leaves by inhibiting sucrose phosphatase is discussed. PMID:4291490

  17. A specific sucrose phosphatase from plant tissues

    PubMed Central

    Hawker, J. S.; Hatch, M. D.

    1966-01-01

    1. A phosphatase that hydrolyses sucrose phosphate (phosphorylated at the 6-position of fructose) was isolated from sugar-cane stem and carrot roots. With partially purified preparations fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, glucose 1-phosphate and fructose 1,6-diphosphate are hydrolysed at between 0 and 2% of the rate for sucrose phosphate. 2. The activity of the enzyme is increased fourfold by the addition of Mg2+ ions and inhibited by EDTA, fluoride, inorganic phosphate, pyrophosphate, Ca2+ and Mn2+ ions. Sucrose (50mm) reduces activity by 60%. 3. The enzyme exhibits maximum activity between pH6·4 and 6·7. The Michaelis constant for sucrose phosphate is between 0·13 and 0·17mm. 4. At least some of the specific phosphatase is associated with particles having the sedimentation properties of mitochondria. 5. A similar phosphatase appears to be present in several other plant species. PMID:4290548

  18. Light availability may control extracellular phosphatase production in turbid environments.

    PubMed

    Rychtecký, Pavel; Řeháková, Klára; Kozlíková, Eliška; Vrba, Jaroslav

    2015-01-01

    Extracellular phosphatase production by phytoplankton was investigated in the moderately eutrophic Lipno reservoir, Czech Republic during 2009 and 2010. We hypothesized that production of extracellular phosphatases is an additional mechanism of phosphorus acquisition enabling producers to survive rather than to dominate the phytoplankton. Hence, we examined the relationship between light availability and phosphatase production, as light plays an important role in polymictic environments. Bulk phosphatase activity was measured using a common fluorometric assay, and the production of phosphatases was studied using the Fluorescently Labelled Enzyme Activity technique, which enabled direct microscopic detection of phosphatase-positive cells. In total, 29 taxa of phytoplankton were identified during both years. Only 17 taxa from the total number of 29 showed production of extracellular phosphatases. Species dominating the phytoplankton rarely produced extracellular phosphatases. In contrast, taxa exhibiting phosphatase activity were present in low biomass in the phytoplankton assemblage. Moreover, there was a significant relationship between the proportion of phosphatase positive species in samples and the Z(eu):Z(mix) ratio (a proxy of light availability). A laboratory experiment with different light intensities confirmed the influence of light on production of phosphatases. Our seasonal study confirmed that extracellular phosphatase production is common in low-abundance populations but not in dominant taxa of the phytoplankton. It also suggested the importance of sufficient light conditions for the production of extracellular phosphatases.

  19. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  20. Phosphatase activities as biosignatures of extant life

    NASA Astrophysics Data System (ADS)

    Kobayashi, K.; Itoh, Y.; Edazawa, Y.; Moroi, A.; Takano, Y.

    It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere high temperature hot springs and stratosphere Possible extraterrestrial biospheres in Mars Europa and Titan are being discussed Many biosignatures or biomarkers have been proposed to detect microbial activities in such extreme environments Phosphate esters are essential for the terrestrial life since they are constituents of nucleic acids and cell mebranes Thus all the terrestrial organisms have phosphatases that are enzymes catalyzing hydrolysis of phosphate esters We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and discussed whether they can be used as biosignatures for extant life Core samples and chimney samples were collected at the Suiyo Seamount Izu-Bonin Arc the Pacific Ocean in 2001 and 2002 and in South Mariana hydrothermal systems the Pacific Oceanas in 2003 both in a part of the Archaean Park Project Phosphatase activity in solid rock samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate pH 8 0 or pH 6 5 as a substrate as follows Pulverized samples were incuvated with substrate solution for an hour and then production rate of p-nitrophenol was calculated with absorbance at 410 nm Phosphatase activity in extracts was measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate Concentration of amino acids and their enantiomeric ratio were determined by HPLC after HF digestion of the

  1. Atomic structure of dual-specificity phosphatase 26, a novel p53 phosphatase.

    PubMed

    Lokareddy, Ravi Kumar; Bhardwaj, Anshul; Cingolani, Gino

    2013-02-01

    Regulation of p53 phosphorylation is critical to control its stability and biological activity. Dual-specificity phosphatase 26 (DUSP26) is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-Ser20 and phospho-Ser37 in the p53 transactivation domain. In this paper, we report the 1.68 Å crystal structure of a catalytically inactive mutant (Cys152Ser) of DUSP26 lacking the first 60 N-terminal residues (ΔN60-C/S-DUSP26). This structure reveals the architecture of a dual-specificity phosphatase domain related in structure to Vaccinia virus VH1. DUSP26 adopts a closed conformation of the protein tyrosine phosphatase (PTP)-binding loop, which results in an unusually shallow active site pocket and buried catalytic cysteine. A water molecule trapped inside the PTP-binding loop makes close contacts both with main chain and with side chain atoms. The hydrodynamic radius (R(H)) of ΔN60-C/S-DUSP26 measured from velocity sedimentation analysis (R(H) ∼ 22.7 Å) and gel filtration chromatography (R(H) ∼ 21.0 Å) is consistent with an ∼18 kDa globular monomeric protein. Instead in crystal, ΔN60-C/S-DUSP26 is more elongated (R(H) ∼ 37.9 Å), likely because of the extended conformation of C-terminal helix α9, which swings away from the phosphatase core to generate a highly basic surface. As in the case of phosphatase MKP-4, we propose that a substrate-induced conformational change, possibly involving rearrangement of helix α9 with respect to the phosphatase core, allows DUSP26 to adopt a catalytically active conformation. The structural characterization of DUSP26 presented in this paper provides the first atomic insight into this disease-associated phosphatase.

  2. The microsomal glucose-6-phosphatase enzyme of pancreatic islets.

    PubMed Central

    Waddell, I D; Burchell, A

    1988-01-01

    Microsomal fractions isolated from pancreatic islet cells were shown to contain high specific glucose-6-phosphatase activity. The islet-cell glucose-6-phosphatase enzyme has the same Mr (36,500), similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme. Images Fig. 1. Fig. 2. PMID:2849415

  3. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  4. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  5. Acid phosphatase production by recombinant Arxula adeninivorans.

    PubMed

    Minocha, Neha; Kaur, Parvinder; Satyanarayana, T; Kunze, G

    2007-08-01

    Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett-Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g(-1) DYB) and laboratory fermenter (18,465 U g(-1) DYB), respectively. PMID:17541580

  6. Regulatory Roles of MAPK Phosphatases in Cancer

    PubMed Central

    Low, Heng Boon

    2016-01-01

    The mitogen-activated protein kinases (MAPKs) are key regulators of cell growth and survival in physiological and pathological processes. Aberrant MAPK signaling plays a critical role in the development and progression of human cancer, as well as in determining responses to cancer treatment. The MAPK phosphatases (MKPs), also known as dual-specificity phosphatases (DUSPs), are a family of proteins that function as major negative regulators of MAPK activities in mammalian cells. Studies using mice deficient in specific MKPs including MKP1/DUSP1, PAC-1/DUSP2, MKP2/DUSP4, MKP5/DUSP10 and MKP7/DUSP16 demonstrated that these molecules are important not only for both innate and adaptive immune responses, but also for metabolic homeostasis. In addition, the consequences of the gain or loss of function of the MKPs in normal and malignant tissues have highlighted the importance of these phosphatases in the pathogenesis of cancers. The involvement of the MKPs in resistance to cancer therapy has also gained prominence, making the MKPs a potential target for anti-cancer therapy. This review will summarize the current knowledge of the MKPs in cancer development, progression and treatment outcomes. PMID:27162525

  7. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  8. Two potential fish glycerol-3-phosphate phosphatases.

    PubMed

    Raymond, James A

    2015-06-01

    Winter-acclimated rainbow smelt (Osmerus mordax Mitchill) produce high levels of glycerol as an antifreeze. A common pathway to glycerol involves the enzyme glycerol-3-phosphate phosphatase (GPP), but no GPP has yet been identified in fish or any other animal. Here, two phosphatases assembled from existing EST libraries (from winter-acclimated smelt and cold-acclimated smelt hepatocytes) were found to resemble a glycerol-associated phosphatase from a glycerol-producing alga, Dunaliella salina, and a recently discovered GPP from a bacterium, Mycobacterium tuberculosis. Recombinant proteins were generated and were found to have GPP activity on the order of a few μMol Pi/mg enzyme/min. The two enzymes have acidic pH optima (~5.5) similar to that previously determined for GPP activity in liver tissue, with about 1/3 of their peak activities at neutral pH. The two enzymes appear to account for the GPP activity of smelt liver, but due to their reduced activities at neutral pH, their contributions to glycerol production in vivo remain unclear. Similar enzymes may be active in a glycerol-producing insect, Dendroctonus ponderosae.

  9. Acid Phosphatase Development during Ripening of Avocado.

    PubMed

    Sacher, J A

    1975-02-01

    The activity and subcellular distribution of acid phosphatase were assayed during ethylene-induced ripening of whole fruit or thick slices of avocado (Persea americana Mill. var. Fuerte and Hass). The activity increased up to 30-fold during ripening in both the supernatant fraction and the Triton X-100 extract of the precipitate of a 30,000g centrifugation of tissue homogenates from whole fruit or slices ripening in moist air. Enzyme activity in the residual precipitate after Triton extraction remained constant. The development of acid phosphatase in thick slices ripened in moist air was similar to that in intact fruit, except that enzyme development and ripening were accelerated about 24 hours in the slices. The increase in enzyme activity that occurs in slices ripening in moist air was inhibited when tissue sections were infiltrated with solutions, by aspiration for 2 minutes or by soaking for 2 hours, anytime 22 hours or more after addition of ethylene. This inhibition was independent of the presence or absence of cycloheximide or sucrose (0.3-0.5m). However, the large decline in enzyme activity in the presence of cycloheximide, as compared with the controls, indicated that synthesis of acid phosphatase was occurring at all stages of ripening.

  10. Role of Protein Tyrosine Phosphatases in Plants

    PubMed Central

    Shankar, Alka; Agrawal, Nisha; Sharma, Manisha; Pandey, Amita; Pandey, Girdhar K.

    2015-01-01

    Reversible protein phosphorylation is a crucial regulatory mechanism that controls many biological processes in eukaryotes. In plants, phosphorylation events primarily occur on serine (Ser) and threonine (Thr) residues, while in certain cases, it was also discovered on tyrosine (Tyr) residues. In contrary to plants, extensive reports on Tyr phosphorylation regulating a large numbers of biological processes exist in animals. Despite of such prodigious function in animals, Tyr phosphorylation is a least studied mechanism of protein regulation in plants. Recently, various chemical analytical procedures have strengthened the view that Tyr phosphorylation is equally prevalent in plants as in animals. However, regardless of Tyr phosphorylation events occuring in plants, no evidence could be found for the existence of gene encoding for Tyr phosphorylation i.e. the typical Tyr kinases. Various methodologies have suggested that plant responses to stress signals and developmental processes involved modifications in protein Tyr phosphorylation. Correspondingly, various reports have established the role of PTPs (Protein Tyrosine Phosphatases) in the dephosphorylation and inactivation of mitogen activated protein kinases (MAPKs) hence, in the regulation of MAPK signaling cascade. Besides this, many dual specificity protein phosphatases (DSPs) are also known to bind starch and regulate starch metabolism through reversible phosphorylation. Here, we are emphasizing the significant progress on protein Tyr phosphatases to understand the role of these enzymes in the regulation of post-translational modification in plant physiology and development. PMID:26962298

  11. Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

    PubMed Central

    Andreeva, Alexandra V; Kutuzov, Mikhail A

    2004-01-01

    Background In eukaryotes, PPP (protein phosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases. Results We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed Shewanella-like phosphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed ApaH-like phosphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I

  12. Digestion and the distribution of acid phosphatase in Blepharisma.

    PubMed

    Dembitzer, H M

    1968-05-01

    Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces.

  13. DIGESTION AND THE DISTRIBUTION OF ACID PHOSPHATASE IN BLEPHARISMA

    PubMed Central

    Dembitzer, Herbert M.

    1968-01-01

    Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces. PMID:4968524

  14. Carboxyarabinitol-1-P phosphatase of Phaseolus vulgaris

    SciTech Connect

    Kobza, J.; Moore, B.d.; Seemann, J.R. )

    1990-05-01

    The activity of carboxyarabinitol-1-P (CA1P) phosphatase was detected in clarified stromal extracts by the generation of {sup 14}C-carboxyarabinitol from {sup 14}C-CA1P. Carboxyribitol-1-P dependent activity was 3% of the CA1P dependent activity, indicating the enzyme was specific for CA1P. Inclusion of DTT in the assay was required for maximum velocity, but it appears that the enzyme is not regulated by thioredoxin in vivo. Activity o f the CA1P phosphatase was stimulated by RuBP, NADPH and FBP, though the latter two metabolites were required at nonphysiological concentrations in order to achieve significant stimulation. Contrary to a previous report on purified tobacco enzyme, ATP stimulated the CA1P phosphatase activity. In the presence of 1 mM RuBP or ATP, rates of 2 or 3 {mu}mol mg{sup {minus}1} Chl h{sup {minus}1}, respectively, were observed at 1 mM CA1P. These rates were 3-4 fold higher than the rate observed in the absence of effectors and are 2-4 times the in vivo rate of degradation of CA1P during dark/light transitions. The rates from bean were about 7 fold higher than rates reported for the enzyme from tobacco. Changes in the levels of ATP and RuBP associated with dark/light transitions could modulate the enzyme activity in vivo, but it remains to be established if this is the only mechanism for the required regulation of the enzyme.

  15. Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

    PubMed Central

    Gould, W. Douglas; Coleman, David C.; Rubink, Amy J.

    1979-01-01

    The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations. PMID:16345390

  16. Low serum alkaline phosphatase activity in Wilson's disease.

    PubMed

    Shaver, W A; Bhatt, H; Combes, B

    1986-01-01

    Low values for serum alkaline phosphatase activity were observed early in the course of two patients with Wilson's disease presenting with the combination of severe liver disease and Coombs' negative acute hemolytic anemia. A review of other cases of Wilson's disease revealed that 11 of 12 patients presenting with hemolytic anemia had values for serum alkaline phosphatase less than their respective sex- and age-adjusted mean values; in eight, serum alkaline phosphatase activity was less than the lower value for the normal range of the test. Low values for serum alkaline phosphatase were much less common in Wilson's disease patients with more chronic forms of presentation. Copper added in high concentration to serum in vitro did not have an important effect on serum alkaline phosphatase activity. The mechanism responsible for the decrease in serum alkaline phosphatase activity in patients is uncertain.

  17. Phosphatase specificity and pathway insulation in signaling networks.

    PubMed

    Rowland, Michael A; Harrison, Brian; Deeds, Eric J

    2015-02-17

    Phosphatases play an important role in cellular signaling networks by regulating the phosphorylation state of proteins. Phosphatases are classically considered to be promiscuous, acting on tens to hundreds of different substrates. We recently demonstrated that a shared phosphatase can couple the responses of two proteins to incoming signals, even if those two substrates are from otherwise isolated areas of the network. This finding raises a potential paradox: if phosphatases are indeed highly promiscuous, how do cells insulate themselves against unwanted crosstalk? Here, we use mathematical models to explore three possible insulation mechanisms. One approach involves evolving phosphatase KM values that are large enough to prevent saturation by the phosphatase's substrates. Although this is an effective method for generating isolation, the phosphatase becomes a highly inefficient enzyme, which prevents the system from achieving switch-like responses and can result in slow response kinetics. We also explore the idea that substrate degradation can serve as an effective phosphatase. Assuming that degradation is unsaturatable, this mechanism could insulate substrates from crosstalk, but it would also preclude ultrasensitive responses and would require very high substrate turnover to achieve rapid dephosphorylation kinetics. Finally, we show that adaptor subunits, such as those found on phosphatases like PP2A, can provide effective insulation against phosphatase crosstalk, but only if their binding to substrates is uncoupled from their binding to the catalytic core. Analysis of the interaction network of PP2A's adaptor domains reveals that although its adaptors may isolate subsets of targets from one another, there is still a strong potential for phosphatase crosstalk within those subsets. Understanding how phosphatase crosstalk and the insulation mechanisms described here impact the function and evolution of signaling networks represents a major challenge for

  18. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  19. Histochemical and electrophoretic studies on phosphatases of some Indian trematodes.

    PubMed

    Haque, M; Siddiqi, A H

    1982-06-01

    The isoenzymes of acid and alkaline phosphatases and their histochemical localization were studied by polyacrylamide disc gel electrophoresis in four species of trematodes: Gigantocotyle explanatum from the liver and Gastrothylax crumenifer from the rumen of water buffalo (Bubalus bubalis) and Echinostoma malayanum and Fasciolopsis buski from the small intestine of the pig (Sus scrofa). Both acid and alkaline phosphatases were present in the tegument, gastrodermis, suckers, testes, ovary, eggs, vitellaria and uterus but alkaline phosphatase activity was demonstrated only in the parenchyma and excretory ducts. Polyacrylamide gel electrophoresis revealed two to four isoenzymes for both acid and alkaline phosphatase.

  20. NUCLEOSIDE PHOSPHATASE ACTIVITIES IN RAT CARDIAC MUSCLE.

    PubMed

    ESSNER, E; NOVIKOFF, A B; QUINTANA, N

    1965-05-01

    Localizations of aldehyde-resistant nucleoside phosphatase activities in frozen sections of rat cardiac muscle have been studied by electron microscopy. Activities are higher after fixation with formaldehyde than with glutaraldehyde. After incubation with adenosine triphosphate or inosine diphosphate at pH 7.2, reaction product is found in the "terminal cisternae" or "transverse sacs" of the sarcoplasmic reticulum, which, together with the "intermediary vesicles" (T system), constitute the "dyads" or "triads". Reaction product is also present at the membranes of micropinocytotic vacuoles which apparently form from the plasma membrane of capillary endothelial cells and from the sarcolemma. In certain regions of the intercalated discs, reaction product is found within the narrow spaces between sarcolemmas of adjacent cells and within micropinocytotic vacuoles that seem to form from the sarcolemma. With inosine diphosphate, reaction product is also found in other parts of the sarcoplasmic reticulum. After incubation with cytidine monophosphate at pH 5, reaction product is present in the transverse sacs of sarcoplasmic reticulum, in micropinocytotic vacuoles in capillary endothelium, and in lysosomes of muscle fibers and capillaries. The possible significance of the sarcoplasmic reticulum phosphatases is discussed in relation to the role the reticulum probably plays in moving calcium ions and thereby controlling contraction and relaxation of the muscle fiber.

  1. Phosphatase Specificity and Pathway Insulation in Signaling Networks

    PubMed Central

    Rowland, Michael A.; Harrison, Brian; Deeds, Eric J.

    2015-01-01

    Phosphatases play an important role in cellular signaling networks by regulating the phosphorylation state of proteins. Phosphatases are classically considered to be promiscuous, acting on tens to hundreds of different substrates. We recently demonstrated that a shared phosphatase can couple the responses of two proteins to incoming signals, even if those two substrates are from otherwise isolated areas of the network. This finding raises a potential paradox: if phosphatases are indeed highly promiscuous, how do cells insulate themselves against unwanted crosstalk? Here, we use mathematical models to explore three possible insulation mechanisms. One approach involves evolving phosphatase KM values that are large enough to prevent saturation by the phosphatase’s substrates. Although this is an effective method for generating isolation, the phosphatase becomes a highly inefficient enzyme, which prevents the system from achieving switch-like responses and can result in slow response kinetics. We also explore the idea that substrate degradation can serve as an effective phosphatase. Assuming that degradation is unsaturatable, this mechanism could insulate substrates from crosstalk, but it would also preclude ultrasensitive responses and would require very high substrate turnover to achieve rapid dephosphorylation kinetics. Finally, we show that adaptor subunits, such as those found on phosphatases like PP2A, can provide effective insulation against phosphatase crosstalk, but only if their binding to substrates is uncoupled from their binding to the catalytic core. Analysis of the interaction network of PP2A’s adaptor domains reveals that although its adaptors may isolate subsets of targets from one another, there is still a strong potential for phosphatase crosstalk within those subsets. Understanding how phosphatase crosstalk and the insulation mechanisms described here impact the function and evolution of signaling networks represents a major challenge for

  2. Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*

    PubMed Central

    Meekins, David A.; Raththagala, Madushi; Auger, Kyle D.; Turner, Benjamin D.; Santelia, Diana; Kötting, Oliver; Gentry, Matthew S.; Vander Kooi, Craig W.

    2015-01-01

    Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. PMID:26231210

  3. Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

    PubMed

    Patni, N J; Dhawale, S W; Aaronson, S

    1977-04-01

    Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.

  4. Biogeochemical drivers of phosphatase activity in salt marsh sediments

    NASA Astrophysics Data System (ADS)

    Freitas, Joana; Duarte, Bernardo; Caçador, Isabel

    2014-10-01

    Although nitrogen has become a major concern for wetlands scientists dealing with eutrophication problems, phosphorous represents another key element, and consequently its biogeochemical cycling has a crucial role in eutrophication processes. Microbial communities are a central component in trophic dynamics and biogeochemical processes on coastal systems, since most of the processes in sediments are microbial-mediated due to enzymatic action, including the mineralization of organic phosphorus carried out by acid phosphatase activity. In the present work, the authors investigate the biogeochemical sediment drivers that control phosphatase activities. Authors also aim to assess biogeochemical factors' influence on the enzyme-mediated phosphorous cycling processes in salt marshes. Plant rhizosediments and bare sediments were collected and biogeochemical features, including phosphatase activities, inorganic and organic phosphorus contents, humic acids content and pH, were assessed. Acid phosphatase was found to give the highest contribution for total phosphatase activity among the three pH-isoforms present in salt marsh sediments, favored by acid pH in colonized sediments. Humic acids also appear to have an important role inhibiting phosphatase activity. A clear relation of phosphatase activity and inorganic phosphorous was also found. The data presented reinforces the role of phosphatase in phosphorous cycling.

  5. Penicillin inhibitors of purple acid phosphatase.

    PubMed

    Faridoon; Hussein, Waleed M; Ul Islam, Nazar; Guddat, Luke W; Schenk, Gerhard; McGeary, Ross P

    2012-04-01

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.

  6. Determination of liver microsomal glucose-6-phosphatase.

    PubMed

    Zak, B; Epstein, E; Baginski, E S

    1977-01-01

    A procedure for the determination of liver microsomal glucose-6-phosphatase is described. Homogenization and ultracentrifrigation were used to prepare a precipitate whose character was defined by monitoring the desire enzyme activity which serves as a marker. Activity of the enzyme was determined by means of a sensitive colorimetric reaction for the product, inorganic phosphate. Non-enzymatic hydrolysis problems with the substrate are minimized in this procedure by the masking action of citrate. The final heteropoly blue color appears to be considerably sensitized by interaction of phosphomolybdous ion with arsenite. The stability of the relatively labile enzyme was ensured by chelating any metals present with ethylene diamine tetraacetic acid. The overall results obtained by the procedure appear to be useful as an aid in the diagnosis of Type I glycogenosis, a glycogen storage disease called Von Gierke's disease. PMID:192125

  7. Intestinal alkaline phosphatase to treat necrotizing enterocolitis

    PubMed Central

    Biesterveld, Ben E.; Koehler, Shannon M.; Heinzerling, Nathan P.; Rentea, Rebecca M.; Fredrich, Katherine; Welak, Scott R.; Gourlay, David M.

    2015-01-01

    Background Intestinal alkaline phosphatase (IAP) activity is decreased in necrotizing enterocolitis (NEC), and IAP supplementation prevents NEC development. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after NEC induction would not reverse intestinal injury. Materials and methods NEC was induced in Sprague–Dawley pups by delivery preterm followed by formula feedings with lipopolysaccharide (LPS) and hypoxia exposure and continued up to 4 d. IAP was added to feeds on day 2 until being sacrificed on day 4. NEC severity was scored based on hematoxylin and eosin-stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and interleukin-6 expression were measured using real time polymerase chain reaction. Results NEC pups' alkaline phosphatase (AP) activity was decreased to 0.18 U/mg compared with controls of 0.57 U/mg (P < 0.01). Discontinuation of LPS and hypoxia after 2 d increased AP activity to 0.36 U/mg (P < 0.01). IAP supplementation in matched groups did not impact total AP activity or expression. Discontinuing LPS and hypoxia after NEC onset improved intestinal injury scores to 1.14 compared with continued stressors, score 2.25 (P < 0.01). IAP supplementation decreased interleukin-6 expression two-fold (P < 0.05), though did not reverse NEC intestinal damage (P = 0.5). Conclusions This is the first work to demonstrate that removing the source of NEC improves intestinal damage and increases AP activity. When used as a rescue treatment, IAP decreased intestinal inflammation though did not impact injury making it likely that IAP is best used preventatively to those neonates at risk. PMID:25840489

  8. Unique structural features of red kidney bean purple acid phosphatase.

    PubMed

    Cashikar, A G; Rao, M N

    1995-06-01

    Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes. PMID:7590853

  9. Discovery of Protein Phosphatase 2C Inhibitors by Virtual Screening

    PubMed Central

    Rogers, Jessica P.; Beuscher, Albert E.; Flajolet, Marc; McAvoy, Thomas; Nairn, Angus C.; Olson, Arthur; Greengard, Paul

    2008-01-01

    Protein phosphatase 2C (PP2C) is an archetype of the PPM Ser/Thr phosphatases, characterized by dependence on divalent magnesium or manganese cofactors, absence of known regulatory proteins, and resistance to all known Ser/Thr phosphatase inhibitors. We have used virtual ligand screening with the AutoDock method and the National Cancer Institute Diversity Set to identify small molecule inhibitors of PP2Cα activity at a protein substrate. These inhibitors are active in the micromolar range, and represent the first non-phosphate-based molecules found to inhibit a type 2C phosphatase. The compounds docked to three recurrent binding sites near the PP2Cα active site and displayed novel Ser/Thr phosphatase selectivity profiles. Common chemical features of these compounds may form the basis for development of a PP2C inhibitor pharmacophore and may facilitate investigation of PP2C control and cellular function. PMID:16509582

  10. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes. PMID:25785438

  11. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  12. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    SciTech Connect

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  13. Resistance of leishmanial phosphatases to inactivation by oxygen metabolites.

    PubMed

    Saha, A K; Das, S; Glew, R H; Gottlieb, M

    1985-09-01

    Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages.

  14. The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells

    PubMed Central

    Frearson, Julie A.; Alexander, Denis R.

    1998-01-01

    Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process

  15. Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

    PubMed Central

    Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

    2012-01-01

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

  16. Elevation of serum acid phosphatase in cancers with bone metastasis

    SciTech Connect

    Tavassoli, M.; Rizo, M.; Yam, L.T.

    1980-05-01

    In patients with nonprostatic cancer, serum acid phosphatase activity is usually elevated when bone metastasis is present but not when bone metastasis is absent. The fraction responsible for serum enzyme elevation is a normal component of serum; it appears in gel electrophoresis as band 5; and is tartrate-resistant. It is suggested that the origin of acid phosphatase elevation is bone osteoclasts rather than cancer tissue, as is the case with prostatic carcinoma. Determination of serum acid phosphatase activity may be useful in the detection of bone metastasis.

  17. Emerging Roles of Human Prostatic Acid Phosphatase

    PubMed Central

    Kong, Hoon Young; Byun, Jonghoe

    2013-01-01

    Prostate cancer is one of the most prevalent non-skin related cancers. It is the second leading cause of cancer deaths among males in most Western countries. If prostate cancer is diagnosed in its early stages, there is a higher probability that it will be completely cured. Prostatic acid phosphatase (PAP) is a non-specific phosphomonoesterase synthesized in prostate epithelial cells and its level proportionally increases with prostate cancer progression. PAP was the biochemical diagnostic mainstay for prostate cancer until the introduction of prostate-specific antigen (PSA) which improved the detection of early-stage prostate cancer and largely displaced PAP. Recently, however, there is a renewed interest in PAP because of its usefulness in prognosticating intermediate to high-risk prostate cancers and its success in the immunotherapy of prostate cancer. Although PAP is believed to be a key regulator of prostate cell growth, its exact role in normal prostate as well as detailed molecular mechanism of PAP regulation is still unclear. Here, many different aspects of PAP in prostate cancer are revisited and its emerging roles in other environment are discussed. PMID:24009853

  18. Studying Protein-Tyrosine Phosphatases in Zebrafish.

    PubMed

    Hale, Alexander James; den Hertog, Jeroen

    2016-01-01

    Protein-tyrosine phosphatases (PTPs) are a large family of signal transduction regulators that have an essential role in normal development and physiology. Aberrant activation or inactivation of PTPs is at the basis of many human diseases. The zebrafish, Danio rerio, is being used extensively to model major aspects of development and disease as well as the mechanism of regeneration of limbs and vital organs, and most classical PTPs have been identified in zebrafish. Zebrafish is an excellent model system for biomedical research because the genome is sequenced, zebrafish produce a large number of offspring, the eggs develop outside the mother and are transparent, facilitating intravital imaging, and transgenesis and (site-directed) mutagenesis are feasible. Together, these traits make zebrafish amenable for the analysis of gene and protein function. In this chapter we cover three manipulations of zebrafish embryos that we have used to study the effects of PTPs in development, regeneration, and biochemistry. Microinjection at the one-cell stage is at the basis of many zebrafish experiments and is described first. This is followed by a description for measuring regeneration of the embryonic caudal fin, a powerful and robust physiological assay. Finally, the considerable but manageable troubleshooting of several complications associated with preparing zebrafish embryos for immunoblotting is explained. Overall, this chapter provides detailed protocols for manipulating zebrafish embryo samples with a compilation of tips collected through extensive experience from the zebrafish research community. PMID:27514815

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  20. Structure and Mechanism of the Phosphotyrosyl Phosphatase Activator

    SciTech Connect

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

    2006-01-01

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

  1. A high-throughput screening for phosphatases using specific substrates.

    PubMed

    Senn, Alejandro M; Wolosiuk, Ricardo A

    2005-04-01

    A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

  2. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses

    PubMed Central

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-01-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events. PMID:27669825

  3. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    PubMed

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events. PMID:27669825

  4. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  5. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  6. Multiple forms of acid phosphatase activity in Gaucher's disease.

    PubMed

    Chambers, J P; Peters, S P; Glew, R H; Lee, R E; McCafferty, L R; Mercer, D W; Wenger, D A

    1978-07-01

    Although the primary genetic defect in all individuals with Gaucher's disease is a deficiency in glucocerebrosidase activity, the finding of marked elevations in splenic and serum acid phosphatase activity is almost as consistent a finding. Gaucher spleen and serum contain at least two forms of acid phosphatase that can be readily separated by chromatography on columns containing the cation exchange resin Sulphopropyl Sephadex. The major species of acid phosphatase (designated SP-I) contained in Triton X-100 (1% v/v) extracts of Gaucher spleen accounts for 65%--95% of the total activity and has the following properties: (1) it does not bind to the cation exchange column; (2) it exhibitis a pH optimum of 4.5--5.0; (3) it is inhibited by sodium fluoride (15 mM), L(+)-tartaric acid (20 mM), and beta-mercaptoethanol (2.1 M), and (4) it is resistant to inhibition by sodium dithionite (10 mM). The minor acid phosphatase activity (designated SP-II) present in extracts of Gaucher spleen has properties similar to those of the major species of acid phosphatase activity contained in serum from patients with Gaucher's disease: (1) it binds firmly to cation exchange columns (eluted by 0.5 M sodium chloride); (2) it exhibits a pH optimum of 5.0--6.0; (3) it is inhibited by sodium fluoride and sodium dithionite; and (4) it is resistant to inhibition by beta-mercaptoethanol (2.1 M) and L(+)-tartaric acid (20 mM). In addition, a second form of acid phosphatase that is tartrate resistant was found to be elevated in Gaucher serum. This form of serum acid phosphatase did not bind to Sulphopropyl Sephadex, was found to be significantly resistant to beta-mercaptoethanol (2.1 M), and was only partially inhibited by sodium dithionite (10 mM). The findings reported here indicate that at least three distinct forms of acid phosphatase activity are elevated in Gaucher's disease. Furthermore, the minor acid phosphatase activity contained in spleen homogenates has properties very similar to

  7. Protein phosphatase 1 is a key player in nuclear events.

    PubMed

    Rebelo, Sandra; Santos, Mariana; Martins, Filipa; da Cruz e Silva, Edgar F; da Cruz e Silva, Odete A B

    2015-12-01

    Reversible protein phosphorylation at serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is among the major regulatory mechanism in eukaryotic cells. The eukaryotic genome encodes many protein kinases and protein phosphatases. However, the localization, activity and specificity towards phosphatase substrates are dictated by a large array of phosphatase binding and regulatory subunits. For protein phosphatase 1 (PP1) more than 200 binding subunits have been described. The various PP1 isoforms and the binding subunits can be located throughout the cell, including in the nucleus. It follows that several nuclear specific PP1 binding proteins (PIPs) have been described and these will be discussed. Among them are PNUTS (phosphatase 1 nuclear targeting subunit), NIPP1 (nuclear inhibitor of PP1) and CREB (cAMP-responsive element-binding protein), which have all been associated with transcription. In fact PP1 can associate with transcription factors fulfilling an important regulatory function, in this respect it can bind to Hox11, human factor C1 (HCF1) and myocyte enhancer factor-2 (MEF2). PP1 also regulates cell cycle progression and centrosome maturation and splitting, again by binding to specific regulatory proteins. Moreover, PP1 together with other protein phosphatases control the entry into mitosis by regulating the activity of mitotic kinases. Thus, PP1, its binding proteins and/or the phosphorylation states of both, directly control a vast array of cell nucleus associated functions, many of which are starting to be unraveled.

  8. Isolation and characterization of a neutral phosphatase from wheat seedlings

    SciTech Connect

    Cheng, H.F.

    1988-01-01

    A neutral phosphatase was purified to homogeneity from wheat seedlings. The enzyme was a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 26 A, and sedimentation coefficient of 3.2 S. That the enzyme was a glycoprotein was surmised from its chromatographic property on Concanavalin A-Sepharose column. The phosphatase activity was assayed using either fructose-2,6-bisphosphate or p-nitrophenyl phosphate as substrate. The phosphatase activity was not affected by high concentrations of chelating agents and did not require the addition of Mg{sup +2} or Ca{sup +2} for its activity. Molybdate, orthovanadate, Zn{sup +2}, and Hg{sup +2} were all potent inhibitors of the phosphatase activity. The inhibition by Hg{sup +2} was reversed by dithiothreitol. The enzyme activity was stimulated by Mn{sup +2} about 2-fold. On the other hand, 3-phosphoglycerate, fructose-6-P and Pi as well as polyamines inhibited the enzyme activity. The ability of the neutral phosphatase to dephosphorylate protein phosphotyrosine was also investigated. The phosphotyrosyl-substrates, such as ({sup 32}P) phosphotyrosyl-poly(Glu, Tyr)n, -alkylated bovine serum albumin, -angiotensin-1, and -band 3 of erythrocytes, were all substrates of the phosphatase. On the other hand, the enzyme had no activity toward protein phosphoserine and protein phosphothreonine.

  9. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  10. Structural elucidation of the NADP(H) phosphatase activity of staphylococcal dual-specific IMPase/NADP(H) phosphatase.

    PubMed

    Bhattacharyya, Sudipta; Dutta, Anirudha; Dutta, Debajyoti; Ghosh, Ananta Kumar; Das, Amit Kumar

    2016-02-01

    NADP(H)/NAD(H) homeostasis has long been identified to play a pivotal role in the mitigation of reactive oxygen stress (ROS) in the intracellular milieu and is therefore critical for the progression and pathogenesis of many diseases. NAD(H) kinases and NADP(H) phosphatases are two key players in this pathway. Despite structural evidence demonstrating the existence and mode of action of NAD(H) kinases, the specific annotation and the mode of action of NADP(H) phosphatases remains obscure. Here, structural evidence supporting the alternative role of inositol monophosphatase (IMPase) as an NADP(H) phosphatase is reported. Crystal structures of staphylococcal dual-specific IMPase/NADP(H) phosphatase (SaIMPase-I) in complex with the substrates D-myo-inositol-1-phosphate and NADP(+) have been solved. The structure of the SaIMPase-I-Ca(2+)-NADP(+) ternary complex reveals the catalytic mode of action of NADP(H) phosphatase. Moreover, structures of SaIMPase-I-Ca(2+)-substrate complexes have reinforced the earlier proposal that the length of the active-site-distant helix α4 and its preceding loop are the predisposing factors for the promiscuous substrate specificity of SaIMPase-I. Altogether, the evidence presented suggests that IMPase-family enzymes with a shorter α4 helix could be potential candidates for previously unreported NADP(H) phosphatase activity.

  11. [Phosphatase activity in Amoeba proteus at low pH].

    PubMed

    Sopina, V A

    2009-01-01

    In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed using PAGE of the supernatant of ameba homogenates obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the ameba supernatant was sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium molybdate, EDTA, EGTA, o-phospho-L-tyrosine, DL-dithiotreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals - Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosome location in the ameba cell, and wide substrate specificity of these two forms, it has been concluded that they belong to nonspecific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosyl-protein phosphatase activitis. Two ecto-phosphatases were revealed in the culture medium, in which amebas were cultivated. One of them was inhibited by the same reagents as the ameba tartrate-sensitive AcP and seems to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed, which was not inhibited by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belong to the ecto-ATPases found in many protists; however, its ability to hydrolyze ATP has not yet been proven.

  12. Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence.

    PubMed

    Keum, Dongil; Kruse, Martin; Kim, Dong-Il; Hille, Bertil; Suh, Byung-Chang

    2016-06-28

    Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P3 is 55-fold slower than 5-phosphatase activity against PI(4,5)P2; thus, PI(4,5)P2 generated more slowly from dephosphorylating PI(3,4,5)P3 might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation

  13. Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase

    SciTech Connect

    Henthorn, P.S.; Raducha, M.; Edwards, Y.H.; Weiss, M.J.; Slaughter, C.; Lafferty, M.A.; Harris, H.

    1987-03-01

    A cDNA clone for human adult intestinal alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1) was isolated from a lambdagt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.

  14. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

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

    PubMed

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

    2015-07-21

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

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

    PubMed Central

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

    2015-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α+/− myocytes resulted in reduced Ca2+ waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca2+ regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart. PMID:26198358

  17. Protein Ser/Thr phosphatases PPEF interact with calmodulin.

    PubMed

    Kutuzov, Mikhail A; Solov'eva, Olga V; Andreeva, Alexandra V; Bennett, Nelly

    2002-05-10

    Regulation of protein dephosphorylation by cytoplasmic Ca(2+) levels and calmodulin (CaM) is well established and considered to be mediated solely by calcineurin. Yet, recent identification of protein phosphatases with EF-hand domains (PPEF/rdgC) point to the existence of another group of Ca(2+)-dependent protein phosphatases. We have recently hypothesised that PPEF/rdgC phosphatases might possess CaM-binding sites of the IQ-type in their N-terminal domains. We now employed yeast two-hybrid system and surface plasmon resonance (SPR) to test this hypothesis. We found that entire human PPEF2 interacts with CaM in the in vivo tests and that its N-terminal domain binds to CaM in a Ca(2+)-dependent manner with nanomolar affinity in vitro. The fragments corresponding to the second exons of PPEF1 and PPEF2, containing the IQ motifs, are sufficient for specific Ca(2+)-dependent interaction with CaM both in vivo and in vitro. These findings demonstrate the existence of mammalian CaM-binding protein Ser/Thr phosphatases distinct from calcineurin and suggest that the activity of PPEF phosphatases may be controlled by Ca(2+) in a dual way: via C-terminal Ca(2+)-binding domain and via interaction of the N-terminal domain with CaM.

  18. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  19. Thermal inactivation of alkali phosphatases under various conditions

    NASA Astrophysics Data System (ADS)

    Atyaksheva, L. F.; Tarasevich, B. N.; Chukhrai, E. S.; Poltorak, O. M.

    2009-02-01

    The thermal inactivation of alkali phosphatases from bacteria Escherichia coli (ECAP), bovine intestines (bovine IAP), and chicken intestines (chicken IAP) was studied in different buffer solutions and in the solid state. The conclusion was made that these enzymes had maximum stability in the solid state, and, in a carbonate buffer solution, their activity decreased most rapidly. It was found that the bacterial enzyme was more stable than animal phosphatases. It was noted that, for ECAP, four intermediate stages preceded the loss of enzyme activity, and, for bovine and chicken IAPs, three intermediate stages were observed. The activation energy of thermal inactivation of ECAP over the range 25-70°C was determined to be 80 kJ/mol; it corresponded to the dissociation of active dimers into inactive monomers. Higher activation energies (˜200 kJ/mol) observed at the initial stage of thermal inactivation of animal phosphatases resulted from the simultaneous loss of enzyme activity caused by dimer dissociation and denaturation. It was shown that the activation energy of denaturation of monomeric animal alkali phosphatases ranged from 330 to 380 kJ/mol depending on buffer media. It was concluded that the inactivation of solid samples of alkali phosphatases at 95°C was accompanied by an about twofold decrease in the content of β structures in protein molecules.

  20. [Granulocyte alkaline phosphatase--a biomarker of chronic benzene exposure].

    PubMed

    Khristeva, V; Meshkov, T

    1994-01-01

    In tracing the cellular population status in the peripheral blood of workers, exposed to benzene, was included and cytochemical determination of the alkaline phosphatase activity in leucocytes. This enzyme is accepted as marker of the neutrophilic granulocytes, as maturation of the cells and their antibacterial activity are parallel to the cytochemical activity of the enzyme. 78 workers from the coke-chemical production from state firm "Kremikovtsi" and 41 workers from the production "Benzene" and "Isopropylbenzene"--Oil Chemical Plant, Burgas are included. The benzene concentrations in the air of the working places in all productions are in the range of 5 to 50 mg/m3. For cytochemical determination of the alkaline phosphatase activity is used the method of L. Kaplow and phosphatase index was calculated. It was established that in 98.4% of all examined the alkaline phosphatase activity is inhibited to different rate, as from 46.5% [61 workers] it is zero. In considerably lower percentage of workers were established and other deviations: leucocytosis or leucopenia, neutropenia, increased percent of band neutrophils and toxic granules. The results of the investigation of the granulocyte population show that from all indices, the activity of granulocyte alkaline phosphatase demonstrates most convincing the early myelotoxic effect of benzene.

  1. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  2. [Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

    PubMed

    Beniaminov, A D; Krasnov, G S; Dmitriev, A A; Puzanov, G A; Snopok, B A; Senchenko, V N; Kashuba, V I

    2016-01-01

    Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation. PMID:27414789

  3. Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium.

    PubMed

    King, B F

    1985-01-01

    The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.

  4. Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

    PubMed

    Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

    2013-03-01

    In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

  5. Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production.

    PubMed

    Standish, Alistair J; Salim, Angela A; Zhang, Hua; Capon, Robert J; Morona, Renato

    2012-01-01

    Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.

  6. Phosphatase activity on the cell wall of Fonsecaea pedrosoi.

    PubMed

    Kneipp, L F; Palmeira, V F; Pinheiro, A A S; Alviano, C S; Rozental, S; Travassos, L R; Meyer-Fernandes, J R

    2003-12-01

    The activity of a phosphatase was characterized in intact mycelial forms of Fonsecaea pedrosoi, a pathogenic fungus that causes chromoblastomycosis. At pH 5.5, this fungus hydrolyzed p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 12.78 +/- 0.53 nmol p-NP per h per mg hyphal dry weight. The values of Vmax and apparent Km for p-NPP hydrolyses were measured as 17.89 +/- 0.92 nmol p-NP per h per mg hyphal dry weight and 1.57 +/- 0.26 mmol/l, respectively. This activity was inhibited at increased pH, a finding compatible with an acid phosphatase. The enzymatic activity was strongly inhibited by classical inhibitors of acid phosphatases such as sodium orthovanadate (Ki = 4.23 micromol/l), sodium molybdate (Ki = 7.53 micromol/l) and sodium fluoride (Ki = 126.78 micromol/l) in a dose-dependent manner. Levamizole (1 mmol/l) and sodium tartrate (10 mmol/l), had no effect on the enzyme activity. Cytochemical localization of the acid phosphatase showed electrondense cerium phosphate deposits on the cell wall, as visualized by transmission electron microscopy. Phosphatase activity in F. pedrosoi seems to be associated with parasitism, as sclerotic cells, which are the fungal forms mainly detected in chromoblastomycosis lesions, showed much higher activities than conidia and mycelia did. A strain of F. pedrosoi recently isolated from a human case of chromoblastomycosis also showed increased enzyme activity, suggesting that the expression of surface phosphatases may be stimulated by interaction with the host.

  7. Structural Basis of Response Regulator Dephosphorylation by Rap Phosphatases

    SciTech Connect

    V Parashar; N Mirouze; D Dubnau; M Neiditch

    2011-12-31

    Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic 'switch' residue to an internal position when the {beta}4-{alpha}4 loop adopts an active-site proximal conformation.

  8. Genetic alterations of protein tyrosine phosphatases in human cancers

    PubMed Central

    Zhao, Shuliang; Sedwick, David; Wang, Zhenghe

    2014-01-01

    Protein tyrosine phosphatases (PTPs) are enzymes that remove phosphate from tyrosine residues in proteins. Recent whole-exome sequencing of human cancer genomes reveals that many PTPs are frequently mutated in a variety of cancers. Among these mutated PTPs, protein tyrosine phosphatase T (PTPRT) appears to be the most frequently mutated PTP in human cancers. Beside PTPN11 which functions as an oncogene in leukemia, genetic and functional studies indicate that most of mutant PTPs are tumor suppressor genes. Identification of the substrates and corresponding kinases of the mutant PTPs may provide novel therapeutic targets for cancers harboring these mutant PTPs. PMID:25263441

  9. Effects of organic dairy manure amendment on soil phosphatase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Organic dairy production is increasing in the U.S. due to concerns over environmental, human, and animal health. It is well known that the application of livestock manure to soil can influence enzyme activities involved in nutrient cycling and soil fertility, such as soil phosphatases; however, orga...

  10. Enzymatic method of determining lead using alkaline phosphatase

    SciTech Connect

    Shekhovtsova, T.N.; Kucheryaeva, V.V.; Dolmanova, I.F.

    1986-03-20

    The purpose of this work was to determine the possibility of using alkaline phosphatase to determine trace amounts of ions of a number of metals - Mg, Ba, Ca, Sr, Cd, Pb - for which there are virtually no sensitive and simple methods of determination.

  11. Methods to distinguish various types of protein phosphatase activity

    SciTech Connect

    Brautigan, D.L.; Shriner, C.L.

    1988-01-01

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on /sup 32/P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of /sup 32/P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble (/sup 32/P)phosphate.

  12. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    ERIC Educational Resources Information Center

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  13. Protein Tyrosine Phosphatases in Hypothalamic Insulin and Leptin Signaling.

    PubMed

    Zhang, Zhong-Yin; Dodd, Garron T; Tiganis, Tony

    2015-10-01

    The hypothalamus is critical to the coordination of energy balance and glucose homeostasis. It responds to peripheral factors, such as insulin and leptin, that convey to the brain the degree of adiposity and the metabolic status of the organism. The development of leptin and insulin resistance in hypothalamic neurons appears to have a key role in the exacerbation of diet-induced obesity. In rodents, this has been attributed partly to the increased expression of the tyrosine phosphatases Protein Tyrosine Phosphatase 1B (PTP1B) and T cell protein tyrosine phosphatase (TCPTP), which attenuate leptin and insulin signaling. Deficiencies in PTP1B and TCPTP in the brain, or specific neurons, promote insulin and leptin signaling and prevent diet-induced obesity, type 2 diabetes mellitus (T2DM), and fatty liver disease. Although targeting phosphatases and hypothalamic circuits remains challenging, recent advances indicate that such hurdles might be overcome. Here, we focus on the roles of PTP1B and TCPTP in insulin and leptin signaling and explore their potential as therapeutic targets.

  14. Structural and functional basis of protein phosphatase 5 substrate specificity

    PubMed Central

    Oberoi, Jasmeen; Dunn, Diana M.; Woodford, Mark R.; Mariotti, Laura; Schulman, Jacqualyn; Bourboulia, Dimitra; Mollapour, Mehdi

    2016-01-01

    The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP–substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors. PMID:27466404

  15. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

    PubMed Central

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S.; Flick, Robert; Wolf, Yuri I.; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D.; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M.; Koonin, Eugene V.; Yakunin, Alexander F.

    2015-01-01

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. PMID:26071590

  16. Selective dephosphorylation of histone H1 by nuclear phosphatases

    SciTech Connect

    Jakes, S.; Schlender, K.K.

    1987-05-01

    The aim of this study was to characterize the sites of H1 phosphorylated by the cAMP-dependent protein kinase (kinase A) and the CaS phospholipid-dependent protein kinase (kinase C) and to study their dephosphorylation by nuclear protein phosphatases. H1 was phosphorylated on a ser residue to approx. 1 mole (TSP)/mole H1 with either kinase A or C. The sites of phosphorylation were differentiated by digestion of the H1 by thrombin or N-bromosuccinimide. Phosphopeptide maps on reversed phase HPLC and gel filtration HPLC clearly showed that the kinase C phosphorylated a different site than the well characterized kinase A site. H1, phosphorylated by kinase C or kinase A, was used as a substrate for the nuclear phosphatases. The nuclear phosphatases were purified from salt extracted rat liver chromatin and separated into 2 forms based on heat-stable inhibitor sensitivity and polycation stimulation. Polycation-stimulated phosphatase rapidly dephosphorylated the kinase C site and slowly dephosphorylated the kinase A site. The inhibitor-sensitive enzyme showed little activity toward either site under standard assay conditions.

  17. Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas.

    PubMed Central

    Galski, H; Fridovich, S E; Weinstein, D; De Groot, N; Segal, S; Folman, R; Hochberg, A A

    1981-01-01

    The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared. PMID:7306029

  18. Phosphatase acitivity as biosignatures in terrestrial extreme environments

    NASA Astrophysics Data System (ADS)

    Kawai, Jun; Nakamoto, Saki; Hara, Masashi; Obayashi, Yumiko; Kaneko, Takeo; Mita, Hajime; Yoshimura, Yoshitaka; Takano, Yoshinori; Kobayashi, Kensei

    Since phosphate esters are essential for the terrestrial life, phosphatase activity can be a can-didate for biosignatures of biological activity. It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere, high temperature hot springs and stratosphere. We analyzed phosphatase activities in the samples obtained in ex-treme environments such as submarine hydrothermal systems and Antarctica , and discussed whether they can be used as biosignatures for extant life. Core samples and chimney samples were collected at Tarama Knoll in Okinawa Trough in 2009, both in a part of the Archaean Park Project. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Alkaline Phosphatase activ-ity in sea water and in soil was measured spectrometrically by using 25 mM p-nitrophenyl phosphate (pH 8.0) as a substrate. Phosphatase activities in extracts were measured fluoro-metrically by using 4-methylumberyferryl phosphate as a substrate. Concentration of amino acids and their enantiomeric ratios were also determined by HPLC . Significant enzymatic ac-tivities were revealed in both some of the hydrothermal sub-vent systems and Antarctica soils, which is crucial evidence of vigorous microbial oasis. It is consistent with the fact that large enantiomeric excess of L-form amino acids were found in the same core sequences. Optimum temperatures of ALP in the chimney, Antarctica soil and YNU campus soil were 353 K, 313 K, and 333 K, respectively. The present results suggested that phosphatase activities,, together with amino acids, can be used as possible biosignatures for extant life.

  19. Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum.

    PubMed

    Pendyala, Prakash Rao; Ayong, Lawrence; Eatrides, Jennifer; Schreiber, Melissa; Pham, Connie; Chakrabarti, Ratna; Fidock, David A; Allen, Charles M; Chakrabarti, Debopam

    2008-03-01

    Isoprenylated proteins have important functions in cell growth and differentiation of eukaryotic cells. Inhibitors of protein prenylation in malaria have recently shown strong promise as effective antimalarials. In studying protein prenylation in the malaria protozoan parasite Plasmodium falciparum, we have shown earlier that the incubation of P. falciparum cells with (3)H-prenol precursors resulted in various size classes of labeled proteins. To understand the physiological function of prenylated proteins of malaria parasites, that are targets of prenyltransferase inhibitors, we searched the PlasmoDB database for proteins containing the C-terminus prenylation motif. We have identified, among other potentially prenylated proteins, an orthologue of a PRL (protein of regenerating liver) subgroup protein tyrosine phosphatases, termed PfPRL. Here, we show that PfPRL is expressed in the parasite's intraerythrocytic stages, where it partially associates with endoplasmic reticulum and within a subcompartment of the food vacuole. Additionally, PfPRL targeting parallels that of apical membrane antigen-1 in developing merozoites. Recombinant PfPRL shows phosphatase activity that is preferentially inhibited by a tyrosine phosphatase inhibitor suggesting that PfPRL functions as a tyrosine phosphatase. Recombinant PfPRL can also be farnesylated in vitro. Inhibition of malarial farnesyltransferase activity can be achieved with the heptapetide RKCHFM, which corresponds to the C-terminus of PfPRL. This study provides the first evidence for expression of enzymatically active PRL-related protein tyrosine phosphatases in malarial parasites, and demonstrates the potential of peptides derived from Plasmodium prenylated proteins as malarial farnesyltransferase inhibitors.

  20. The relationship between the MMP system, adrenoceptors and phosphoprotein phosphatases

    PubMed Central

    Rietz, A; Spiers, JP

    2012-01-01

    The MMPs and their inhibitors [tissue inhibitor of MMPs (TIMPs) ] form the mainstay of extracellular matrix homeostasis. They are expressed in response to numerous stimuli including cytokines and GPCR activation. This review highlights the importance of adrenoceptors and phosphoprotein phosphatases (PPP) in regulating MMPs in the cardiovascular system, which may help explain some of the beneficial effects of targeting the adrenoceptor system in tissue remodelling and will establish emerging crosstalk between these three systems. Although α- and β-adrenoceptor activation increases MMP but decreases TIMP expression, MMPs are implicated in the growth stimulatory effects of adrenoceptor activation through transactivation of epidermal growth factor receptor. Furthermore, they have recently been found to catalyse the proteolysis of β-adrenoceptors and modulate vascular tone. While the mechanisms underpinning these effects are not well defined, reversible protein phosphorylation by kinases and phosphatases may be key. In particular, PPP (Ser/Thr phosphatases) are not only critical in resensitization and internalization of adrenoceptors but also modulate MMP expression. The interrelationship is complex as isoprenaline (ISO) inhibits okadaic acid [phosphoprotein phosphatase type 1/phosphoprotein phosphatase type 2A (PP2A) inhibitor]-mediated MMP expression. While this may be simply due to its ability to transiently increase PP2A activity, there is evidence for MMP-9 that ISO prevents okadaic acid-mediated expression of MMP-9 through a β-arrestin, NF-κB-dependent pathway, which is abolished by knock-down of PP2A. It is essential that crosstalk between MMPs, adrenoceptors and PPP are investigated further as it will provide important insight into how adrenoceptors modulate cardiovascular remodelling, and may identify new targets for pharmacological manipulation of the MMP system. PMID:22364165

  1. Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C.

    PubMed

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D; Nairn, Angus C; Bibb, James A

    2006-08-25

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

  2. Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2

    SciTech Connect

    Yang, S.D.; Fong, Y.L.; Benovic, J.L.; Sibley, D.R.; Caron, M.G.; Lefkowitz, R.J.

    1988-06-25

    Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as (/sup 32/P)phosphorylase a and (/sup 32/P)myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells. These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.

  3. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    NASA Technical Reports Server (NTRS)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  4. Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates.

    PubMed

    Spinelli, Laura; Leslie, Nicholas R

    2016-01-01

    PTEN is a one of the most frequently mutated tumor suppressors in human cancers. It is essential for regulating diverse biological processes and through its lipid phosphatase activity regulates the PI 3-Kinase signaling pathway. Sensitive phosphatase assays are employed to study the catalytic activity of PTEN against phospholipid substrates. Here we describe protocols to assay PTEN lipid phosphatase activity using either purified enzyme (purified PTEN lipid phosphatase assay) or PTEN immunopurified from tissues or cultured cells (cellular IP PTEN lipid phosphatase assay) against vesicles containing radiolabeled PIP3 substrate. PMID:27514802

  5. Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

    PubMed

    Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

    2005-01-12

    Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

  6. phoD Alkaline Phosphatase Gene Diversity in Soil.

    PubMed

    Ragot, Sabine A; Kertesz, Michael A; Bünemann, Else K

    2015-10-01

    Phosphatase enzymes are responsible for much of the recycling of organic phosphorus in soils. The PhoD alkaline phosphatase takes part in this process by hydrolyzing a range of organic phosphoesters. We analyzed the taxonomic and environmental distribution of phoD genes using whole-genome and metagenome databases. phoD alkaline phosphatase was found to be spread across 20 bacterial phyla and was ubiquitous in the environment, with the greatest abundance in soil. To study the great diversity of phoD, we developed a new set of primers which targets phoD genes in soil. The primer set was validated by 454 sequencing of six soils collected from two continents with different climates and soil properties and was compared to previously published primers. Up to 685 different phoD operational taxonomic units were found in each soil, which was 7 times higher than with previously published primers. The new primers amplified sequences belonging to 13 phyla, including 71 families. The most prevalent phoD genes identified in these soils were affiliated with the orders Actinomycetales (13 to 35%), Bacillales (1 to 29%), Gloeobacterales (1 to 18%), Rhizobiales (18 to 27%), and Pseudomonadales (0 to 22%). The primers also amplified phoD genes from additional orders, including Burkholderiales, Caulobacterales, Deinococcales, Planctomycetales, and Xanthomonadales, which represented the major differences in phoD composition between samples, highlighting the singularity of each community. Additionally, the phoD bacterial community structure was strongly related to soil pH, which varied between 4.2 and 6.8. These primers reveal the diversity of phoD in soil and represent a valuable tool for the study of phoD alkaline phosphatase in environmental samples.

  7. phoD Alkaline Phosphatase Gene Diversity in Soil

    PubMed Central

    Kertesz, Michael A.; Bünemann, Else K.

    2015-01-01

    Phosphatase enzymes are responsible for much of the recycling of organic phosphorus in soils. The PhoD alkaline phosphatase takes part in this process by hydrolyzing a range of organic phosphoesters. We analyzed the taxonomic and environmental distribution of phoD genes using whole-genome and metagenome databases. phoD alkaline phosphatase was found to be spread across 20 bacterial phyla and was ubiquitous in the environment, with the greatest abundance in soil. To study the great diversity of phoD, we developed a new set of primers which targets phoD genes in soil. The primer set was validated by 454 sequencing of six soils collected from two continents with different climates and soil properties and was compared to previously published primers. Up to 685 different phoD operational taxonomic units were found in each soil, which was 7 times higher than with previously published primers. The new primers amplified sequences belonging to 13 phyla, including 71 families. The most prevalent phoD genes identified in these soils were affiliated with the orders Actinomycetales (13 to 35%), Bacillales (1 to 29%), Gloeobacterales (1 to 18%), Rhizobiales (18 to 27%), and Pseudomonadales (0 to 22%). The primers also amplified phoD genes from additional orders, including Burkholderiales, Caulobacterales, Deinococcales, Planctomycetales, and Xanthomonadales, which represented the major differences in phoD composition between samples, highlighting the singularity of each community. Additionally, the phoD bacterial community structure was strongly related to soil pH, which varied between 4.2 and 6.8. These primers reveal the diversity of phoD in soil and represent a valuable tool for the study of phoD alkaline phosphatase in environmental samples. PMID:26253682

  8. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  9. Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

    NASA Astrophysics Data System (ADS)

    Wang, Yiyan; Sun, Hushan; Wang, Yanjie; Yan, Dongchun; Wang, Lei

    2015-03-01

    In this study, a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase, and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules (YGI, YGII, and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage, morula, blastula, gastrula, trochophore, and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation, indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI (free of acid phosphatase reaction) with YGII (rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.

  10. Alkaline Phosphatase-Mimicking Peptide Nanofibers for Osteogenic Differentiation.

    PubMed

    Gulseren, Gulcihan; Yasa, I Ceren; Ustahuseyin, Oya; Tekin, E Deniz; Tekinay, Ayse B; Guler, Mustafa O

    2015-07-13

    Recognition of molecules and regulation of extracellular matrix synthesis are some of the functions of enzymes in addition to their catalytic activity. While a diverse array of enzyme-like materials have been developed, these efforts have largely been confined to the imitation of the chemical structure and catalytic activity of the enzymes, and it is unclear whether enzyme-mimetic molecules can also be used to replicate the matrix-regulatory roles ordinarily performed by natural enzymes. Self-assembled peptide nanofibers can provide multifunctional enzyme-mimetic properties, as the active sequences of the target enzymes can be directly incorporated into the peptides. Here, we report enhanced bone regeneration efficiency through peptide nanofibers carrying both catalytic and matrix-regulatory functions of alkaline phosphatase, a versatile enzyme that plays a critical role in bone formation by regulating phosphate homeostasis and calcifiable bone matrix formation. Histidine presenting peptide nanostructures were developed to function as phosphatases. These molecules are able to catalyze phosphate hydrolysis and serve as bone-like nodule inducing scaffolds. Alkaline phosphatase-like peptide nanofibers enabled osteogenesis for both osteoblast-like and mesenchymal cell lines.

  11. An alkaline phosphatase reporter for use in Clostridium difficile.

    PubMed

    Edwards, Adrianne N; Pascual, Ricardo A; Childress, Kevin O; Nawrocki, Kathryn L; Woods, Emily C; McBride, Shonna M

    2015-04-01

    Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.

  12. The role of serine/threonine protein phosphatases in exocytosis.

    PubMed Central

    Sim, Alistair T R; Baldwin, Monique L; Rostas, John A P; Holst, Jeff; Ludowyke, Russell I

    2003-01-01

    Modulation of exocytosis is integral to the regulation of cellular signalling, and a variety of disorders (such as epilepsy, hypertension, diabetes and asthma) are closely associated with pathological modulation of exocytosis. Emerging evidence points to protein phosphatases as key regulators of exocytosis in many cells and, therefore, as potential targets for the design of novel therapies to treat these diseases. Diverse yet exquisite regulatory mechanisms have evolved to direct the specificity of these enzymes in controlling particular cell processes, and functionally driven studies have demonstrated differential regulation of exocytosis by individual protein phosphatases. This Review discusses the evidence for the regulation of exocytosis by protein phosphatases in three major secretory systems, (1) mast cells, in which the regulation of exocytosis of inflammatory mediators plays a major role in the respiratory response to antigens, (2) insulin-secreting cells in which regulation of exocytosis is essential for metabolic control, and (3) neurons, in which regulation of exocytosis is perhaps the most complex and is essential for effective neurotransmission. PMID:12749763

  13. Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphatase

    SciTech Connect

    Ou, Zhonghui; Felts, Richard L.; Reilly, Thomas J.; Nix, Jay C.; Tanner, John J.

    2006-05-01

    Lipoprotein e (P4) is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host–pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7 Å resolution native X-ray diffraction data set. The space group is P4{sub 2}2{sub 1}2, with unit-cell parameters a = 65.6, c = 101.4 Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase.

  14. Discovery and Development of Small Molecule SHIP Phosphatase Modulators

    PubMed Central

    Viernes, Dennis R.; Choi, Lydia B.

    2016-01-01

    Inositol phospholipids play an important role in the transfer of signaling information across the cell membrane in eukaryotes. These signals are often governed by the phosphorylation patterns on the inositols, which are mediated by a number of inositol kinases and phosphatases. The src homology 2 (SH2) – containing inositol 5-phosphatase (SHIP) plays a central role in these processes, influencing signals delivered through the PI3K/Akt/mTOR pathway. SHIP modulation by small molecules has been implicated as a treatment in a number of human disease states, including cancer, inflammatory diseases, diabetes, atherosclerosis, and Alzheimer's disease. In addition, alteration of SHIP phosphatase activity may provide a means to facilitate bone marrow transplantation and increase blood cell production. This review discusses the cellular signaling pathways and protein-protein interactions that provide the molecular basis for targeting the SHIP enzyme in these disease states. In addition, a comprehensive survey of small molecule modulators of SHIP1 and SHIP2 is provided, with a focus on the structure, potency, selectivity and solubility properties of these compounds. PMID:24302498

  15. The role of phosphatases in the initiation of skeletal mineralization.

    PubMed

    Millán, José Luis

    2013-10-01

    Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PPi), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl(-/-)) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the Pi needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.

  16. Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.

    PubMed

    Hovestad-Bijl, Liesbeth; van Ameijde, Jeroen; Pijnenburg, Dirk; Hilhorst, Riet; Liskamp, Rob; Ruijtenbeek, Rob

    2016-01-01

    A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0-5.0 μg of protein from a cell or tissue lysate or 0.1-2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases. PMID:27514800

  17. Uncoupling of 3'-phosphatase and 5'-kinase functions in budding yeast. Characterization of Saccharomyces cerevisiae DNA 3'-phosphatase (TPP1).

    PubMed

    Vance, J R; Wilson, T E

    2001-05-01

    Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

  18. Tyrosine phosphatases as key regulators of StAR induction and cholesterol transport: SHP2 as a potential tyrosine phosphatase involved in steroid synthesis.

    PubMed

    Cooke, Mariana; Mele, Pablo; Maloberti, Paula; Duarte, Alejandra; Poderoso, Cecilia; Orlando, Ulises; Paz, Cristina; Cornejo Maciel, Fabiana; Podestá, Ernesto J

    2011-04-10

    The phospho-dephosphorylation of intermediate proteins is a key event in the regulation of steroid biosynthesis. In this regard, it is well accepted that steroidogenic hormones act through the activation of serine/threonine (Ser/Thr) protein kinases. Although many cellular processes can be regulated by a crosstalk between different kinases and phosphatases, the relationship of Ser/Thr phosphorylation and tyrosine (Tyr)-dephosphorylation is a recently explored field in the regulation of steroid synthesis. Indeed in steroidogenic cells, one of the targets of hormone-induced Ser/Thr phosphorylation is a protein tyrosine phosphatase. Whereas protein tyrosine phosphatases were initially regarded as household enzymes with constitutive activity, dephosphorylating all the substrates they encountered, evidence is now accumulating that protein tyrosine phosphatases are tightly regulated by various mechanisms. Here, we will describe the role of protein tyrosine phosphatases in the regulation of steroid biosynthesis, relating them to steroidogenic acute regulatory protein, arachidonic acid metabolism and mitochondrial rearrangement.

  19. Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development

    PubMed Central

    Stumpf, Miriam; den Hertog, Jeroen

    2016-01-01

    The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten. PMID:26848951

  20. Structural basis for the glucan phosphatase activity of Starch Excess4

    SciTech Connect

    Vander Kooi, Craig W.; Taylor, Adam O.; Pace, Rachel M.; Meekins, David A.; Guo, Hou-Fu; Kim, Youngjun; Gentry, Matthew S.

    2010-11-12

    Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.

  1. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    SciTech Connect

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

    1987-05-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to US -adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of (TSP)C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of TS(P). Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein.

  2. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

    Robert J. Martinez; Melanie J. Beazley; Samuel M. Webb; Martial Taillefert; and Patricia A. Sobecky

    2007-04-19

    The overall objective of this project is to examine the activity of nonspecific phosphohydrolases present in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, we hypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbial release and/or accumulation of PO4 3- as a means to detoxify radionuclides and heavy metals. An experimental approach was designed to determine the extent of phosphatase activity in bacteria previously isolated from contaminated subsurface soils collected at the ERSP Field Research Center (FRC) in Oak Ridge, TN. Screening of 135 metal resistant isolates for phosphatase activity indicated the majority (75 of 135) exhibited a phosphatase-positive phenotype. During this phase of the project, a PCR based approach has also been designed to assay FRC isolates for the presence of one or more classes of the characterized non-specific acid phophastase (NSAP) genes likely to be involved in promoting U(VI) precipitation. Testing of a subset of Pb resistant (Pbr) Arthrobacter, Bacillus and Rahnella strains indicated 4 of the 9 Pbr isolates exhibited phosphatase phenotypes suggestive of the ability to bioprecipitate U(VI). Two FRC strains, a Rahnella sp. strain Y9602 and a Bacillus sp. strain Y9-2, were further characterized. The Rahnella sp. exhibited enhanced phosphatase activity relative to the Bacillus sp. Whole-cell enzyme assays identified a pH optimum of 5.5, and inorganic phosphate accumulated in pH 5.5 synthetic groundwater (designed to mimic FRC conditions) incubations of both strains in the presence of a model organophosphorus substrate provided as the sole C and P source. Kinetic experiments showed that these two organisms can grow in the presence of 200 μM dissolved uranium and that Rahnella is much more efficient in precipitating U(VI) than Bacillus sp. The

  3. Phosphate solubilization potential and phosphatase activity of rhizospheric trichoderma spp.

    PubMed

    Anil, Kapri; Lakshmi, Tewari

    2010-07-01

    Trichoderma sp., a well known biological control agent against several phytopathogens, was tested for its phosphate (P) solubilizing potential. Fourteen strains of Trichoderma sp. were isolated from the forest tree rhizospheres of pinus, deodar, bamboo, guava and oak on Trichoderma selective medium. The isolates were tested for their in-vitro P-solubilizing potential using National Botanical Research Institute Phosphate (NBRIP) broth containing tricalcium phosphate (TCP) as the sole P source, and compared with a standard culture of T. harzianum. All the cultures were found to solubilize TCP but with varying potential. The isolate DRT-1 showed maximum amount of soluble phosphate (404.07 εg.ml(-1)), followed by the standard culture of T. harzianum (386.42 εg.ml(-1)) after 96 h of incubation at 30±1(0)C. Extra-cellular acid and alkaline phosphatases of the fungus were induced only in the presence of insoluble phosphorus source (TCP). High extra-cellular alkaline phosphatase activity was recorded for the isolate DRT-1 (14.50 U.ml(-1)) followed by the standard culture (13.41 U.ml(-1)) at 72h. The cultures showed much lesser acid phosphatase activities. Under glasshouse conditions, Trichoderma sp. inoculation increased chickpea (Cicer arietinum) growth parameters including shoot length, root length, fresh and dry weight of shoot as well as roots, in P-deficient soil containing only bound phosphate (TCP). Shoot weight was increased by 23% and 33% by inoculation with the isolate DRT-1 in the soil amended with 100 and 200 mg TCP kg(-1) soil, respectively, after 60 d of sowing. The study explores high P-solubilizing potential of Trichoderma sp., which can be exploited for the solubilization of fixed phosphates present in the soil, thereby enhancing soil fertility and plant growth.

  4. Graphical techniques for kinetic data analyses of alkaline phosphatase

    SciTech Connect

    Frazer, J.W.; Brand, H.R.

    1980-09-01

    The use of an automated reactor for the experimentation and on-line graphics for the rapid and exhaustive analysis of experimental data is described. Traditional (linear) methods are used for selecting the most promising model for the alkaline phosphatase catalyzed reaction from a set of ten models under consideration. Then, nonlinear techniques for model selection are used and compared with traditional techniques. In both approaches, interactive graphics techniques are used to advantage for evaluating various models and for examining the quality of the experimental data.

  5. Structural Basis for Protein Phosphatase 1 Regulation and Specificity

    PubMed Central

    Peti, Wolfgang; Nairn, Angus C.; Page, Rebecca

    2012-01-01

    The ubiquitous Ser/Thr Protein Phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity. PMID:22284538

  6. A description of alkaline phosphatases from marine organisms

    NASA Astrophysics Data System (ADS)

    Tian, Jiyuan; Jia, Hongbing; Yu, Juan

    2016-07-01

    Alkaline phosphatases (APs) are non-specific phosphohydrolases, and they are widely used in clinical diagnostics and biological studies. APs are widespread in nature and exhibit different structural formulations. Based on the diversity of biogenetic sources, APs exhibit temperature-propensity traits, and they are classified as psychrophilic, mesophilic, and thermophilic. In this article, the characteristics of psychrophilic APs from marine organisms were described, accompanied by a simple description of APs from other organisms. This review will facilitate better utilization of marine APs in the biotechnology field.

  7. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    SciTech Connect

    Martinez, Robert J.; Beazley, Melanie J.; Wilson, Jarad J.; Taillefert, Martial; Sobecky, Patricia A.

    2005-04-05

    The overall goal of this project is to examine the role of nonspecific phosphohydrolases present in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, we hypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbial release and/or accumulation of PO{sub 4}{sup 3-}. During this phase of the project we have been conducting assays to determine the effects of pH, inorganic anions and organic ligands on U(VI) mineral formation and precipitation when FRC bacterial isolates were grown in simulated groundwater medium. The molecular characterization of FRC isolates has also been undertaken during this phase of the project. Analysis of a subset of gram-positive FRC isolates cultured from FRC soils (Areas 1, 2 and 3) and background sediments have indicated a higher percentage of isolates exhibiting phosphatase phenotypes (i.e., in particular those surmised to be PO{sub 4}{sup 3-}-irrepressible) relative to isolates from the reference site. A high percentage of strains that exhibited such putatively PO{sub 4}{sup 3-}-irrepressible phosphatase phenotypes were also resistant to the heavy metals lead and cadmium. Previous work on FRC strains, including Arthrobacter, Bacillus and Rahnella spp., has demonstrated differences in tolerance to U(VI) toxicity (200 {micro}M) in the absence of organophosphate substrates. For example, Arthrobacter spp. exhibited the greatest tolerance to U(VI) while the Rahnella spp. have been shown to facilitate the precipitation of U(VI) from solution and the Bacillus spp. demonstrate the greatest sensitivity to acidic conditions and high concentrations of U(VI). PCR-based detection of FRC strains are being conducted to determine if non-specific acid phosphatases of the known molecular classes [i.e., classes A, B and C] are present in these FRC isolates. Additionally, these

  8. How Important Is the Phosphatase Activity of Sensor Kinases?

    PubMed Central

    Kenney, Linda J.

    2010-01-01

    In two-component signaling systems, phosphorylated response regulators (RRs) are often dephosphorylated by their partner kinases in order to control the in vivo concentration of phospho-RR (RR~P). This activity is easily demonstrated in vitro, but these experiments have typically used very high concentrations of the histidine kinase (HK) compared to the RR~P. Many two-component systems exhibit exquisite control over the ratio of HK to RR in vivo. The question thus arises as to whether the phosphatase activity of HKs is significant in vivo. This topic will be explored in the present review. PMID:20223700

  9. Regulation of Eye Development by Protein Serine/Threonine Phosphatases-1 and -2A.

    PubMed

    Wang, L; Yang, Y; Gong, X-D; Huang, Z-X; Nie, Q; Wang, Z-F; Ji, W-K; Hu, X-H; Hu, W-F; Gong, L-L; Zhang, L; Huang, S; Qi, R-L; Yang, T-H; Chen, Z-G; Liu, W-B; Liu, Y-Z; Li, D W-C

    2015-01-01

    The protein serine/threonine phosphatases-1 and -2A are major cellular phosphatases, playing a fundamental role in organisms from prokaryotes to eukaryotes. They contribute to 90% dephosphorylation in eukaryote proteins. In the eye, both phosphatases are highly expressed and display important functions in regulating normal eye development. Moreover, they are implicated in pathogenesis through modulation of stress-induced apoptosis. Here we review the recent progresses on these aspects.

  10. Regulation of Eye Development by Protein Serine/Threonine Phosphatases-1 and -2A.

    PubMed

    Wang, L; Yang, Y; Gong, X-D; Huang, Z-X; Nie, Q; Wang, Z-F; Ji, W-K; Hu, X-H; Hu, W-F; Gong, L-L; Zhang, L; Huang, S; Qi, R-L; Yang, T-H; Chen, Z-G; Liu, W-B; Liu, Y-Z; Li, D W-C

    2015-01-01

    The protein serine/threonine phosphatases-1 and -2A are major cellular phosphatases, playing a fundamental role in organisms from prokaryotes to eukaryotes. They contribute to 90% dephosphorylation in eukaryote proteins. In the eye, both phosphatases are highly expressed and display important functions in regulating normal eye development. Moreover, they are implicated in pathogenesis through modulation of stress-induced apoptosis. Here we review the recent progresses on these aspects. PMID:26592247

  11. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    PubMed

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete.

  12. Regan isoenzyme of alkaline phosphatase as a tumour marker for renal cell carcinoma.

    PubMed

    Bukowczan, J; Pattman, S; Jenkinson, F; Quinton, R

    2014-09-01

    Alkaline phosphatase is an enzyme present in all tissues of the human body. Several isoforms of this enzyme have been described with different catalytic nature, stability and antigenic structure. Rises in the activity of alkaline phosphatase are recognised in various states including bone diseases, liver disease, pregnancy, hyperthyroidism and malignant processes. The Regan isoenzyme, a rare variant of placental alkaline phosphatase, has been identified circulating in association with various tumours. The reported case describes a rising Regan isoform of alkaline phosphatase concentrations that led to a new diagnosis of occult renal cell carcinoma and persistently elevated activity postoperatively signposting persistent or recurrent disease.

  13. Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii.

    PubMed Central

    Quisel, J D; Wykoff, D D; Grossman, A R

    1996-01-01

    We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinhardtii in response to phosphorus deprivation. Phosphorus-deprived cells increase extra-cellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the Alkaline phosphatase activity. This phosphatase has optimal activity at pH 9.5 and a Km of 120 to 262 microns for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells. PMID:8754684

  14. Hepatic Src Homology Phosphatase 2 Regulates Energy Balance in Mice

    PubMed Central

    Nagata, Naoto; Matsuo, Kosuke; Bettaieb, Ahmed; Bakke, Jesse; Matsuo, Izumi; Graham, James; Xi, Yannan; Liu, Siming; Tomilov, Alexey; Tomilova, Natalia; Gray, Susan; Jung, Dae Young; Ramsey, Jon J.; Kim, Jason K.; Cortopassi, Gino; Havel, Peter J.

    2012-01-01

    The Src homology 2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) is a negative regulator of hepatic insulin action in mice fed regular chow. To investigate the role of hepatic Shp2 in lipid metabolism and energy balance, we determined the metabolic effects of its deletion in mice challenged with a high-fat diet (HFD). We analyzed body mass, lipid metabolism, insulin sensitivity, and glucose tolerance in liver-specific Shp2-deficient mice (referred to herein as LSHKO) and control mice fed HFD. Hepatic Shp2 protein expression is regulated by nutritional status, increasing in mice fed HFD and decreasing during fasting. LSHKO mice gained less weight and exhibited increased energy expenditure compared with control mice. In addition, hepatic Shp2 deficiency led to decreased liver steatosis, enhanced insulin-induced suppression of hepatic glucose production, and impeded the development of insulin resistance after high-fat feeding. At the molecular level, LSHKO exhibited decreased hepatic endoplasmic reticulum stress and inflammation compared with control mice. In addition, tyrosine and serine phosphorylation of total and mitochondrial signal transducer and activator of transcription 3 were enhanced in LSHKO compared with control mice. In line with this observation and the increased energy expenditure of LSHKO, oxygen consumption rate was higher in liver mitochondria of LSHKO compared with controls. Collectively, these studies identify hepatic Shp2 as a novel regulator of systemic energy balance under conditions of high-fat feeding. PMID:22619361

  15. Protein kinase and phosphatase activities of thylakoid membranes

    SciTech Connect

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg/sup 2 +/ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg/sup 2 +/ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs.

  16. Inhibition of lymphoid tyrosine phosphatase by benzofuran salicylic acids.

    PubMed

    Vang, Torkel; Xie, Yuli; Liu, Wallace H; Vidović, Dusica; Liu, Yidong; Wu, Shuangding; Smith, Deborah H; Rinderspacher, Alison; Chung, Caty; Gong, Gangli; Mustelin, Tomas; Landry, Donald W; Rickert, Robert C; Schürer, Stephan C; Deng, Shi-Xian; Tautz, Lutz

    2011-01-27

    The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds. PMID:21190368

  17. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    PubMed

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  18. Protein phosphatase Z modulates oxidative stress response in fungi.

    PubMed

    Leiter, Éva; González, Asier; Erdei, Éva; Casado, Carlos; Kovács, László; Ádám, Csaba; Oláh, Judit; Miskei, Márton; Molnar, Monika; Farkas, Ilona; Hamari, Zsuzsanna; Ariño, Joaquín; Pócsi, István; Dombrádi, Viktor

    2012-09-01

    The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.

  19. Spatial control of protein phosphatase 2A (de)methylation

    SciTech Connect

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A{sub C}) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A{sub C} antibodies revealed a good correlation with the methylation status of PP2A{sub C}, demethylated PP2A{sub C} being substantially nuclear. Throughout mitosis, demethylated PP2A{sub C} is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A{sub C} in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.

  20. Protein Phosphatase 1α Interacting Proteins in the Human Brain

    PubMed Central

    Esteves, Sara L.C.; Domingues, Sara C.; da Cruz e Silva, Odete A.B.; da Cruz e Silva, Edgar F.

    2012-01-01

    Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved. PMID:22321011

  1. Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids.

    PubMed

    Bobkova, Ekaterina V; Liu, Wallace H; Colayco, Sharon; Rascon, Justin; Vasile, Stefan; Gasior, Carlton; Critton, David A; Chan, Xochella; Dahl, Russell; Su, Ying; Sergienko, Eduard; Chung, Thomas D Y; Mustelin, Tomas; Page, Rebecca; Tautz, Lutz

    2011-02-01

    Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.

  2. Protein phosphatase 1 suppresses androgen receptor ubiquitylation and degradation.

    PubMed

    Liu, Xiaming; Han, Weiwei; Gulla, Sarah; Simon, Nicholas I; Gao, Yanfei; Cai, Changmeng; Yang, Hongmei; Zhang, Xiaoping; Liu, Jihong; Balk, Steven P; Chen, Shaoyong

    2016-01-12

    The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4-2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC).

  3. Plant species richness increases phosphatase activities in an experimental grassland

    NASA Astrophysics Data System (ADS)

    Hacker, Nina; Wilcke, Wolfgang; Oelmann, Yvonne

    2014-05-01

    Plant species richness has been shown to increase aboveground nutrient uptake requiring the mobilization of soil nutrient pools. For phosphorus (P) the underlying mechanisms for increased P release in soil under highly diverse grassland mixtures remain obscure because aboveground P storage and concentrations of inorganic and organic P in soil solution and differently reactive soil P pools are unrelated (Oelmann et al. 2011). The need of plants and soil microorganisms for P can increase the exudation of enzymes hydrolyzing organically bound P (phosphatases) which might represent an important release mechanism of inorganic P in a competitive environment such as highly diverse grassland mixtures. Our objectives were to test the effects of i) plant functional groups (legumes, grasses, non-leguminous tall and small herbs), and of (ii) plant species richness on microbial P (Pmic) and phosphatase activities in soil. In autumn 2013, we measured Pmic and alkaline phosphomonoesterase and phosphodiesterase activities in soil of 80 grassland mixtures comprising different community compositions and species richness (1, 2, 4, 8, 16, 60) in the Jena Experiment. In general, Pmic and enzyme activities were correlated (r = 0.59 and 0.46 for phosphomonoesterase and phosphodiesterase activities, respectively; p

  4. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

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

    SciTech Connect

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

    1995-07-20

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

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

    PubMed Central

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

    2015-01-01

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

  7. Environmental photoinactivation of extracellular phosphatases and the effects of dissolved organic matter.

    PubMed

    Janssen, Elisabeth M L; McNeill, Kristopher

    2015-01-20

    Alkaline phosphatases are ubiquitous extracellular enzymes in aquatic systems and play a central role in the biogeochemical cycling of phosphorus. Yet, the photochemical stability of phosphatase and effects of natural organic matter (DOM) are not completely understood. We demonstrate that phosphatase activity in natural biofilm samples decreased during sunlight exposure similar to well-defined bacterial phosphatase solutions. Direct photoinactivation was slowed by more than 50% in the presence of redox-active dissolved organic matter (DOM, 10 mgC L(–1)) or a model antioxidant (esculetin, 50 μM), even after light screening effects had been accounted for. Thus, DOM can not only inhibit enzymes (in the dark) or sensitize photodegradation by producing photochemically produced reactive intermediates but can also significantly quench direct photoinactivation of phosphatase. Our data further suggest that direct photooxidation of tryptophan residues within the protein structure are significantly involved in the photoinactivation of phosphatase because a loss of tryptophan-like fluorescence paralleled photoinactivation kinetics and because DOM acted as an antioxidant toward photoinactivation, a phenomenon recently established for the photooxidation of freely dissolved tryptophan. Thus, photoinactivation of phosphatase can be significantly slowed in the presence of naturally occurring antioxidants like DOM. The mechanistic link between tryptophan photooxidation and inactivation of phosphatase may have applicability to other extracellular enzymes but remains to be established.

  8. Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

    PubMed

    Płachno, B J; Adamec, L; Lichtscheidl, I K; Peroutka, M; Adlassnig, W; Vrba, J

    2006-11-01

    A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients.

  9. Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

    PubMed

    Thaller, M C; Schippa, S; Rossolini, G M

    1998-07-01

    Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.

  10. Identification of a Photosystem II Phosphatase Involved in Light Acclimation in Arabidopsis[W

    PubMed Central

    Samol, Iga; Shapiguzov, Alexey; Ingelsson, Björn; Fucile, Geoffrey; Crèvecoeur, Michèle; Vener, Alexander V.; Rochaix, Jean-David; Goldschmidt-Clermont, Michel

    2012-01-01

    Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree. PMID:22706287

  11. Enhancing Potato System Sustainability: Crop Rotation Impacts on Soil Phosphatase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is a species with a low efficiency of acquiring soil P. Rotation crops may potentially influence P uptake by potato by increasing soil organic acids, phosphatase activity, and microbial biomass. However, this kind of information is very limited. We measured the activities of acid phosphatase,...

  12. Protein tyrosine and serine–threonine phosphatases in the sea urchin, Strongylocentrotus purpuratus: Identification and potential functions

    PubMed Central

    Byrum, C.A.; Walton, K.D.; Robertson, A.J.; Carbonneau, S.; Thomason, R.T.; Coffman, J.A.; McClay, D.R.

    2011-01-01

    Protein phosphatases, in coordination with protein kinases, play crucial roles in regulation of signaling pathways. To identify protein tyrosine phosphatases (PTPs) and serine–threonine (ser–thr) phosphatases in the Strongylocentrotus purpuratus genome, 179 annotated sequences were studied (122 PTPs, 57 ser–thr phosphatases). Sequence analysis identified 91 phosphatases (33 conventional PTPs, 31 dual specificity phosphatases, 1 Class III Cysteine-based PTP, 1 Asp-based PTP, and 25 ser–thr phosphatases). Using catalytic sites, levels of conservation and constraint in amino acid sequence were examined. Nine of 25 receptor PTPs (RPTPs) corresponded to human, nematode, or fly homologues. Domain structure revealed that sea urchin-specific RPTPs including two, PTPRLec and PTPRscav, may act in immune defense. Embryonic transcription of each phosphatase was recorded from a high-density oligonucleotide tiling microarray experiment. Most RPTPs are expressed at very low levels, whereas nonreceptor PTPs (NRPTPs) are generally expressed at moderate levels. High expression was detected in MAP kinase phosphatases (MKPs) and numerous ser–thr phosphatases. For several expressed NRPTPs, MKPs, and ser–thr phosphatases, morpholino antisense-mediated knockdowns were performed and phenotypes obtained. Finally, to assess roles of annotated phosphatases in endomesoderm formation, a literature review of phosphatase functions in model organisms was superimposed on sea urchin developmental pathways to predict areas of functional activity. PMID:17087928

  13. Methods to monitor classical protein-tyrosine phosphatase oxidation

    PubMed Central

    Karisch, Robert; Neel, Benjamin G.

    2012-01-01

    SUMMARY Reactive oxygen species (ROS), particularly H2O2, act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling. PMID:22577968

  14. Intramolecular dynamics of structure of alkaline phosphatase from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Mazhul, Vladimir M.; Mjakinnik, Igor V.; Volkova, Alena N.

    1995-01-01

    The luminescent analysis with nano- and millisecond time resolution of intramolecular dynamics of Escherichia coli alkaline phosphatase was carried out. The effect of pH within the range 7.2 - 9.0, thermal inactivation, limited proteolysis by trypsin, binding of pyrophosphate, interconversion of enzyme and apoenzyme, the replacement of Zn2+ and Mg2+ in the active site by Cd2+ and Ni2+ on the spectral and kinetic parameters of luminescence was investigated. The essential changes of the level of nano- and millisecond dynamics of protein structure were found to correlate with the shift of enzymatic activity. The importance of small- and large-scale flexibility of protein structure for the act of enzymatic catalysis realization was shown.

  15. The influence of complexing pharmaceutical compositions on alkaline phosphatase

    NASA Astrophysics Data System (ADS)

    Atyaksheva, L. F.; Chukhrai, E. S.; Stepina, N. D.; Novikova, N. N.; Yur'eva, E. A.

    2011-06-01

    It is established that the pharmaceutical compositions xydiphon, medifon, succimer, and EDTA, which are used as complexing agents for accelerating the excretion of heavy metals from human organism, at certain concentrations inhibit enzyme alkaline phosphatase (AP). It is concluded that xydiphon and EDTA have a noticeable effect on AP activity at concentrations over 0.01 mM; medifon and succimer, at concentrations of over 0.3-0.5 mM. The enzyme's inhibition constants and type of inhibition are determined. Xydiphon is found to manifest the highest affinity to AP ( K I = 0.35 mM). It is shown by kinetic analysis that dissociative chemoinactivation of the enzyme takes place under the action of complexing agents. The corresponding kinetic parameters are calculated.

  16. Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk

    PubMed Central

    Chon, Jung-Whan; Kim, Hyunsook; Kim, Kwang-Yup

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk. PMID:27194927

  17. Significantly Elevated Liver Alkaline Phosphatase in Congestive Heart Failure

    PubMed Central

    Shamban, Leonid; Patel, Brijesh; Williams, Michael

    2014-01-01

    Congestive hepatopathy can have a mildly elevated liver profile, which should normalize with appropriate therapy. Liver specific alkaline phosphatase (ALP) in decompensated heart failure (HF) can be mildly elevated. The levels exceeding beyond the expected rise should be a concern and lead to further investigation. The literature reports insubstantial number of cases regarding significantly elevated levels of ALP and congestive hepatopathy. We report a case of a 45-year-old female with known history of severe cardiomyopathy that had persistently elevated levels of ALP. The extensive workup was negative for any specific pathology. The liver biopsy was consistent with congestive hepatopathy. The patient’s ALP levels decreased with aggressive diuretic therapy but still remained elevated.

  18. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    PubMed

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  19. Utilizing ultrafiltration to remove alkaline phosphatase from clinical analyzer water.

    PubMed

    Bôle, Julien; Mabic, Stéphane

    2006-01-01

    Alkaline phosphatase (ALP) conjugated to antibodies is often used in enzyme immunoassays (EIAs). These assays are notably sensitive to experimental conditions. A possible source of interference is bacterial ALP, which is released when bacterial contamination occurs in clinical analyzers. Preliminary experiments led to the selection of a detection kit, ALP source, and specific types of tubes for collecting water samples and performing assays. The release of ALP from various strains of bacteria identified in pure water was demonstrated (10-30 x 10(6) cfu/mL released 6-10 microU/microL). It was shown that ultrafiltration is totally efficient in removing ALP from water, while residual ALP activity (2.21 microU/microL after filtration of an ALP solution of 6.22 microU/microL) was observed after filtration using a 0.22-mum filter.

  20. Covalent Docking Predicts Substrates for Haloalkanoate Dehalogenase Superfamily Phosphatases

    PubMed Central

    2015-01-01

    Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill-suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the haloalkanoate dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding modes of known cocrystal structures and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, noncovalent docking of high-energy intermediates yielded nonproductive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides. PMID:25513739

  1. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  2. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells

    PubMed Central

    Abbasian, Nima; Burton, James O.; Herbert, Karl E.; Tregunna, Barbara-Emily; Brown, Jeremy R.; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J.; Goodall, Alison H.

    2015-01-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  3. Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.

    PubMed

    Abbasian, Nima; Burton, James O; Herbert, Karl E; Tregunna, Barbara-Emily; Brown, Jeremy R; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J; Goodall, Alison H; Bevington, Alan

    2015-09-01

    Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. PMID:25745026

  4. Phosphatase Under-Producer Mutants Have Altered Phosphorus Relations1

    PubMed Central

    Tomscha, Jennifer L.; Trull, Melanie C.; Deikman, Jill; Lynch, Jonathan P.; Guiltinan, Mark J.

    2004-01-01

    Phosphorus (P) acquisition and partitioning are essential for plant homeostasis. P is available for plant uptake when in its inorganic form (H2PO4−, or Pi), but Pi is often limiting in soils. Plants secrete acid phosphatases (APases) into the apoplastic space, which may be important for obtaining Pi from organic P sources; however, the relative importance of these enzymes for plant P nutrition has yet to be determined. We demonstrate that the root-associated APase pool is increased in Arabidopsis when Pi is limiting and document five APase isoforms secreted from Arabidopsis roots. Previously, we presented the identification of the phosphatase under-producer (pup) mutants, which have decreased in vivo root APase staining when grown under low P conditions. Here, we present the characterization of one of these, pup3, and further studies with pup1. pup3 has 49%, 38%, and 37% less specific APase activity in exudates, roots, and shoots, respectively. Root-associated APase activity is decreased by 16% in pup1 and 25% in pup3, regardless of P treatment. Two APase activity isoforms are reduced in pup3 exudates, and root and shoot isoforms are also affected. One of the two exudate isoforms is recognized by a polyclonal antibody raised to an Arabidopsis purple APase recombinant protein (AtPAP12); however, AtPAP12 transcript levels are unaffected in the mutant. The pup3 mutation was mapped to 68.4 ± 6.0 centimorgans on chromosome 5. Although P concentrations were not altered in pup1 and pup3 tissues when grown in nutrient solution in which Pi was the sole source of P, the mutants had 10% (pup1) and 17% (pup3) lower shoot P concentrations when grown in a peat-vermiculite mix in which the majority of the total P was present as organic P. Therefore, the pup defects, which include secreted APases, are functionally important for plant P nutrition. PMID:15122033

  5. Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.

    PubMed

    Yang, C; Chang, J; Gorospe, M; Passaniti, A

    1996-02-01

    Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.

  6. Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity

    PubMed Central

    Wong, J. T.; Kim, P. T. W.; Peacock, J. W.; Yau, T. Y.; Mui, A. L.-F.; Chung, S. W.; Sossi, V.; Doudet, D.; Green, D.; Ruth, T. J.; Parsons, R.; Verchere, C. B.

    2006-01-01

    Aims/hypothesis Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake. Materials and methods Insulin sensitivity in Pten heterozygous (Pten+/−) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten+/− mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3β (GSK3β), a substrate of PKB/Akt, was determined by western immunoblotting. Results Following i.p. insulin challenge, blood glucose levels in Pten+/− mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten+/− mice. Enhanced glucose uptake was observed both in Pten+/− myocytes and in skeletal muscle of Pten+/− mice by PET. PKB and GSK3β phosphorylation was enhanced and prolonged in Pten+/− myocytes. Conclusions/interpretation Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten+/− mice. PMID:17195063

  7. Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

    PubMed

    Kent, C; Vagelos, P R

    1976-06-17

    Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

  8. Cortisol modification of HeLa 65 alkaline phosphatase. Decreased phosphate content of the induced enzyme.

    PubMed

    Bazzell, K L; Price, G; Tu, S; Griffin, M

    1976-01-15

    Alkaline phosphatase activity of HeLa cells is increased 5-20-fold during growth in medium with cortisol. The increase in enzyme activity is due to an enhanced catalytic efficiency rather than an increase in alkaline phosphatase protein in induced cells. In the present study the chemical composition of control and induced forms of alkaline phosphatase were investigated to determine the enzyme modification that may be responsible for the increased catalytic activity. HeLa alkaline phosphatase is a phosphoprotein and the induced form of the enzyme has approximately one-half of the phosphate residues associated with control enzyme. The decrease in phosphate residues of the enzyme apparently alters its catalytic activity. Other chemical components of purified alkaline phosphatase from control and induced cells are similar; these include sialic acid, hexosamine and sulfhydryl residues. PMID:1248469

  9. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

  10. Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus.

    PubMed Central

    Weinberg, R A; Zusman, D R

    1990-01-01

    One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and

  11. Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription

    PubMed Central

    Ammosova, Tatyana; Washington, Kareem; Debebe, Zufan; Brady, John; Nekhai, Sergei

    2005-01-01

    Background HIV-1 Tat protein recruits human positive transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to HIV-1 transactivation response (TAR) RNA. CDK9 is maintained in dephosphorylated state by TFIIH and undergo phosphorylation upon the dissociation of TFIIH. Thus, dephosphorylation of CDK9 prior to its association with HIV-1 preinitiation complex might be important for HIV-1 transcription. Others and we previously showed that protein phosphatase-2A and protein phosphatase-1 regulates HIV-1 transcription. In the present study we analyze relative contribution of PP2A and PP1 to dephosphorylation of CDK9 and to HIV-1 transcription in vitro and in vivo. Results In vitro, PP2A but not PP1 dephosphorylated autophosphorylated CDK9 and reduced complex formation between P-TEFb, Tat and TAR RNA. Inhibition of PP2A by okadaic acid inhibited basal as well as Tat-induced HIV-1 transcription whereas inhibition of PP1 by recombinant nuclear inhibitor of PP1 (NIPP1) inhibited only Tat-induced transcription in vitro. In cultured cells, low concentration of okadaic acid, inhibitory for PP2A, only mildly inhibited Tat-induced HIV-1 transcription. In contrast Tat-mediated HIV-1 transcription was strongly inhibited by expression of NIPP1. Okadaic acid induced phosphorylation of endogenous as well transiently expressed CDK9, but this induction was not seen in the cells expressing NIPP1. Also the okadaic acid did not induce phosphorylation of CDK9 with mutation of Thr 186 or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues involved in autophosphorylation of CDK9. Conclusion Our results indicate that although PP2A dephosphorylates autophosphorylated CDK9 in vitro, in cultured cells PP1 is likely to dephosphorylate CDK9 and contribute to the regulation of activated HIV-1 transcription. PMID:16048649

  12. Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

    PubMed

    Cesari, I M; Ballén, D E; Mendoza, L; Ferrer, A; Pointier, J-P; Kombila, M; Richard-Lenoble, D; Théron, A

    2014-04-01

    To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

  13. Three new pigment protein tyrosine phosphatases inhibitors from the insect parasite fungus Cordyceps gracilioides: terreusinone A, pinophilin C and cryptosporioptide A.

    PubMed

    Wei, Pei-Yao; Liu, Lin-Xia; Liu, Ting; Chen, Chuan; Luo, Du-Qiang; Shi, Bao-Zhong

    2015-01-01

    Three new pigment compounds--terreusinone A (1), pinophilin C (2) and cryptosporioptide A (3)-were isolated from a solid culture of Cordyceps gracilioides. The structures of these compounds were determined by extensive spectroscopic analysis including HRESIMS, 1D- and 2D-NMR. The structure of terreusinone A (1) was further confirmed by single-crystal X-ray crystallographic diffraction analysis. In an in vitro activity assay, 1, 2 and 3 exhibited high inhibitory activity against PTP1B, SHP2, CDC25B, LAR and SHP1. Terreusinone A (1) inhibited PTP1B, SHP2, CDC25B, LAR and SHP1 enzyme with IC50 values 12.5, >50, 4.1, 10.6, 5.6 µg/mL, respectively; pinophilin C (2) with IC50 values 6.8, 8.0, 4.5, 4.7, 3.4 µg/mL, respectively; and cryptosporioptide A (3) with IC50 values 7.3, 5.7, 7.6, >50, 4.9 µg/mL, respectively. PMID:25849805

  14. The dynamics of alkaline phosphatase activity during operculum regeneration in the polychaete Pomatoceros lamarckii.

    PubMed

    Szabó, Réka; Ferrier, David E K

    2014-01-01

    Alkaline phosphatase enzymes are found throughout the living world and fulfil a variety of functions. They have been linked to regeneration, stem cells and biomineralisation in a range of animals. Here we describe the pattern of alkaline phosphatase activity in a spiralian appendage, the operculum of the serpulid polychaete Pomatoceros lamarckii. The P. lamarckii operculum is reinforced by a calcified opercular plate and is capable of rapid regeneration, making it an ideal model system to study these key processes in annelids. Alkaline phosphatase activity is present in mesodermal tissues of both intact and regenerating opercular filaments, in a strongly regionalised pattern correlated with major morphological features. Based on the lack of epidermal activity and the broad distribution of staining in mesodermal tissues, calcification- or stem cell-specific roles are unlikely. Transcriptomic data reveal that at least four distinct genes contribute to the detected activity. Opercular alkaline phosphatase activity is sensitive to levamisole. Phylogenetic analysis of metazoan alkaline phosphatases indicates homology of the P. lamarckii sequences to other annelid alkaline phosphatases, and shows that metazoan alkaline phosphatase evolution was characterised by extensive lineage-specific duplications. PMID:25690977

  15. Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay.

    PubMed

    Sugiyama, Yasunori; Yamashita, Sho; Uezato, Yuuki; Senga, Yukako; Katayama, Syouichi; Goshima, Naoki; Shigeri, Yasushi; Sueyoshi, Noriyuki; Kameshita, Isamu

    2016-11-15

    To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca(2+)/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities. PMID:27565380

  16. Alkaline phosphatase activity in salivary gland cells of Rhodnius neglectus and R. prolixus (Hemiptera, Triatominae).

    PubMed

    Lima-Oliveira, A P M; Alevi, K C C; Anhê, A C B; Azeredo-Oliveira, M T V

    2016-07-29

    Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease.

  17. Alkaline phosphatase activity in salivary gland cells of Rhodnius neglectus and R. prolixus (Hemiptera, Triatominae).

    PubMed

    Lima-Oliveira, A P M; Alevi, K C C; Anhê, A C B; Azeredo-Oliveira, M T V

    2016-01-01

    Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease. PMID:27525888

  18. WIP1 phosphatase as a potential therapeutic target in neuroblastoma.

    PubMed

    Richter, Mark; Dayaram, Tajhal; Gilmartin, Aidan G; Ganji, Gopinath; Pemmasani, Sandhya Kiran; Van Der Key, Harjeet; Shohet, Jason M; Donehower, Lawrence A; Kumar, Rakesh

    2015-01-01

    The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with

  19. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    NASA Astrophysics Data System (ADS)

    Martinez, R.; Wu, C. H.; Beazley, M. J.; Andersen, G. L.; Hazen, T. C.; Taillefert, M.; Sobecky, P. A.

    2011-12-01

    Soils and groundwater contaminated with heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. Due to the scale of environmental contamination, in situ sequestration of heavy metals and radionuclides remain the most cost-effective strategy for remediation. We are currently investigating a remediation approach that utilizes periplasmic and extracellular microbial phosphatase activity of soil bacteria capable promoting in situ uranium phosphate sequestration. Our studies focus on the contaminated soils from the DOE Field Research Center (ORFRC) in Oak Ridge, TN. We have previously demonstrated that ORFRC strains with phosphatase-positive phenotypes were capable of promoting the precpitation of >95% U(VI) as a low solubility phosphate mineral during growth on glycerol phosphate as a sole carbon and phosphorus source. Here we present culture-independent soil slurry studies aimed at understanding microbial community dynamics resulting from exogenous organophosphate additions. Soil slurries containing glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P) and nitrate as the sole C, P and N sources were incubated under oxic growth conditions at pH 5.5 or pH 6.8. Following treatments, total DNA was extracted and prokaryotic diversity was assessed using high-density 16S oligonucleotide microarray (PhyloChip) analysis. Treatments at pH 5.5 and pH 6.8 amended with G2P required 36 days to accumulate 4.8mM and 2.2 mM phosphate, respectively. In contrast, treatments at pH 5.5 and pH 6.8 amended with G3P accumulated 8.9 mM and 8.7 mM phosphate, respectively, after 20 days. A total of 2120 unique taxa representing 46 phyla, 66 classes, 110 orders, and 186 families were detected among all treatment conditions. The phyla that significantly (P<0.05) increased in abundance relative to incubations lacking organophosphate amendments included: Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria. Members from the classes Bacteroidetes

  20. The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

    NASA Technical Reports Server (NTRS)

    Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

  1. Purification of prostatic acid phosphatase (PAP) for structural and functional studies.

    PubMed

    Herrala, Annakaisa M; Quintero, Ileana B; Vihko, Pirkko T

    2013-01-01

    High-scale purification methods are required for several protein studies such as crystallography, mass spectrometry, circular dichroism, and function. Here we describe a purification method for PAP based on anion exchange, L-(+)-tartrate affinity, and gel filtration chromatographies. Acid phosphatase activity and protein concentration were measured for each purification step, and to collect the fractions with the highest acid phosphatase activity the p-nitrophenyl phosphate method was used. The purified protein obtained by the procedure described here was used for the determination of the first reported three-dimensional structure of prostatic acid phosphatase.

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

    NASA Astrophysics Data System (ADS)

    Fujiki, Hirota; Suganuma, Masami

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

  3. Allosteric substrate switching in a voltage sensing lipid phosphatase

    PubMed Central

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  4. Zn2Mg alkaline phosphatase in an early ptolemeic mummy.

    PubMed

    Kaup, Y; Baumer, U; Koller, J; Hedges, R E; Werner, H; Hartmann, H J; Etspüler, H; Weser, U

    1994-01-01

    Bone samples of a ptolemeic mummy have been employed to study the mode of conservation on the intactness of Zn2Mg alkaline phosphatase in both structure and catalytic activity. A protein of M(r) = 190 +/- 10 kDa being identical to the 200 kDa enzyme of fresh human bones was successfully isolated. Regardless of age 200 kDa protein bands and a distinct subunit at 60 kDa were seen in SDS-PAGE electrophoresis. The 200 kDa band was also monitored by activity staining. The specific activity was 120 mU/mg and 65% of the respective activity obtained in the identical preparation using fresh human tibia or rib. The enzymic activity was inhibited in the presence of 1,10-phenanthroline and L-homoarginine. Radiocarbon dating supported the assignment of the mummy to the early ptolemeic period. Among the many bactericidal and fungicidal components employed for mummification were aromatic alcohols, mono- and sesquiterpenes. Pistachio resin was the major balm resin used. The microbiological sterility of the bone surface was ascertained by independent bacterial and fungal examinations.

  5. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function.

    PubMed

    Kamceva, Marija; Benedict, Jessie; Nairn, Angus C; Lombroso, Paul J

    2016-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity. PMID:27190655

  6. Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

    PubMed Central

    Frezza, Ludivine; Sandtner, Walter

    2013-01-01

    Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing. PMID:24127524

  7. Expression of acid phosphatase in the seminiferous epithelium of vertebrates.

    PubMed

    Peruquetti, R L; Taboga, S R; Azeredo-Oliveira, M T V

    2010-01-01

    Acid phosphatases (AcPs) are known to provide phosphate to tissues that have high energy requirements, especially during development, growth and maturation. During spermatogenesis AcP activity is manifested in heterophagous lysosomes of Sertoli cells. This phagocytic function appears to be hormone-independent. We examined the expression pattern of AcP during the reproductive period of four species belonging to different vertebrate groups: Tilapia rendalli (Teleostei, Cichlidae), Dendropsophus minutus (Amphibia, Anura), Meriones unguiculatus (Mammalia, Rodentia), and Oryctolagus cuniculus (Mammalia, Lagomorpha). To demonstrate AcP activity, cryosections were processed for enzyme histochemistry by a modification of the method of Gömöri. AcP activity was similar in the testes of these four species. Testes of T. rendalli, D. minutus and M. unguiculatus showed an intense reaction in the Sertoli cell region. AcP activity was detected in the testes of D. minutus and O. cuniculus in seminiferous epithelium regions, where cells are found in more advanced stages of development. The seminiferous epithelium of all four species exhibited AcP activity, mainly in the cytoplasm of either Sertoli cells or germ cells. These findings reinforce the importance of AcP activity during the spermatogenesis process in vertebrates. PMID:20391346

  8. Reliable Digital Single Molecule Electrochemistry for Ultrasensitive Alkaline Phosphatase Detection.

    PubMed

    Wu, Zhen; Zhou, Chuan-Hua; Pan, Liang-Jun; Zeng, Tao; Zhu, Lian; Pang, Dai-Wen; Zhang, Zhi-Ling

    2016-09-20

    Single molecule electrochemistry (SME) has gained much progress in fundamental studies, but it is difficult to use in practice due to its less reliability. We have solved the reliability of single molecule electrochemical detection by integration of digital analysis with efficient signal amplification of enzyme-induced metallization (EIM) together with high-throughput parallelism of microelectrode array (MA), establishing a digital single molecule electrochemical detection method (dSMED). Our dSMED has been successfully used for alkaline phosphatase (ALP) detection in the complex sample of liver cancer cells. Compared to direct measurement of the oxidation current of enzyme products, EIM can enhance signals by about 100 times, achieving signal-to-background ratio high enough for single molecule detection. The integration of digital analysis with SME can further decrease the detection limit of ALP to 1 aM relative to original 50 aM, enabling dSMED to be sensitively, specifically and reliably applied in liver cancer cells. The presented dSMED is enormously promising in exploring physical and chemical properties of single molecules, single biomolecular detection, or single-cell analysis.

  9. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    PubMed

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis. PMID:26878552

  10. Structural basis of protein phosphatase 2A stable latency

    PubMed Central

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

    2013-01-01

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

  11. Structure of the Protein Phosphatase 2A Holoenzyme

    SciTech Connect

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

    2006-01-01

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

  12. Phosphatase and Tensin Homologue: Novel Regulation by Developmental Signaling

    PubMed Central

    Jerde, Travis J.

    2015-01-01

    Phosphatase and tensin homologue (PTEN) is a critical cell endogenous inhibitor of phosphoinositide signaling in mammalian cells. PTEN dephosphorylates phosphoinositide trisphosphate (PIP3), and by so doing PTEN has the function of negative regulation of Akt, thereby inhibiting this key intracellular signal transduction pathway. In numerous cell types, PTEN loss-of-function mutations result in unopposed Akt signaling, producing numerous effects on cells. Numerous reports exist regarding mutations in PTEN leading to unregulated Akt and human disease, most notably cancer. However, less is commonly known about nonmutational regulation of PTEN. This review focuses on an emerging literature on the regulation of PTEN at the transcriptional, posttranscriptional, translational, and posttranslational levels. Specifically, a focus is placed on the role developmental signaling pathways play in PTEN regulation; this includes insulin-like growth factor, NOTCH, transforming growth factor, bone morphogenetic protein, wnt, and hedgehog signaling. The regulation of PTEN by developmental mediators affects critical biological processes including neuronal and organ development, stem cell maintenance, cell cycle regulation, inflammation, response to hypoxia, repair and recovery, and cell death and survival. Perturbations of PTEN regulation consequently lead to human diseases such as cancer, chronic inflammatory syndromes, developmental abnormalities, diabetes, and neurodegeneration. PMID:26339505

  13. An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily

    PubMed Central

    van Loo, Bert; Jonas, Stefanie; Babtie, Ann C.; Benjdia, Alhosna; Berteau, Olivier; Hyvönen, Marko; Hollfelder, Florian

    2010-01-01

    We report a catalytically promiscuous enzyme able to efficiently promote the hydrolysis of six different substrate classes. Originally assigned as a phosphonate monoester hydrolase (PMH) this enzyme exhibits substantial second-order rate accelerations ((kcat/KM)/kw), ranging from 107 to as high as 1019, for the hydrolyses of phosphate mono-, di-, and triesters, phosphonate monoesters, sulfate monoesters, and sulfonate monoesters. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature, and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half-lives) range from 200 days to 105 years under near neutrality. The substantial rate accelerations for a set of relatively difficult reactions suggest that efficient catalysis is not necessarily limited to efficient stabilization of just one transition state. The crystal structure of PMH identifies it as a member of the alkaline phosphatase superfamily. PMH encompasses four of the native activities previously observed in this superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. The functional links between superfamily activities can be presumed to have played a role in functional evolution by gene duplication. PMID:20133613

  14. Nanoceria particles as catalytic amplifiers for alkaline phosphatase assays.

    PubMed

    Hayat, Akhtar; Andreescu, Silvana

    2013-11-01

    We propose a novel system to enhance detection sensitivity of alkaline phosphatase (ALP) in electrochemical assays by using nanoceria particles as redox active catalytic amplifiers of ALP signals. The catalytic activity of nanoceria particles attributed to their dual oxidation state Ce(4+)/Ce(3+) and high oxygen mobility enabled oxidation of the products of the ALP-catalyzed reaction. A suite of spectroscopic and electrochemical methods, including UV-vis spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and cyclic voltammetry (CV) were used to characterize the interaction of nanoceria with the ALP-generated products. Spectrometric experiments demonstrate change in the oxidation state of nanoceria upon exposure to the hydrolytic products of ALP. Three enzymatically generated products of commonly used ALP substrates were detected at a screen printing electrode surface in the presence of nanoceria. Electrochemical experiments demonstrate signal amplification of the ALP activity assay by nanoceria for all three products, demonstrating remarkable sensitivity of this assay. The assay was optimized with respect to pH and buffer composition. Analytical characterization of the nanoceria-based ALP activity assay was established using a 1-naphthyl phosphate substrate. The proposed strategy can find widespread applications in sensing schemes involving ALP. PMID:24053108

  15. Interplay between intestinal alkaline phosphatase, diet, gut microbes and immunity.

    PubMed

    Estaki, Mehrbod; DeCoffe, Daniella; Gibson, Deanna L

    2014-11-14

    Intestinal alkaline phosphatase (IAP) plays an essential role in intestinal homeostasis and health through interactions with the resident microbiota, diet and the gut. IAP's role in the intestine is to dephosphorylate toxic microbial ligands such as lipopolysaccharides, unmethylated cytosine-guanosine dinucleotides and flagellin as well as extracellular nucleotides such as uridine diphosphate. IAP's ability to detoxify these ligands is essential in protecting the host from sepsis during acute inflammation and chronic inflammatory conditions such as inflammatory bowel disease. Also important in these complications is IAP's ability to regulate the microbial ecosystem by forming a complex relationship between microbiota, diet and the intestinal mucosal surface. Evidence reveals that diet alters IAP expression and activity and this in turn can influence the gut microbiota and homeostasis. IAP's ability to maintain a healthy gastrointestinal tract has accelerated research on its potential use as a therapeutic agent against a multitude of diseases. Exogenous IAP has been shown to have beneficial effects when administered during ulcerative colitis, coronary bypass surgery and sepsis. There are currently a handful of human clinical trials underway investigating the effects of exogenous IAP during sepsis, rheumatoid arthritis and heart surgery. In light of these findings IAP has been marked as a novel agent to help treat a variety of other inflammatory and infectious diseases. The purpose of this review is to highlight the essential characteristics of IAP in protection and maintenance of intestinal homeostasis while addressing the intricate interplay between IAP, diet, microbiota and the intestinal epithelium.

  16. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  17. Regulation of receptor protein-tyrosine phosphatase dimerization.

    PubMed

    van der Wijk, Thea; Blanchetot, Christophe; den Hertog, Jeroen

    2005-01-01

    Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.

  18. Phosphoglycolate phosphatase of spinach acts as a phosphoenzyme

    SciTech Connect

    Rose, Z.B.; Seal, S.N.

    1987-05-01

    When /sup 32/P-glycolate and phosphoglycolate phosphatase from spinach are mixed, /sup 32/P is incorporated into acid precipitated protein. Properties that relate this phosphorylation to the enzyme are: The K/sub m/ value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the /sup 32/P appearing in the phosphoenzyme is diluted by unlabeled P-glycolate or the alternative substrate, ethyl-P; the activator Cl/sup -/ enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of /sup 32/P-enzyme; and /sup 32/P is lost from the enzyme when /sup 32/P-glycolate is consumed. The acid denatured phosphorylated protein is a molecule of 34,000 Da, which is half of the molecular weight of the native protein and is similar in size to the labeled band that is seen on SDS-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acyl-phosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by /sup 32/P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms.

  19. Pregnancy-secreted Acid phosphatase, uteroferrin, enhances fetal erythropoiesis.

    PubMed

    Ying, Wei; Wang, Haiqing; Bazer, Fuller W; Zhou, Beiyan

    2014-11-01

    Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage. PMID:25093463

  20. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    PubMed Central

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  1. Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface

    SciTech Connect

    Taillefert, Martial

    2015-04-01

    This project investigated the geochemical and microbial processes associated with the biomineralization of radionuclides in subsurface soils. During this study, it was determined that microbial communities from the Oak Ridge Field Research subsurface are able to express phosphatase activities that hydrolyze exogenous organophosphate compounds and result in the non-reductive bioimmobilization of U(VI) phosphate minerals in both aerobic and anaerobic conditions. The changes of the microbial community structure associated with the biomineralization of U(VI) was determined to identify the main organisms involved in the biomineralization process, and the complete genome of two isolates was sequenced. In addition, it was determined that both phytate, the main source of natural organophosphate compounds in natural environments, and polyphosphate accumulated in cells could also be hydrolyzed by native microbial population to liberate enough orthophosphate and precipitate uranium phosphate minerals. Finally, the minerals produced during this process are stable in low pH conditions or environments where the production of dissolved inorganic carbon is moderate. These findings suggest that the biomineralization of U(VI) phosphate minerals is an attractive bioremediation strategy to uranium bioreduction in low pH uranium-contaminated environments. These efforts support the goals of the SBR long-term performance measure by providing key information on "biological processes influencing the form and mobility of DOE contaminants in the subsurface".

  2. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    SciTech Connect

    Sobecky, Patricia A.

    2015-04-06

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  3. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function

    PubMed Central

    Lombroso, Paul J.

    2016-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity. PMID:27190655

  4. Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

    Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

  5. Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.

    PubMed

    Montgomery, D M; Dean, A C; Wiffen, P; Macaskie, L E

    1995-10-01

    The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO4(2-) liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii ('training'), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.

  6. Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

    PubMed

    Uhrig, R Glen; Moorhead, Greg B

    2011-12-01

    Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

  7. A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase--a dual-specific protein tyrosine phosphatase.

    PubMed

    Jeong, Myeong Seon; Kim, Eunha; Kang, Hyo Jin; Choi, Eun Joung; Cho, Alvin R; Chung, Sang J; Park, Seung Bum

    2012-07-01

    We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).

  8. Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate.

    PubMed

    Lountos, George T; Cherry, Scott; Tropea, Joseph E; Waugh, David S

    2015-02-01

    4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in complex with pNPP. The structure illuminates the molecular basis for substrate binding and may also facilitate the structure-assisted development of DUSP22 inhibitors.

  9. Structure of human dual-specificity phosphatase 27 at 2.38 Å resolution

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2011-05-01

    The X-ray crystal structure of human dual-specificity phosphatase 27 (DUSP27) is reported at 2.38 Å resolution. There are over 100 genes in the human genome that encode protein tyrosine phosphatases (PTPs) and approximately 60 of these are classified as dual-specificity phosphatases (DUSPs). Although many dual-specificity phosphatases are still not well characterized, novel functions have been discovered for some of them that have led to new insights into a variety of biological processes and the molecular basis for certain diseases. Indeed, as the functions of DUSPs continue to be elucidated, a growing number of them are emerging as potential therapeutic targets for diseases such as cancer, diabetes and inflammatory disorders. Here, the overexpression, purification and structure determination of DUSP27 at 2.38 Å resolution are presented.

  10. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria.

    PubMed

    Macaskie, L E; Bonthrone, K M; Rouch, D A

    1994-08-15

    A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.

  11. Cloning and sequencing of human intestinal alkaline phosphatase cDNA

    SciTech Connect

    Berger, J.; Garattini, E.; Hua, J.C.; Udenfriend, S.

    1987-02-01

    Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme.

  12. Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase

    SciTech Connect

    Howard, A.D.; Berger, J.; Gerber, L.; Familletti, P.; Udenfriend, S.

    1987-09-01

    Placental alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because, they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with (/sup 14/C)ethanolamine, (/sup 14/C)myristic acid, or myo(/sup 3/H)inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase.

  13. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    SciTech Connect

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-08-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G (IgG) complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table.

  14. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  15. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.

    PubMed

    Rininsland, Frauke; Xia, Wensheng; Wittenburg, Shannon; Shi, Xiaobo; Stankewicz, Casey; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-10-26

    An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation.

  16. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. )

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  17. Protein phosphatase 2A in stretch-induced endothelial cell proliferation

    NASA Technical Reports Server (NTRS)

    Murata, K.; Mills, I.; Sumpio, B. E.

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  18. Structure of human dual-specificity phosphatase 7, a potential cancer drug target

    PubMed Central

    Lountos, George T.; Austin, Brian P.; Tropea, Joseph E.; Waugh, David S.

    2015-01-01

    Human dual-specificity phosphatase 7 (DUSP7/Pyst2) is a 320-residue protein that belongs to the mitogen-activated protein kinase phosphatase (MKP) subfamily of dual-specificity phosphatases. Although its precise biological function is still not fully understood, previous reports have demonstrated that DUSP7 is overexpressed in myeloid leukemia and other malignancies. Therefore, there is interest in developing DUSP7 inhibitors as potential therapeutic agents, especially for cancer. Here, the purification, crystallization and structure determination of the catalytic domain of DUSP7 (Ser141–Ser289/C232S) at 1.67 Å resolution are reported. The structure described here provides a starting point for structure-assisted inhibitor-design efforts and adds to the growing knowledge base of three-dimensional structures of the dual-specificity phosphatase family. PMID:26057789

  19. Stabilization of human prostate acid phosphatase by cross-linking with diimidoesters.

    PubMed

    Wasylewska, E; Dulińska, J; Trubetskoy, V S; Torchilin, V P; Ostrowski, W S

    1987-01-01

    1. Modification of dimeric human prostate acid phosphatase (EC 3.1.3.2) by diimidoesters leads to the formation of water-soluble preparations of high enzymatic activity, resistant to denaturing agents. 2. Monomeric, dimeric, trimeric and tetrameric species were found in SDS-polyacrylamide gel electrophoresis of the phosphatase cross-linked with dimethyl-suberimidate, and dimeric, trimeric and tetrameric enzymatically active species on thin-layer Sephadex 200 gel filtration. This molecular pattern evidenced formation of the inter-subunit covalent linkages. All molecular forms are immunoreactive against the polyclonal rabbit anti-phosphatase antibodies. 3. The catalytic properties of the modified phosphatase are almost the same as those of the native enzyme. Differences in the optical properties between the modified and the native enzymes point to slight conformational transitions in the modified enzyme.

  20. EX VIVIO DETECTION OF KINASE AND PHOSPHATASE ACTIVITIES IN HUMAN BRONCHIAL BIOPSIES

    EPA Science Inventory

    Protein phosphorylation is a posttranslational modification involved in every aspect cellular function. Levels of protein phosphotyrosine, phosphoserine and phosphothreonine are regulated by the opposing activities of kinases and phosphatases, the expression of which can be alt...

  1. Identification of a mammalian-type phosphatidylglycerophosphate phosphatase in the Eubacterium Rhodopirellula baltica.

    PubMed

    Teh, Phildrich G; Chen, Mark J; Engel, James L; Worby, Carolyn A; Manning, Gerard; Dixon, Jack E; Zhang, Ji

    2013-02-15

    Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs. PMID:23293031

  2. Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity.

    PubMed

    Ip, Laura Ren Huey; Gewinner, Christina Anja

    2016-01-01

    Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.

  3. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.

    PubMed

    Hirschi, Alexander; Cecchini, Matthew; Steinhardt, Rachel C; Schamber, Michael R; Dick, Frederick A; Rubin, Seth M

    2010-09-01

    The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking. PMID:20694007

  4. Control of placental alkaline phosphatase gene expression in HeLa cells: induction of synthesis by prednisolone and sodium butyrate

    SciTech Connect

    Chou, J.Y.; Takahashi, S.

    1987-06-16

    HeLa S/sub 3/ cells produce an alkaline phosphatase indistinguishable from the enzyme from human term placenta. The phosphatase activity in these cells was induced by both prednisolone and sodium butyrate. Both agents stimulated de novo synthesis of the enzyme. The increase in phosphatase activity paralleled the increase in immunoactivity and biosynthesis of placental alkaline phosphatase. The fully processed phosphatase monomer in control, prednisolone-treated or butyrate-treated cells was a 64.5 K polypeptide, measured by both incorporation of L-(/sup 35/S)methionine into enzyme protein and active-site labeling. The 64.5K polypeptide was formed by the incorporation of additional N-acetylneuraminic acid moieties to a precursor polypeptide of 61.5K. However, this biosynthetic pathway was identified only in butyrate-treated cells. In prednisolone-treated cells, the processing of 61.5K to 64.5K monomer was accelerated, and the presence of the 61.5 precursor could only be detected by either neuraminidase or monensin treatment. Phosphatase mRNA which comigrated with the term placental alkaline phosphatase mRNA of 2.7 kilobases was induced in the presence of either prednisolone or butyrate. Alkaline phosphatase mRNA is untreated HeLa S/sub 3/ cells migrated slightly faster than the term placental alkaline phosphatase mRNA. Butyrate also induced a second still faster migrating alkaline phosphatase mRNA. Both prednisolone and butyrate increased the steady-state levels of placental alkaline phosphatase mRNA. The data indicate that the increase in phosphatase mRNA by prednisolone and butyrate resulted in the induction of alkaline phosphatase activity and biosynthesis in HeLa S/sub 3/ cells. Furthermore, both agents induced the expression of different alkaline phosphatase gene transcripts without altering its protein product.

  5. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    PubMed

    Lallès, Jean-Paul

    2016-07-01

    The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

  6. Dairy products and the French paradox: Could alkaline phosphatases play a role?

    PubMed

    Lallès, Jean-Paul

    2016-07-01

    The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

  7. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

    PubMed

    Smyth, Susan S; Sciorra, Vicki A; Sigal, Yury J; Pamuklar, Zehra; Wang, Zuncai; Xu, Yong; Prestwich, Glenn D; Morris, Andrew J

    2003-10-31

    Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production. PMID:12909631

  8. The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

    PubMed

    Wadham, Carol; Gamble, Jennifer R; Vadas, Mathew A; Khew-Goodall, Yeesim

    2003-06-01

    Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion. PMID:12808048

  9. Serum alkaline phosphatase negatively affects endothelium-dependent vasodilation in naïve hypertensive patients.

    PubMed

    Perticone, Francesco; Perticone, Maria; Maio, Raffaele; Sciacqua, Angela; Andreucci, Michele; Tripepi, Giovanni; Corrao, Salvatore; Mallamaci, Francesca; Sesti, Giorgio; Zoccali, Carmine

    2015-10-01

    Tissue nonspecific alkaline phosphatase, promoting arterial calcification in experimental models, is a powerful predictor of total and cardiovascular mortality in general population and in patients with renal or cardiovascular diseases. For this study, to evaluate a possible correlation between serum alkaline phosphatase levels and endothelial function, assessed by strain gauge plethysmography, we enrolled 500 naïve hypertensives divided into increasing tertiles of alkaline phosphatase. The maximal response to acetylcholine was inversely related to alkaline phosphatase (r=−0.55; P<0.001), and this association was independent (r=−0.61; P<0.001) of demographic and classical risk factors, body mass index, estimated glomerular filtration rate, serum phosphorus and calcium, C-reactive protein, and albuminuria. At multiple logistic regression analysis, the risk of endothelial dysfunction was ≈3-fold higher in patients in the third tertile than that of patients in the first tertile. We also tested the combined role of alkaline phosphatase and serum phosphorus on endothelial function. The steepness of the alkaline phosphatase/vasodilating response to acetylcholine relationship was substantially attenuated (P<0.001) in patients with serum phosphorus above the median value when compared with patients with serum phosphorus below the median (−5.0% versus −10.2% per alkaline phosphatase unit, respectively), and this interaction remained highly significant (P<0.001) after adjustment of all the previously mentioned risk factors. Our data support a strong and significant inverse relationship between alkaline phosphatase and endothelium-dependent vasodilation, which was attenuated by relatively higher serum phosphorus levels.

  10. Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

    PubMed

    Daum, G; Schmid, B; MacKintosh, C; Cohen, P; Hofer, H W

    1992-07-13

    In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a. PMID:1321672

  11. Cancerous inhibitor of protein phosphatase 2A determines bortezomib-induced apoptosis in leukemia cells

    PubMed Central

    Liu, Chun-Yu; Shiau, Chung-Wai; Kuo, Hsin-Yu; Huang, Hsiang-Po; Chen, Ming-Huang; Tzeng, Cheng-Hwai; Chen, Kuen-Feng

    2013-01-01

    The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib’s downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells. PMID:22983581

  12. Effects of multivalent cations on cell wall-associated acid phosphatase activity

    SciTech Connect

    Tu, S.I.; Brouillette, J.N.; Nagahashi, G.; Kumosinski, T.F.

    1988-09-01

    Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu/sup 2 +/, Mg/sup 2 +/, Za/sup 2 +/, and Mn/sup 2 +/; unaffected by Ba/sup 2 +/, Cd/sup 2 +/, and Pb/sup 2 +/; and inhibited by Al/sup 3 +/. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg/sup 2 +/. On the other hand, in the case of corn root cells walls (CCW), only inhibition of the bound acid phosphatase by Al/sup 3 +/ and Hg/sup 2 +/ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg/sup 2 +/. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca/sup 2 +/ significantly reduced the effects of Hg/sup 2 +/ or Al/sup 3 +/, but not Mg/sup 2 +/, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg/sup 2 +/ or Al/sup 3 +/ which caused a Ca/sup 2 +/-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.

  13. Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is ‘RHA’ unlike ‘RHG’ found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. Results Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. Conclusion Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic

  14. Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C.

    PubMed

    Low, M G; Finean, J B

    1977-10-01

    Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant.

  15. Protein-tyrosine phosphatases: biological function, structural characteristics, and mechanism of catalysis.

    PubMed

    Zhang, Z Y

    1998-01-01

    The protein-tyrosine phosphatases (PTPases) superfamily consists of tyrosine-specific phosphatases, dual specificity phosphatases, and the low-molecular-weight phosphatases. They are modulators of signal transduction pathways that regulate numerous cell functions. Malfunction of PTPases have been linked to a number of oncogenic and metabolic disease states, and PTPases are also employed by microbes and viruses for pathogenicity. There is little sequence similarity among the three subfamilies of phosphatases. Yet, three-dimensional structural data show that they share similar conserved structural elements, namely, the phosphate-binding loop encompassing the PTPase signature motif (H/V)C(X)5R(S/T) and an essential general acid/base Asp residue on a surface loop. Biochemical experiments demonstrate that phosphatases in the PTPase superfamily utilize a common mechanism for catalysis going through a covalent thiophosphate intermediate that involves the nucleophilic Cys residue in the PTPase signature motif. The transition states for phosphoenzyme intermediate formation and hydrolysis are dissociative in nature and are similar to those of the solution phosphate monoester reactions. One strategy used by these phosphatases for transition state stabilization is to neutralize the developing negative charge in the leaving group. A conformational change that is restricted to the movement of a flexible loop occurs during the catalytic cycle of the PTPases. However, the relationship between loop dynamics and enzyme catalysis remains to be established. The nature and identity of the rate-limiting step in the PTPase catalyzed reaction requires further investigation and may be dependent on the specific experimental conditions such as temperature, pH, buffer, and substrate used. In-depth kinetic and structural analysis of a representative number of phosphatases from each group of the PTPase superfamily will most likely continue to yield insightful mechanistic information that may be

  16. Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

    PubMed

    Silver, Dylan M; Silva, Leslie P; Issakidis-Bourguet, Emmanuelle; Glaring, Mikkel A; Schriemer, David C; Moorhead, Greg B G

    2013-01-01

    Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

  17. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

    PubMed

    Chen, Xi'en; Lü, Shumin; Zhang, Yalin

    2014-01-01

    Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests. PMID:24823652

  18. The development of ribonuclease and acid phosphatase during germination of Pisum arvense.

    PubMed

    Barker, G R; Bray, C M; Walter, T J

    1974-08-01

    1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.

  19. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

    PubMed

    Chen, Xi'en; Lü, Shumin; Zhang, Yalin

    2014-01-01

    Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.

  20. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

    PubMed Central

    Davidson, Lindsay; Maccario, Helene; Perera, Nevin M.; Yang, Xuesong; Spinelli, Laura; Tibarewal, Priyanka; Glancy, Ben; Gray, Alex; Weijer, Cornelis J.; Downes, C. Peter; Leslie, Nick R.

    2009-01-01

    PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion. PMID:19915616

  1. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-01

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au+ complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe3+ with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe3+, and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs.A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au+ complexes, and then a class of ~2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ~1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by

  2. Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

    PubMed Central

    Domenech, Carlos Eduardo; Otero, Lisandro Horacio; Beassoni, Paola Rita; Lisa, Angela Teresita

    2011-01-01

    Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O)2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure. PMID:21915373

  3. Imaging of alkaline phosphatase activity in bone tissue.

    PubMed

    Gade, Terence P; Motley, Matthew W; Beattie, Bradley J; Bhakta, Roshni; Boskey, Adele L; Koutcher, Jason A; Mayer-Kuckuk, Philipp

    2011-01-01

    The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications. PMID:21799916

  4. Modeling catalytic promiscuity in the alkaline phosphatase superfamily

    PubMed Central

    Duarte, Fernanda; Amrein, Beat Anton

    2013-01-01

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein

  5. Cloning of the canine glucose-6-phosphatase gene

    SciTech Connect

    Kishnani, P.; Bao, Y.; Brix, A.E.

    1994-09-01

    Two Maltese puppies with massive hepatomegaly and failure to thrive were found to have a markedly reduced Glucose-6-phosphatase (G-6-Pase) activity in the liver and kidney. Deficiency of G-6-Pase activity causes type 1a glycogen storage disease in humans. To further study the mutation responsible for the disease in dog, we cloned G-6-Pase canine cDNA from normal mixed breed dog liver RNA using reverse transcriptase and PCR amplification using primers derived from the published murine G-6-Pase gene sequence. Sequencing revealed an open reading frame of 1071 nucleotides that encodes a predicted 357 amino acid polypeptide in the canine G-6-Pase gene, same as mouse and human. We found more than 90% sequence homology between dog and human G-6-Pase sequence. Hydropathy analysis of the deduced canine G-6-Pase polypeptide shows six transmembrane-spanning segments similar to those seen in human and mouse. Endoplasmic reticulum (ER) localization is similarly predicted by the presence of the ER protein retention signal KK positioned 3 and 4 amino acids from the carboxy terminal. Potential asparagine-linked glycosylation sites are identified at positions 96, 203, and 276. Northern blot analysis revealed increased G-6-Pase mRNA in the deficient dog liver compared to control. This could possibly reflect upregulation of transcription due to the persistent hypoglycemic state. Further studies are directed at the identification of the mutation involved in this deficient dog strain. Characterization of the G-6-Pase gene and protein in the deficient dog model can pave the way for new understanding in the pathophysiology of this disease and for the trials of novel therapeutic approaches including gene therapy.

  6. Downscaling Alkaline Phosphatase Activity in a Subtropical Reservoir

    NASA Astrophysics Data System (ADS)

    Tseng, Y.

    2011-12-01

    This research was conducted by downscaling study to understand phosphorus (P)-deficient status of different plankton and the role of alkaline phosphatase activity (APA) in subtropical Feitsui Reservoir. Results from field survey showed that bulk APA (1.6~95.2 nM h-1) was widely observed in the epilimnion (0~20 m) with an apparent seasonal variations, suggesting that plankton in the system were subjected to P-deficient seasonally. Mixed layer depth (an index of phosphate availability) is the major factor influencing the variation of bulk APA and specific APA (124~1,253 nmol mg C-1 h-1), based on multiple linear regression analysis. Size-fractionated APA assays showed that picoplankton (size 0.2~3 um) contributed most of the bulk APA in the system. In addition, single-cell APA detected by enzyme-labeled fluorescence (ELF) assay indicated that heterotrophic bacteria are the major contributors of APA. Thus, we can infer that bacteria play an important role in accelerating P-cycle within P-deficient systems. Light/nutrient manipulation bioassays showed that bacterial growth was directly controlled by phosphate, while picocyanobacterial growth is controlled by light and can out-compete bacteria under P-limited condition with the aid of light. Further analysis revealed that the strength of summer typhoon is a factor responsible for the inter-annual variability of bulk and specific APA. APA study demonstrated the episodic events (e.g. strong typhoon and extreme precipitation) had significant influence on APA variability in sub-tropical to tropical aquatic ecosystems. Hence, the results herein will allow future studies on monitoring typhoon disturbance (intensity and frequency) as well as the APA of plankton during summer-to-autumn in subtropical systems.

  7. Synthesis of functionalized fluorescent gold nanoclusters for acid phosphatase sensing.

    PubMed

    Sun, Jian; Yang, Fan; Yang, Xiurong

    2015-10-21

    A novel and convenient one-pot but two-step synthesis of fluorescent gold nanoclusters, incorporating glutathione (GSH) and 11-mercaptoundecanoic acid (MUA) as the functionalized ligands (i.e. AuNCs@GSH/MUA), is demonstrated. Herein, the mixing of HAuCl4 and GSH in aqueous solution results in the immediate formation of non-fluorescent GSH-Au(+) complexes, and then a class of ∼2.6 nm GSH-coated AuNCs (AuNCs@GSH) with mild orange-yellow fluorescence after several days. Interestingly, the intense orange-red emitting ∼1.7 nm AuNCs@GSH/MUA can be synthesized within seconds by introducing an alkaline aqueous solution of MUA into the GSH-Au(+) complexes or AuNC@GSH solution. Subsequently, a reliable AuNC@GSH/MUA-based real-time assay of acid phosphatase (ACP) is established for the first time, inspired by the selective coordination of Fe(3+) with surface ligands of AuNCs, the higher binding affinity between the pyrophosphate ion (PPi) and Fe(3+), and the hydrolysis of PPi into orthophosphate by ACP. Our fluorescent chemosensor can also be applied to assay ACP in a real biological sample and, furthermore, to screen the inhibitor of ACP. This report paves a new avenue for synthesizing AuNCs based on either the bottom-up reduction or top-down etching method, establishing real-time fluorescence assays for ACP by means of PPi as the substrate, and further exploring the sensing applications of fluorescent AuNCs. PMID:26391420

  8. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    PubMed

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform. PMID:18717264

  9. Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Bharatham, Kavitha; Bharatham, Nagakumar; Lee, Keun Woo

    2007-05-01

    A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors.

  10. Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate

    PubMed Central

    Hamarneh, Sulaiman R.; Mohamed, Mussa M. Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N.; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S.; Narisawa, Sonoko; Millán, José Luis; Warren, H. Shaw; Hohmann, Elizabeth; Malo, Madhu S.; Hodin, Richard A.

    2013-01-01

    Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP. PMID:23306083

  11. Pyruvate dehydrogenase/sub b/ phosphatase inhibition by NADH and dihydrolipoamide along with effects of and capacity for binding the phosphatase to the bovine kidney transacetylase-protein X subcomplex

    SciTech Connect

    Roche, T.E.; Rahmatullah, M.; Maher, J.

    1986-05-01

    NADH inhibits PDH/sub b/ phosphatase activity when /sup 32/P-PDH is associated with the intact complex but not when /sup 32/P-PDH is prepared free of other components of the complex. Addition of the transacetylase-protein X (E2-X) subcomplex both activated the phosphatase and restored NADH inhibition. Low levels of dihydrolipoyl dehydrogenase associated with the subcomplex might be required for NADH inhibition. Dihydrolipoamide gave inhibition of the phosphatase equivalent to NADH and the combination did not give additional inhibition suggesting a common mechanism. Pretreatment of phosphorylated complex and phosphatase with 2.0 mM dithiothreitol nearly eliminated inhibition of the phosphatase by NADH or dihydrolipoamide. Strong arsenite inhibition of phosphatase activity occurred only in the presence of NADH suggesting modification of thiols reduced by NADH can alter phosphatase activity. Only about 6 molecules of purified phosphatase could be activated by 1 molecule of E2-X subcomplex (initial velocities measured in 15s period). Since that corresponded to the number of protein X rather than E2 subunits, protein X may contribute to the Ca/sup 2 +/-dependent binding of the phosphatase. Since protein X also contains a lipoyl moiety, it may also contribute to NADH inhibition of the phosphatase.

  12. The first structure of a bacterial class B Acid phosphatase reveals further structural heterogeneity among phosphatases of the haloacid dehalogenase fold.

    PubMed

    Calderone, Vito; Forleo, Costantino; Benvenuti, Manuela; Cristina Thaller, Maria; Rossolini, Gian Maria; Mangani, Stefano

    2004-01-16

    AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. The crystal structure of AphA has been determined at 2.2A and its resolution extended to 1.7A on an AuCl(3) derivative. This represents the first crystal structure of a class B bacterial phosphatase. Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold. This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases. The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers. The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions. The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.

  13. Generic phosphatase activity detection using zinc mediated aggregation modulation of polypeptide-modified gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Selegård, Robert; Enander, Karin; Aili, Daniel

    2014-11-01

    A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme. Phosphatase activity generates inorganic phosphate that forms an insoluble complex with Zn2+. In a sample containing a preset concentration of Zn2+, phosphatase activity will markedly reduce the concentration of dissolved Zn2+ from the original value, which in turn affects the aggregation of gold nanoparticles functionalized with a designed Zn2+ responsive polypeptide. The change in nanoparticle stability thus provides a rapid and sensitive readout of the phosphatase activity. The assay is not limited to a particular enzyme or enzyme substrate, which is demonstrated using three completely different phosphatases and five different substrates, and thus constitutes a highly interesting system for drug screening and diagnostics.A challenge in the design of plasmonic nanoparticle-based colorimetric assays is that the change in colloidal stability, which generates the colorimetric response, is often directly linked to the biomolecular recognition event. New assay strategies are hence required for every type of substrate and enzyme of interest. Here, a generic strategy for monitoring of phosphatase activity is presented where substrate recognition is completely decoupled from the nanoparticle stability modulation mechanism, which enables detection of a wide range of enzymes using different natural substrates with a single simple detection scheme

  14. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    PubMed Central

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  15. Expression of human protein phosphatase-1 in Saccharomyces cerevisiae highlights the role of phosphatase isoforms in regulating eukaryotic functions.

    PubMed

    Gibbons, Jennifer A; Kozubowski, Lukasz; Tatchell, Kelly; Shenolikar, Shirish

    2007-07-27

    Human (PP1) isoforms, PP1alpha, PP1beta, PP1gamma1, and PP1gamma2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1alpha-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1alpha-expressing yeast accumulated less glycogen than PP1beta-or PP1gamma1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1alpha/beta subunits highlighted a critical role for their unique N termini in defining PP1alpha and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.

  16. Effects of precipitation on soil acid phosphatase activity in three successional forests in Southern China

    NASA Astrophysics Data System (ADS)

    Huang, W.; Liu, J.; Zhou, G.; Zhang, D.; Deng, Q.

    2011-01-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of P supply to ecosystems. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment of precipitation treatments (no precipitation, natural precipitation and doubled precipitation) in three forests of early-, mid- and advanced-successional stages in Southern China was carried out. Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, no precipitation treatment depressed soil acid phosphatase activity, while doubled precipitation treatment exerted no positive effects on it, and even significantly lowered it in the advanced forest. These indicate the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. The negative responses of soil acid phosphatase activity to precipitation suggest that P supply in subtropical ecosystems might be reduced if there was a drought in a whole year or more rainfall in the wet season in the future. NP, no precipitation; Control, natural precipitation; DP, double precipitation.

  17. Cloning and characterization of three Eimeria tenella lipid phosphate phosphatases.

    PubMed

    Guo, Aijiang; Cai, Jianping; Luo, Xuenong; Zhang, Shaohua; Hou, Junling; Li, Hui; Cai, Xuepeng

    2015-01-01

    Although lipid phosphate phosphatases (LPPs) play an important role in cellular signaling in addition to lipid biosynthesis, little is thus far known about parasite LPPs. In this study, we characterized three Eimeria tenella cDNA clones encoding LPP named EtLPP1, EtLPP2 and EtLPP3. Key structural features previously described in LPPs, including the three conserved domains proposed as catalytic sites, a single conserved N-glycosylation site, and putative transmembrane domains were discovered in the three resulting EtLPP amino acid sequences. Expression of His6-tagged EtLPP1, -2, and -3 in HEK293 cells produced immunoreactive proteins with variable molecular sizes, suggesting the presence of multiple forms of each of the three EtLPPs. The two faster-migrating protein bands below each of the three EtLPP proteins were found to be very similar to the porcine 35-kDa LPP enzyme in their molecular size and the extent of their N-glycosylation, suggesting that the three EtLPPs are partially N-glycosylated. Kinetic analyses of the activity of the three enzymes against PA, LPA, C1P and S1P showed that Km values for each of the substrates were (in μM) 284, 46, 28, and 22 for EtLPP1; 369, 179, 237, and 52 for EtLPP2; and 355, 83, and 260 for EtLPP3. However, EtLPP3 showed negligible activity on S1P. These results confirmed that the three EtLPPs have broad substrate specificity. The results also indicated that despite structural similarities, the three EtLPPs may play distinct functions through their different models of substrate preference. Furthermore, particularly high expression levels of the three EtLPP genes were detected in the sporozoite stage of the E. tenella life cycle (p<0.001), suggesting that their encoded proteins might play an important biological function in the sporozoite stage. PMID:25861032

  18. Associations between Renal Hyperfiltration and Serum Alkaline Phosphatase

    PubMed Central

    Oh, Se Won; Han, Kum Hyun; Han, Sang Youb

    2015-01-01

    Renal hyperfiltration, which is associated with renal injury, occurs in diabetic or obese individuals. Serum alkaline phosphatase (ALP) level is also elevated in patients with diabetes (DM) or metabolic syndrome (MS), and increased urinary excretion of ALP has been demonstrated in patients who have hyperfiltration and tubular damage. However, little was investigated about the association between hyperfiltration and serum ALP level. A retrospective observational study of the 21,308 adults in the Korea National Health and Nutrition Examination Survey IV-V databases (2008–2011) was performed. Renal hyperfiltration was defined as exceeding the age- and sex-specific 97.5th percentile. We divided participants into 4 groups according to their estimated glomerular filtration rate (eGFR): >120, 90–119, 60–89, and <60 mL/min/1.73 m2. The participants with eGFR >120 mL/min/1.73 m2 showed the highest risk for MS, in the highest ALP quartiles (3.848, 95% CI, 1.876–7.892), compared to the lowest quartile. Similarly, the highest risk for DM, in the highest ALP quartiles, was observed in participants with eGFR >120 ml/min/1.73 m2 (2.166, 95% CI, 1.084–4.329). ALP quartiles were significantly associated with albuminuria in participants with eGFR ≥ 60 ml/min/1.73m2. The highest ALP quartile had a 1.631-fold risk elevation for albuminuria with adjustment of age and sex. (95% CI, 1.158-2.297, P = 0.005). After adjustment, the highest ALP quartile had a 1.624-fold risk elevation, for renal hyperfiltration (95% CI, 1.204–2.192, P = 0.002). In addition, hyperfiltration was significantly associated with hemoglobin, triglyceride, white blood cell count, DM, smoking, and alcohol consumption (P<0.05). The relationship between serum ALP and metabolic disorders is stronger in participants with an upper-normal range of eGFR. Higher ALP levels are significantly associated with renal hyperfiltration in Korean general population. PMID:25853240

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

    PubMed Central

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

    1996-01-01

    In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA

  20. Root surface acid phosphatases and their role in phosphorus assimilation by Eriophorum vaginatum

    SciTech Connect

    Kroehler, C.J.; Linkins, A.E.

    1988-01-01

    Eriophorum vaginatum is a dominant plant in much of the arctic tundra ecosystem where phosphorus is frequently a limiting nutrient. The mineralization of this organic phosphorus was thought to be principally controlled by microbial respiration, however, more recent work shows that extracellular soil phosphatases are the principal regulators. The existence of plant root and mycorrhizal surface phosphatases which are capable of hydrolyzing organic phosphorus compounds, suggests that soil organic phosphorus may be directly utilized by plants. Since E. vaginatum is a tussock forming sedge with a very dense annually produced rooting system which can exploit most of the tussock soil volume, its surface phosphatases may play a dominant role in organic phosphorus hydrolysis into inorganic phosphorus. Of equal significance would be the potential for this activity to contribute to the phosphorus nutrition through the coupling of phosphorus hydrolysis on the root and root uptake of the resultant inorganic phosphorus. Phosphatase activity was investigated and found to be uniformly distributed along the surface of the root. Kinetic analysis of the enzyme gave estimates of 9.23 mM for the apparent Km and 1.61 * 10/sup -3/ ..mu..moles mm-2 hr/sup -1/ for the apparent Vmax. Saturation values for E. vaginatum phosphatases are about 3 times higher than average soil solution organic phosphorus concentrations. 12 refs., 4 figs.

  1. Root-surface phosphatase activity in shrublands across a European gradient: effects of warming.

    PubMed

    Estiarte, M; Peuuelas, J; Sardans, J; Emmett, B A; Sowerby, A; Beier, C; Schmidt, I K; Tietema, A; Van Meeteren, M J M; Kovacs-Lang, E; Mathe, P; De Angelis, P; De Dato, G

    2008-01-01

    Root-surface phosphatase activities were measured in natural and semi-natural shrublands across an European climatic gradient of temperature and rainfall including Wales (WL), Denmark (DK), Netherlands (NL), Hungary (HU), Italy (IT) and Spain (SP). In each site a warming experiment was conducted since 1999 or 2001 by means of passive night-time warming using reflective curtains that covered the vegetation at night. The treatments increased yearly average soil temperatures around 0. 8 degrees C in most of sites. Root-surface phosphatase activity values ranged between 56 mg PNP g(-1) h(-1) in IT and 3.5 mg PNP g(-1) h(-1) in HU. Warming had no effect on root-surface phosphatase activity across the sites and only in Hungary a slight increase was detected. Plants at Mediterranean sites (IT, SP) showed a higher root-surface phosphatase activity than plants at temperate sites (WL, NL, DK). We suggest it might be an adaptation of plant species evolved under Mediterranean climate that allows them a) to compensate in wet period for the decrease in phosphatase activity, and thus P uptake, during drought periods, and/or b) to benefit from soluble organic P flushes following the frequent drying-rewetting episodes experienced by soils in Mediterranean ecosystems. PMID:18831327

  2. Demonstration of protein tyrosine phosphatase activity in the second of two homologous domains of CD45.

    PubMed

    Tan, X; Stover, D R; Walsh, K A

    1993-04-01

    It has been reported that alteration of deletion of critical residues within one of the two homologous protein tyrosine phosphatase (PTPase)-like domains of CD45 completely abolishes all activity, suggesting that only the more N-terminal domain is catalytically active. However, we now demonstrate, by two independent techniques, that the second (C-terminal) domain is also a viable phosphatase. Limited proteolysis by endoproteinase Lys-C or trypsin increased the phosphatase activity toward reduced, carboxymethylated, and maleylated lysozyme approximately 8-fold. A 50-kDa fragment, isolated by ion exchange chromatography, was found to be responsible for this activity. N-terminal sequencing revealed that this fragment includes less than half of the first phosphatase domain and most, if not all, of the second. In a second experiment, 109 residues, including the presumed catalytic region, were removed from domain I by site-directed mutagenesis. Expression of this construct in a mammalian cell line resulted in increased PTPase activity over nontransfected control cells. Isolation of the recombinant CD45 by immunoprecipitation and immunoaffinity chromatography revealed that it had phosphatase activity. Both of these experimental approaches demonstrate that the second conserved PTPase domain of CD45 is a functioning PTPase, but that external regulation may be required to express its activity in the context of the native molecule. PMID:8463207

  3. Alkaline phosphatase: a possible treatment for sepsis-associated acute kidney injury in critically ill patients.

    PubMed

    Peters, Esther; Heemskerk, Suzanne; Masereeuw, Rosalinde; Pickkers, Peter

    2014-06-01

    Acute kidney injury (AKI) is a common disease in the intensive care unit and accounts for high morbidity and mortality. Sepsis, the predominant cause of AKI in this setting, involves a complex pathogenesis in which renal inflammation and hypoxia are believed to play an important role. A new therapy should be aimed at targeting both these processes, and the enzyme alkaline phosphatase, with its dual mode of action, might be a promising candidate. First, alkaline phosphatase is able to reduce inflammation through dephosphorylation and thereby detoxification of endotoxin (lipopolysaccharide), which is an important mediator of sepsis. Second, adenosine triphosphate, released during cellular stress caused by inflammation and hypoxia, has detrimental effects but can be converted by alkaline phosphatase into adenosine with anti-inflammatory and tissue-protective effects. These postulated beneficial effects of alkaline phosphatase have been confirmed in animal experiments and two phase 2a clinical trials showing that kidney function improved in critically ill patients with sepsis-associated AKI. Because renal inflammation and hypoxia also are observed commonly in AKI induced by other causes, it would be of interest to investigate the therapeutic effect of alkaline phosphatase in these nephropathies as well.

  4. An alkaline phosphatase transport mechanism in the pathogenesis of Alzheimer's disease and neurodegeneration.

    PubMed

    Pike, Adrianne F; Kramer, Nynke I; Blaauboer, Bas J; Seinen, Willem; Brands, Ruud

    2015-01-25

    Systemic inflammation is associated with loss of blood-brain barrier integrity and neuroinflammation that lead to the exacerbation of neurodegenerative diseases. It is also associated specifically with the characteristic amyloid-β and tau pathologies of Alzheimer's disease. We have previously proposed an immunosurveillance mechanism for epithelial barriers involving negative feedback-regulated alkaline phosphatase transcytosis as an acute phase anti-inflammatory response that hangs in the balance between the resolution and the progression of inflammation. We now extend this model to endothelial barriers, particularly the blood-brain barrier, and present a literature-supported mechanistic explanation for Alzheimer's disease pathology with this system at its foundation. In this mechanism, a switch in the role of alkaline phosphatase from its baseline duties to a stopgap anti-inflammatory function results in the loss of alkaline phosphatase from cell membranes into circulation, thereby decreasing blood-brain barrier integrity and functionality. This occurs with impairment of both amyloid-β efflux and tau dephosphorylating activity in the brain as alkaline phosphatase is replenished at the barrier by receptor-mediated transport. We suggest systemic alkaline phosphatase administration as a potential therapy for the resolution of inflammation and the prevention of Alzheimer's disease pathology as well as that of other inflammation-related neurodegenerative diseases.

  5. Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

    PubMed

    Stefanoni Rubio, P J; Godoy, M S; Della Mónica, I F; Pettinari, M J; Godeas, A M; Scervino, J M

    2016-01-01

    The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

  6. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    PubMed

    Majumder, Shyam Prasad; Das, Amal Chandra

    2016-04-01

    An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil.

  7. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    PubMed

    Majumder, Shyam Prasad; Das, Amal Chandra

    2016-04-01

    An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil. PMID:26720809

  8. An alkaline phosphatase transport mechanism in the pathogenesis of Alzheimer's disease and neurodegeneration.

    PubMed

    Pike, Adrianne F; Kramer, Nynke I; Blaauboer, Bas J; Seinen, Willem; Brands, Ruud

    2015-01-25

    Systemic inflammation is associated with loss of blood-brain barrier integrity and neuroinflammation that lead to the exacerbation of neurodegenerative diseases. It is also associated specifically with the characteristic amyloid-β and tau pathologies of Alzheimer's disease. We have previously proposed an immunosurveillance mechanism for epithelial barriers involving negative feedback-regulated alkaline phosphatase transcytosis as an acute phase anti-inflammatory response that hangs in the balance between the resolution and the progression of inflammation. We now extend this model to endothelial barriers, particularly the blood-brain barrier, and present a literature-supported mechanistic explanation for Alzheimer's disease pathology with this system at its foundation. In this mechanism, a switch in the role of alkaline phosphatase from its baseline duties to a stopgap anti-inflammatory function results in the loss of alkaline phosphatase from cell membranes into circulation, thereby decreasing blood-brain barrier integrity and functionality. This occurs with impairment of both amyloid-β efflux and tau dephosphorylating activity in the brain as alkaline phosphatase is replenished at the barrier by receptor-mediated transport. We suggest systemic alkaline phosphatase administration as a potential therapy for the resolution of inflammation and the prevention of Alzheimer's disease pathology as well as that of other inflammation-related neurodegenerative diseases. PMID:25500268

  9. [Inhibition of alkaline phosphatase I of Pichia guilliermondii yeast in vitro and in vivo].

    PubMed

    Sibirnyi, A A; Shavlovskii, G M

    1978-01-01

    The rate of p-nitrophenyl phosphate and flavin mononucleotide (FMN) hydrolysis by the partially purified preparation of alkaline phosphatase I of Pichia guilliermondii flavinogenic yeast was studied as affected by different substrates and inorganic ions. Their Km was established to be 2.0 X 10(-4) m and 2.5 X 10(-4) M, respectively. Dephosphorylation of p-nitrophenylphosphate and FMN was inhibited competitively by beta-glycerophosphate (Ki = 3.1 X 10(-3) M, respectively). The presence of inorganic phosphate ions in the reaction mixture decreases or removes inhibition of these compounds hydrolysis by other substrates of alkaline phosphatase I. The activity of alkaline phosphatase I increases in the presence of Mg2+ and was strongly inhibited in the presence of Be2+, Cu2+, Zn2+, Cd2+ and inorganic phosphate, the mixture of Be2+ and F- being the most effective. This mixture inhibited the phosphatase activity of the partially purified preparation of alkaline phosphatase I of the cell-free extract as well as of intact cells in both the alkaline and acid zones of pH (8.6 and 5.5, respectively). Incubation of the washed iron-deficient P. guilliermondii cells in the presence of Be2+ and F- did not result in accumulation of FMN in the yeast culture. A possible role of nonspecific phosphomonoesterases in hydrolysis of FMN in vivo is discussed. PMID:208203

  10. A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

    PubMed

    Cox, W G; Singer, V L

    1999-11-01

    We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)

  11. Structure of Human PIR1, an Atypical Dual-Specificity Phosphatase

    PubMed Central

    2015-01-01

    PIR1 is an atypical dual-specificity phosphatase (DSP) that dephosphorylates RNA with a higher specificity than phosphoproteins. Here we report the atomic structure of a catalytically inactive mutant (C152S) of the human PIR1 phosphatase core (PIR1-core, residues 29–205), refined at 1.20 Å resolution. PIR1-core shares structural similarities with DSPs related to Vaccinia virus VH1 and with RNA 5′-phosphatases such as the baculovirus RNA triphosphatase and the human mRNA capping enzyme. The PIR1 active site cleft is wider and deeper than that of VH1 and contains two bound ions: a phosphate trapped above the catalytic cysteine C152 exemplifies the binding mode expected for the γ-phosphate of RNA, and ∼6 Å away, a chloride ion coordinates the general base R158. Two residues in the PIR1 phosphate-binding loop (P-loop), a histidine (H154) downstream of C152 and an asparagine (N157) preceding R158, make close contacts with the active site phosphate, and their nonaliphatic side chains are essential for phosphatase activity in vitro. These residues are conserved in all RNA 5′-phosphatases that, analogous to PIR1, lack a “general acid” residue. Thus, a deep active site crevice, two active site ions, and conserved P-loop residues stabilizing the γ-phosphate of RNA are defining features of atypical DSPs that specialize in dephosphorylating 5′-RNA. PMID:24447265

  12. Partial purification and characterization of an enzyme from pea nuclei with protein tyrosine phosphatase activity.

    PubMed

    Guo, Y L; Roux, S J

    1995-01-01

    A pea (Pisum sativum L.) nuclear enzyme with protein tyrosine phosphatase activity has been partially purified and characterized. The enzyme has a molecular mass of 90 kD as judged by molecular sieve column chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Like animal protein tyrosine phosphatases it can be inhibited by low concentrations of molybdate and vanadate. It is also inhibited by heparin and spermine but not by either the acid phosphatase inhibitors citrate and tartrate or the protein serine/threonine phosphatase inhibitor okadaic acid. The enzyme does not require Ca2+, Mg2+, or Mn2+ for its activity but is stimulated by ethylenediaminetetraacetate and by ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid. It dephosphorylates phosphotyrosine residues on the four different 32P-tyrosine-labeled peptides tested but not the phosphoserine/threonine residues on casein and histone. Like some animal protein tyrosine phosphatases, it has a variable pH optimum depending on the substrate used: the optimum is 5.5 when the substrate is [32P]tyrosine-labeled lysozyme, but it is 7.0 when the substrate is [32P]tyrosine-labeled poly(glutamic acid, tyrosine). It has a Km of 4 microM when the lysozyme protein is used as a substrate.

  13. The Sac1 Phosphoinositide Phosphatase Regulates Golgi Membrane Morphology and Mitotic Spindle Organization in Mammals

    PubMed Central

    Liu, Yang; Boukhelifa, Malika; Tribble, Emily; Morin-Kensicki, Elizabeth; Uetrecht, Andrea; Bear, James E.

    2008-01-01

    Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties. PMID:18480408

  14. The Sac1 phosphoinositide phosphatase regulates Golgi membrane morphology and mitotic spindle organization in mammals.

    PubMed

    Liu, Yang; Boukhelifa, Malika; Tribble, Emily; Morin-Kensicki, Elizabeth; Uetrecht, Andrea; Bear, James E; Bankaitis, Vytas A

    2008-07-01

    Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties.

  15. [Role of protein phosphatase 2A in renal interstitial fibrosis].

    PubMed

    Xi, Yiyun; Li, Hua; Li, Jun; Li, Ying; Liu, Yuping; You, Yanhua; Duan, Shaobin; Liu, Hong; Sun, Lin; Peng, Youming; Liu, Fuyou

    2015-06-01

    目的:探讨蛋白磷酸酶2A(protein phosphatase 2A,PP2A)在大鼠单侧输尿管梗阻(unilateral ureteral obstruction,UUO)及TGF-β1刺激的人近端肾小管上皮细胞-2(human kidney proximal tubular epithelial-2,HK-2)的肾纤维化模型中的作用。方法:1)15只雄性SD大鼠随机分成假手术组( sham组)、模型组(UUO组)和UUO+冈田酸(okadaic acid,OA)干预组(OA组),每组各5只。术后OA组每日给予1.8%酒精稀释的OA 30 μg/kg,胃管饲喂72 h,对照组和模型组给予相等体积的1.8%酒精胃管饲喂,72 h后处死大鼠,收集血和肾组织,检测肾功能并采用免疫组织化学、Western印迹和RT-PCR法检测肾组织PP2A的c亚基(PP2Ac)、纤维连接蛋白(fibronectin,FN)、胶原-I(collagen-I,Col-I)、E-钙黏蛋白(E-cadherin,E-cad)和α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的蛋白及mRNA的表达。2)采用台盼蓝排斥实验及MTT法找出适宜的OA浓度。常规培养HK-2细胞,随机分为对照组、TGF-β1组(TGF-β1 5 ng/mL干预24 h)、TGF-β1+OA组(TGF-β1 5 ng/mL+OA 40 nmol/L,同时干预24 h),Western印迹检测肾小管上皮细胞PP2Ac,FN,Col-I,E-cad和α-SMA 蛋白的表达。结果:1)肾功能表明UUO组尿素氮和肌酐较sham组升高,OA组尿素氮、肌酐均比UUO组下降(均P<0.05)。免疫组织化学、Western印迹和RT-PCR均显示:与sham组比较,UUO组PP2Ac,FN,Col-I和α-SMA表达升高,而E-cad表达下降(均P<0.05);与UUO组比较,OA组PP2Ac,FN,Col-I和α-SMA表达下降,E-cad表达升高(均P<0.05);2) OA 40 nmol/L为最适宜的实验质量浓度;Western印迹显示:与对照组比较,TGF-β1组PP2Ac,FN,Col-I和α-SMA表达升高,E-cad表达下降(均P<0.05);与TGF-β1组比较,TGF-β1+OA组PP2Ac,FN,Col-I和α-SMA表达下降,E-cad表达升高(均P<0.05)。结论:PP2A能促进肾间质纤维化。.

  16. Role of bone-type tissue-nonspecific alkaline phosphatase and PHOSPO1 in vascular calcification.

    PubMed

    Bobryshev, Yuri V; Orekhov, Alexander N; Sobenin, Igor; Chistiakov, Dimitry A

    2014-01-01

    Matrix vesicle (MV)-mediated mineralization is important for bone ossification. However, under certain circumstances such as atherosclerosis, mineralization may occur in the arterial wall. Bone-type tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes inorganic pyrophosphate (PPi) and generates inorganic phosphate (Pi), which is essential for MV-mediated hydroxyapatite formation. MVs contain another phosphatase, PHOSPHO1, that serves as an additional supplier of Pi. Activation of bone-type tissue-nonspecific alkaline phosphatase (TNAP) in vascular smooth muscle cells precedes vascular calcification. By degrading PPi, TNAP plays a procalcific role changing the Pi/PPi ratio toward mineralization. A pathologic role of bone-type TNAP and PHOSPHO1 make them to be attractive targets for cardiovascular therapy.

  17. The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

    PubMed

    Srivastava, Pramod Kumar; Anand, Asha

    2015-01-01

    Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

  18. Structure of human dual-specificity phosphatase 27 at 2.38 Å resolution

    PubMed Central

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2011-01-01

    There are over 100 genes in the human genome that encode protein tyrosine phosphatases (PTPs) and approximately 60 of these are classified as dual-specificity phosphatases (DUSPs). Although many dual-specificity phosphatases are still not well characterized, novel functions have been discovered for some of them that have led to new insights into a variety of biological processes and the molecular basis for certain diseases. Indeed, as the functions of DUSPs continue to be elucidated, a growing number of them are emerging as potential therapeutic targets for diseases such as cancer, diabetes and inflammatory disorders. Here, the overexpression, purification and structure determination of DUSP27 at 2.38 Å resolution are presented. PMID:21543850

  19. The calcineurin signaling network evolves via conserved kinase-phosphatase modules that transcend substrate identity.

    PubMed

    Goldman, Aaron; Roy, Jagoree; Bodenmiller, Bernd; Wanka, Stefanie; Landry, Christian R; Aebersold, Ruedi; Cyert, Martha S

    2014-08-01

    To define a functional network for calcineurin, the conserved Ca(2+)/calmodulin-regulated phosphatase, we systematically identified its substrates in S. cerevisiae using phosphoproteomics and bioinformatics, followed by copurification and dephosphorylation assays. This study establishes new calcineurin functions and reveals mechanisms that shape calcineurin network evolution. Analyses of closely related yeasts show that many proteins were recently recruited to the network by acquiring a calcineurin-recognition motif. Calcineurin substrates in yeast and mammals are distinct due to network rewiring but, surprisingly, are phosphorylated by similar kinases. We postulate that corecognition of conserved substrate features, including phosphorylation and docking motifs, preserves calcineurin-kinase opposition during evolution. One example we document is a composite docking site that confers substrate recognition by both calcineurin and MAPK. We propose that conserved kinase-phosphatase pairs define the architecture of signaling networks and allow other connections between kinases and phosphatases to develop that establish common regulatory motifs in signaling networks.

  20. Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

    PubMed Central

    Pereira, Sandro F. F.; Goss, Lindsie; Dworkin, Jonathan

    2011-01-01

    Summary: Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms. PMID:21372323

  1. The baculovirus uses a captured host phosphatase to induce enhanced locomotory activity in host caterpillars.

    PubMed

    Katsuma, Susumu; Koyano, Yasue; Kang, Wonkyung; Kokusho, Ryuhei; Kamita, Shizuo George; Shimada, Toru

    2012-01-01

    The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme.

  2. ASSOCIATION OF THE ALKALINE PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES WITH THE MEMBRANE OF THE SPECIFIC GRANULES

    PubMed Central

    Bretz, Ursula; Baggiolini, Marco

    1973-01-01

    The localization of alkaline phosphatase in the specific granules of rabbit polymorphonuclear leukocytes was investigated. The results obtained suggest very strongly that alkaline phosphatase is a component of the granule membrane. The enzyme remains attached to the membrane upon disruption of the granules by the use of detergents or by hypotonic shock and subsequent extraction with sodium sulfate, and can be isolated together with fragments of the granule membrane by isopycnic equilibration. Treatment of the granules with high amounts of Triton-X-100, sodium deoxycholate, or hexadecyltrimethylammonium bromide releases the enzyme in soluble form. In polymorphonuclear leukocyte homogenates, lysis of the granules is needed in order to render alkaline phosphatase fully accessible to substrates. This suggests that the catalytic site of the enzyme is exposed at the inner face of the granule membrane. PMID:4761336

  3. New pleiotropic alkaline phosphatase-negative mutants of Escherichia coli K-12.

    PubMed Central

    Heyde, M; Portalier, R

    1982-01-01

    Escherichia coli K-12 mutants showing reduced alkaline phosphatase activity were isolated as 5-fluorouracil-plus-adenosine-resistant derivatives of a upp pho (either phoS or phoT) strain. One class of these mutants displayed a temperature-sensitive alkaline phosphatase-negative phenotype, a pleiotropic defect for growth on some substrates, an increased sensitivity to toxic compounds (e.g., EDTA, mitomycin, and chloramphenicol), and alterations in the expression of some membrane proteins. It phenotypically differed from previously described mutants. The mutation was located at min 8.5 close to the phoA gene and defines a new genetic locus we called napA (for negative alkaline phosphatase pleiotropic phenotype). As these mutants have lost the ability to grow on lactose and galactose, Lac+ and Gal+ revertants were isolated that simultaneously recovered the parental phenotype. PMID:7047492

  4. The Baculovirus Uses a Captured Host Phosphatase to Induce Enhanced Locomotory Activity in Host Caterpillars

    PubMed Central

    Katsuma, Susumu; Koyano, Yasue; Kang, WonKyung; Kokusho, Ryuhei; Kamita, Shizuo George; Shimada, Toru

    2012-01-01

    The baculovirus is a classic example of a parasite that alters the behavior or physiology of its host so that progeny transmission is maximized. Baculoviruses do this by inducing enhanced locomotory activity (ELA) that causes the host caterpillars to climb to the upper foliage of plants. We previously reported that this behavior is not induced in silkworms that are infected with a mutant baculovirus lacking its protein tyrosine phosphatase (ptp) gene, a gene likely captured from an ancestral host. Here we show that the product of the ptp gene, PTP, associates with baculovirus ORF1629 as a virion structural protein, but surprisingly phosphatase activity associated with PTP was not required for the induction of ELA. Interestingly, the ptp knockout baculovirus showed significantly reduced infectivity of larval brain tissues. Collectively, we show that the modern baculovirus uses the host-derived phosphatase to establish adequate infection for ELA as a virion-associated structural protein rather than as an enzyme. PMID:22496662

  5. [Appearance of phosphatase reticular cells in the spleen parenchyme of normal and tumor-bearing mice].

    PubMed

    Catayée, G; Chalet, M

    1976-01-01

    The splenic mouse parenchyma presents 3 reticular cells types revealed by histo-enzymatic technics: the phagocytic cells show a strong phosphatasic acid activity, the endothelial cells possess a phosphatasic alcalin or adenosine-triphosphatasic reaction, the perithelial cells are 5' nucleotidasic. These different cells are distributed by forming specific topographic structures in the splenic tissue. The phosphatasic alcalin reticular cells seem to be, with their distribution, characteristic elements of mouse spleen. Indeed the modifications in tumoral animal interest chiefly this cell category. In this case, the reticular cells form a deep and large membrane between marginal zone and perivascular lymphoid sheath of the white pulp. These different reticular cells probably react for the defense system of the animal.

  6. Protein Tyrosine Phosphatases: From Housekeeping Enzymes to Master-Regulators of Signal Transduction

    PubMed Central

    Tonks, Nicholas K.

    2013-01-01

    There are many misconceptions surrounding the roles of protein phosphatases in the regulation of signal transduction, perhaps the most damaging of which is the erroneous view that these enzymes exert their effects merely as constitutively active housekeeping enzymes. On the contrary, the phosphatases are critical, specific regulators of signaling in their own right and serve an essential function, in a coordinated manner with the kinases, to determine the response to a physiological stimulus. This review is a personal perspective on the development of our understanding of the protein tyrosine phosphatase (PTP) family of enzymes. I have discussed various aspects of the structure, regulation and function of the PTP family, which I hope will illustrate the fundamental importance of these enzymes to the control of signal transduction. PMID:23176256

  7. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2.

    PubMed

    Gyuris, J; Golemis, E; Chertkov, H; Brent, R

    1993-11-19

    We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases. PMID:8242750

  8. Alkaline phosphatase protein increases in response to prednisolone in HeLa cells.

    PubMed Central

    Hanford, W C; Kottel, R H; Fishman, W H

    1981-01-01

    Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state. Images Fig. 1. Fig. 2. PMID:7340849

  9. Phosphatase activity against neurofilament proteins from bovine spinal cord: effect of aluminium and neuropsychoactive drugs.

    PubMed

    Shetty, K T; Veeranna; Guru, S C

    1992-03-16

    Protein phosphatase activity associated with neurofilament (NF) rich (Triton X-100 insoluble) fraction was extracted and partially characterised by using known inhibitors of protein phosphatases such as vanadate and fluoride. Protein phosphatase activity was demonstrated with reference to the dephosphorylation of endogenous substrate, NF protein and exogenous protein substrates, casein and phosvitin. Phosphoamino acids and beta-glycerophosphate were found to be poor substrates. Further, new observations have been made regarding the in vitro inhibitory effect of aluminium and the differential effects of some of the neuropsychoactive drugs. The findings could possibly lead to studies explaining the biochemical basis of aluminium induced neurotoxicity as well as the side effects associated with the long term medication of neuropsychoactive drugs. PMID:1320755

  10. Multiple Phosphatases Regulate Carbon Source-Dependent Germination and Primary Metabolism in Aspergillus nidulans.

    PubMed

    de Assis, Leandro José; Ries, Laure Nicolas Annick; Savoldi, Marcela; Dinamarco, Taisa Magnani; Goldman, Gustavo Henrique; Brown, Neil Andrew

    2015-03-11

    Aspergillus nidulans is an important mold and a model system for the study of fungal cell biology. In addition, invasive A. nidulans pulmonary infections are common in humans with chronic granulomatous disease. The morphological and biochemical transition from dormant conidia into active, growing, filamentous hyphae requires the coordination of numerous biosynthetic, developmental, and metabolic processes. The present study exhibited the diversity of roles performed by seven phosphatases in regulating cell cycle, development, and metabolism in response to glucose and alternative carbon sources. The identified phosphatases highlighted the importance of several signaling pathways regulating filamentous growth, the action of the pyruvate dehydrogenase complex as a metabolic switch controlling carbon usage, and the identification of the key function performed by the α-ketoglutarate dehydrogenase during germination. These novel insights into the fundamental roles of numerous phosphatases in germination and carbon sensing have provided new avenues of research into the identification of inhibitors of fungal germination, with implications for the food, feed, and pharmaceutical industries.

  11. Allosteric Activation of the Phosphoinositide Phosphatase Sac1 by Anionic Phospholipids

    PubMed Central

    2012-01-01

    Sac family phosphoinositide phosphatases comprise an evolutionarily conserved family of enzymes in eukaryotes. Our recently determined crystal structure of the Sac phosphatase domain of yeast Sac1, the founding member of the Sac family proteins, revealed a unique conformation of the catalytic P-loop and a large positively charged groove at the catalytic site. We now report a unique mechanism for the regulation of its phosphatase activity. Sac1 is an allosteric enzyme that can be activated by its product phosphatidylinositol or anionic phospholipid phosphatidylserine. The activation of Sac1 may involve conformational changes of the catalytic P-loop induced by direct binding with the regulatory anionic phospholipids in the large cationic catalytic groove. These findings highlight the fact that lipid composition of the substrate membrane plays an important role in the control of Sac1 function. PMID:22452743

  12. Structure of human dual-specificity phosphatase 27 at 2.38 Å resolution

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2012-03-26

    There are over 100 genes in the human genome that encode protein tyrosine phosphatases (PTPs) and approximately 60 of these are classified as dual-specificity phosphatases (DUSPs). Although many dual-specificity phosphatases are still not well characterized, novel functions have been discovered for some of them that have led to new insights into a variety of biological processes and the molecular basis for certain diseases. Indeed, as the functions of DUSPs continue to be elucidated, a growing number of them are emerging as potential therapeutic targets for diseases such as cancer, diabetes and inflammatory disorders. Here, the overexpression, purification and structure determination of DUSP27 at 2.38 {angstrom} resolution are presented.

  13. Probing Mechanistic Similarities between Response Regulator Signaling Proteins and Haloacid Dehalogenase Phosphatases.

    PubMed

    Immormino, Robert M; Starbird, Chrystal A; Silversmith, Ruth E; Bourret, Robert B

    2015-06-01

    Response regulator signaling proteins and phosphatases of the haloacid dehalogenase (HAD) superfamily share strikingly similar folds, active site geometries, and reaction chemistry. Proteins from both families catalyze the transfer of a phosphoryl group from a substrate to one of their own aspartyl residues, and subsequent hydrolysis of the phosphoprotein. Notable differences include an additional Asp that functions as an acid/base catalyst and an active site well-structured prior to phosphorylation in HAD phosphatases. Both features contribute to reactions substantially faster than those for response regulators. To investigate mechanisms underlying the functional differences between response regulators and HAD phosphatases, we characterized five double mutants of the response regulator CheY designed to mimic HAD phosphatases. Each mutant contained the extra Asp paired with a phosphatase-inspired substitution to potentially position the Asp properly. Only CheY DR (Arg as the anchor) exhibited enhanced rates of both autophosphorylation with phosphoramidate and autodephosphorylation compared to those of wild-type CheY. Crystal structures of CheY DR complexed with MoO4(2-) or WO4(2-) revealed active site hydrogen bonding networks similar to those in HAD·substrate complexes, with the extra Asp positioned for direct interaction with the leaving group (phosphorylation) or nucleophile (dephosphorylation). However, CheY DR reaction kinetics did not exhibit the pH sensitivities expected for acid/base catalysis. Biochemical analysis indicated CheY DR had an enhanced propensity to adopt the active conformation without phosphorylation, but a crystal structure revealed unphosphorylated CheY DR was not locked in the active conformation. Thus, the enhanced reactivity of CheY DR reflected partial acquisition of catalytic and structural features of HAD phosphatases. PMID:25928369

  14. Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

    PubMed

    Seifried, Annegrit; Bergeron, Alexandre; Boivin, Benoit; Gohla, Antje

    2016-08-01

    Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.

  15. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    SciTech Connect

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  16. Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases.

    PubMed

    Grant, Philip; Pant, Harish C

    2004-03-01

    In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid-sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X-100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment-specific multimeric complexes.

  17. Identification and characterization of a novel human PP1 phosphatase complex.

    PubMed

    Lee, Jeong-Heon; You, Jinsam; Dobrota, Erika; Skalnik, David G

    2010-08-01

    Mammalian Wdr82 is a regulatory component of the Setd1a and Setd1b histone H3-lysine 4 methyltransferase complexes and is implicated in the tethering of Setd1 complexes to transcriptional start sites of active genes. In the studies reported here, immunoprecipitation and mass spectrometry analyses reveal that Wdr82 additionally associates with multiple protein complexes, including an RNA polymerase II complex, four distinct histone H3-Lys(4) methyltransferase complexes, protein phosphatase 1 (PP1)-associated proteins, a chaperonin-containing Tcp1 complex, and other uncharacterized proteins. Further characterization of the PP1-associated proteins identified a stable multimeric complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and a PP1 catalytic subunit (denoted as the PTW/PP1 phosphatase complex). The PTW/PP1 complex exhibits in vitro phosphatase activity in a PP1-dependent manner. Analysis of protein-protein interactions reveals that PNUTS mediates phosphatase complex formation by providing a binding platform to each component. The PNUTS and Tox4 subunits are predominantly associated with the PTW/PP1 phosphatase complex in HEK293 cells, and the integrity of this complex remains intact throughout cell cycle progression. Inducible expression of a PP1 interaction-defective form of PNUTS (W401A) or small interfering RNA-mediated depletion of PNUTS in HEK293 cells causes cell cycle arrest at mitotic exit and apoptotic cell death. PNUTS (W401A) shows normal association with chromosomes but causes defects in the process of chromosome decondensation at late telophase. These data reveal that mammalian Wdr82 functions in a variety of cellular processes and reveal a potential role of the PTW/PP1 phosphatase complex in the regulation of chromatin structure during the transition from mitosis into interphase. PMID:20516061

  18. Effects of precipitation on soil acid phosphatase activity in three successional forests in southern China

    NASA Astrophysics Data System (ADS)

    Huang, W.; Liu, J.; Zhou, G.; Zhang, D.; Deng, Q.

    2011-07-01

    Phosphorus (P) is often a limiting nutrient for plant growth in tropical and subtropical forests. Global climate change has led to alterations in precipitation in the recent years, which inevitably influences P cycling. Soil acid phosphatase plays a vital role in controlling P mineralization, and its activity reflects the capacity of organic P mineralization potential in soils. In order to study the effects of precipitation on soil acid phosphatase activity, an experiment with precipitation treatments (no precipitation, natural precipitation and doubled precipitation) in three successional forests in southern China was carried out. The three forests include Masson pine forest (MPF), coniferous and broad-leaved mixed forest (MF) and monsoon evergreen broad-leaved forest (MEBF). Results showed that driven by seasonality of precipitation, changes in soil acid phosphatase activities coincided with the seasonal climate pattern, with significantly higher values in the wet season than in the dry season. Soil acid phosphatase activities were closely linked to forest successional stages, with enhanced values in the later stages of forest succession. In the dry season, soil acid phosphatase activities in the three forests showed a rising trend with increasing precipitation treatments. In the wet season, soil acid phosphatase activity was depressed by no precipitation treatment in the three forests. However, doubled precipitation treatment exerted a significantly negative effect on it only in MEBF. These results indicate that the potential transformation rate of organic P might be more dependent on water in the dry season than in the wet season. A decrease in organic P turnover would occur in the three forests if there was a drought in a whole year in the future. More rainfall in the wet season would also be adverse to organic P turnover in MEBF due to its high soil moisture.

  19. Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

    PubMed

    Seifried, Annegrit; Bergeron, Alexandre; Boivin, Benoit; Gohla, Antje

    2016-08-01

    Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells. PMID:27179418

  20. 2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

    SciTech Connect

    Stankiewicz, P.J.; Gresser, M.J.; Tracey, A.S.; Hass, L.F.

    1987-03-10

    The binding of inorganic vanadate (V/sub i/) to rabbit muscle phosphoglycerate mutase (PGM), studied by using /sup 51/V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating (V/sub i/). The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 x 10/sup -6/ M. The relevance of this result to other studies which have shown that the V/sub i/-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on (V/sub i/) with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 ..mu..M V/sub i/ has little effect on the phosphatase activity. However, in the presence of 25 ..mu..M V/sub i/, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.

  1. Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase.

    PubMed

    Mamber, S W; Mikkilineni, A B; Pack, E J; Rosser, M P; Wong, H; Ueda, Y; Forenza, S

    1995-08-01

    Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization. PMID:7636751

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

    PubMed

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

    2016-09-01

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

  3. The "manganese(III)-containing" purple acid phosphatase from sweet potatoes is an iron enzyme.

    PubMed

    Hefler, S K; Averill, B A

    1987-08-14

    An improved purification of the purple acid phosphatase from sweet potatoes has been developed, and the properties of the enzyme have been reexamined. Contrary to previous reports, (e.g., Y. Sugiura, et al., J. Biol. Chem., 256, 10664-10670 (1981) ), the enzyme contains two moles of iron and insignificant amounts of manganese. The specific activity of the iron-containing preparations is ca. 14 times higher than that reported previously for the purported "Mn(III)" enzyme. The sweet potato purple acid phosphatase does indeed bind manganese, but it can be removed by dialysis with no changes in specific activity or spectral properties.

  4. Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase

    SciTech Connect

    Butler-Ransohoff, J.E.; Kendall, D.A.; Freeman, S.; Knowles, J.R.; Kaiser, E.T.

    1988-06-28

    The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using /sup 31/P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (R/sub P/)-/sup 17/O, /sup 16/O, /sup 18/O)phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

  5. Catalytic Signature of a Heat-Stable, Chimeric Human Alkaline Phosphatase with Therapeutic Potential

    PubMed Central

    Kiffer-Moreira, Tina; Sheen, Campbell R.; Gasque, Kellen Cristina da Silva; Bolean, Mayte; Ciancaglini, Pietro; van Elsas, Andrea; Hoylaerts, Marc F.; Millán, José Luis

    2014-01-01

    Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn2+ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury. PMID:24586729

  6. Autosomal dominant aniridia: probable linkage to acid phosphatase-1 locus on chromosome 2.

    PubMed Central

    Ferrell, R E; Chakravarti, A; Hittner, H M; Riccardi, V M

    1980-01-01

    Maximum likelihood analysis for linkage between autosomal dominant aniridia and 12 biochemical and serological markers in a single large family showed a probable linkage between autosomal dominant aniridia and the enzyme acid phosphatase-1. The presence of an autosomal dominant aniridia gene linked to acid phosphatase-1 on chromosome arm 2p and the existence of an aniridia syndrome resulting from deletion of band 13 of the short arm of chromosome 11 establishes a chromosome basis for genetic heterogeneity of aniridia phenotypes. PMID:6929510

  7. AN ANALYSIS OF CELLULAR AND SUBCELLULAR SYSTEMS WHICH TRANSFORM THE SPECIES CHARACTER OF ACID PHOSPHATASE IN ACETABULARIA.

    PubMed

    KECK, K; CHOULES, E A

    1963-08-01

    Several species-specific molecular forms of acid phosphatase are known to exist in the unicellular green alga Acetabularia. In graft combinations between cells of Acetabularia mediterranea (med) and Acicularia Schenckii (acic) the expression of the med phosphatase is dominant over acic phosphatase. There is good evidence that in such grafts the preexisting acic phosphatase is converted on the molecular level via an intermediate form to the med phosphatase. This conversion can be initiated by the transplantation of a med cell nucleus to an anucleate acic cell, but will also take place in grafts between anucleate med and anucleate acic cells, indicating that the direct participation of a cell nucleus is not required. An incomplete conversion of acic phosphatase, which terminates at the intermediate stage, is induced in acic cells by injection of a concentrated homogenate of med cytoplasm. A similar partial conversion occurs in vitro in a mixture of homogenates from med and acic cells. Subcellular particles, such as chloroplasts or mitochondria, can be removed from the homogenates by centrifugation without impairing the reactions leading to the intermediate phosphatase type. Experimental evidence suggests that the transformation of phosphatase types is enzymatically catalyzed and may involve the conjugation of small molecules with the phosphatase protein. It was shown, however, that sialic acid is not involved, since the incubation of med or acic homogenates with neuraminidase did not modify the electrophoretic mobility of either enzyme type. Another type of phosphatase, which occurs in Acetabularia erenulata (cren) and can be distinguished electrophoretically from the aforementioned types, is not subject to interaction. In various mono- and multi-nucleate graft combinations between cren cells on one hand, and med or acic cells on the other hand, the cren phosphatase is synthesized independently of the enzyme of the graft partner.

  8. Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR is essential for PROTEIN PHOSPHATASE 2A holoenzyme assembly and plays important roles in hormone signaling, salt stress response, and plant development.

    PubMed

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

    2014-11-01

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

  9. Detection of Extant Life in Extreme Environmentsby Phosphatase ActivitiesDetection of Extant Life in Extreme Environments by Measuring Phosphatase Activities

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kensei; Sato, Shuji; Naganawa, Kazuki; Itoh, Yuki; Kurihara, Hironari; Kaneko, Takeo; Takano, Yoshinori; Yoshimura, Yoshitaka; Kawasaki, Yukishige

    Since phosphate esters are essential for the terrestrial life, phosphatase activity can be a candidate for biosignatures of biological activity. It has been recognized that terrestrial biosphere expands to such extreme environments as deep subsurface lithosphere, high temperature hot springs and stratosphere. We analyzed phosphatase activities in the samples obtained in extreme environments such as submarine hydrothermal systems and Antarctica soils, and discussed whether they can be used as biosignatures for extant life. Core samples and chimney samples were collected at the Suiyo Seamount, Izu-Bonin Arc, the Pacific Ocean in 2001 and 2002, and in South Mariana hydrothermal systems, the Pacific Ocean in 2003, both in a part of the Archaean Park Project. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Alkaline (or acid) Phosphatase activity in solid samples was measured spectrometrically by using 25 mM p-nitrophenyl phosphate (pH 8.0 (or pH 6.5)) as a substrate. Phosphatase activities in extracts were measured fluorometrically by using 4-methylumberyferryl phosphate as a substrate. Concentration of amino acids and their enantiomeric ratios were also determined by HPLC and GC/MS. Significant enzymatic activities were revealed in both some of the hydrothermal sub-vent systems and Antarctica soils, which is crucial evidence of vigorous microbial oasis. It is consistent with the fact that large enantiomeric excess of L-form amino acids were found in the same core sequences. The ALP activity was diminished with EDTA and was recovered with addition of zinc ion. The present results showed that zinc-containing metalloenzymes are present in such environments as hydrothermal vent chimneys and Antarctica soils. Optimum temperatures of ALP in the chimney, Antarctica soil and YNU campus soil were 353 K, 313 K, and 333 K, respectively. The present results suggested that phosphatase

  10. Identification of protein tyrosine phosphatases and dual-specificity phosphatases in mammalian spermatozoa and their role in sperm motility and protein tyrosine phosphorylation.

    PubMed

    González-Fernández, L; Ortega-Ferrusola, C; Macias-Garcia, B; Salido, G M; Peña, F J; Tapia, J A

    2009-06-01

    Protein tyrosine kinases have important roles in spermatozoa; however, little is known about the presence and regulation in these cells of their counterparts in signaling, namely, protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). The objectives of the present study were to identify PTPs and DSPs in boar, stallion, and dog spermatozoa; to characterize their subcellular distribution; and to investigate the roles of tyrosine phosphatases in maintenance of protein tyrosine phosphorylation level and in sperm motility. Using Western blotting with specific antibodies in boar and stallion sperm lysates, we unequivocally identified two PTPs (PTPRB and PTPN11) and two DSPs (DUSP3 and DUSP4). In dog sperm lysates, only PTPN11, DUSP3, and DUSP4 were detected. In all these species, we did not detect the specific signal with anti-PTPRC (CD45), CDKN3, DUSP1, DUSP2, DUSP6, DUSP9, PTPN1, PTPN3, PTPN6, PTPN7, PTPN13, PTPRA, PTPRG, PTPRJ, PTPRK, or PTPRZ antibodies. Positive matches were further investigated by indirect immunofluorescence and confocal microscopy. Results showed that PTPRB was associated with the plasma membrane in the head and tail of boar and stallion spermatozoa. In agreement with Western blotting results, PTPRB antibodies did not show immunoreactivity in dog sperm analyzed by immunofluorescence. In the three species, DUSP4 was mainly found in the tail of spermatozoa, with little or no immunoreactivity in the head. PTPN11 was mainly located in the postacrosomal region in the head, whereas DUSP3 immunoreactivity was extended within the acrosome. PTPN11 and DUSP3 showed immunoreactivity in the tail that was restricted to the midpiece. Finally, we incubated boar, stallion, and dog spermatozoa with pervanadate and sodium orthovanadate, two PTP inhibitors, and analyzed overall protein tyrosine phosphorylation and assessed sperm motility. Sodium orthovanadate and pervanadate showed concentration-dependent inhibition of sperm motility that was

  11. ISOZYMES OF ACID PHOSPHATASE IN NORMAL AND CALMETTE-GUÉRIN BACILLUS-INDUCED RABBIT ALVEOLAR MACROPHAGES

    PubMed Central

    Axline, S. G.

    1968-01-01

    The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed. PMID:4878908

  12. Extreme Elevation of Alkaline Phosphatase in a Pregnancy Complicated by Gestational Diabetes and Infant with Neonatal Alloimmune Thrombocytopenia.

    PubMed

    Lozo, Svjetlana; Atabeygi, Amir; Healey, Michael

    2016-01-01

    There have been few case reports of isolated elevation of alkaline phosphatase beyond the normal physiologic amount with subsequent return to baseline after delivery. Here we present a similar case of extreme elevation of alkaline phosphatase in a pregnancy complicated by gestational diabetes and subsequently by neonatal alloimmune thrombocytopenia (NAIT). PMID:27610256

  13. Extreme Elevation of Alkaline Phosphatase in a Pregnancy Complicated by Gestational Diabetes and Infant with Neonatal Alloimmune Thrombocytopenia

    PubMed Central

    Healey, Michael

    2016-01-01

    There have been few case reports of isolated elevation of alkaline phosphatase beyond the normal physiologic amount with subsequent return to baseline after delivery. Here we present a similar case of extreme elevation of alkaline phosphatase in a pregnancy complicated by gestational diabetes and subsequently by neonatal alloimmune thrombocytopenia (NAIT).

  14. Extreme Elevation of Alkaline Phosphatase in a Pregnancy Complicated by Gestational Diabetes and Infant with Neonatal Alloimmune Thrombocytopenia

    PubMed Central

    Healey, Michael

    2016-01-01

    There have been few case reports of isolated elevation of alkaline phosphatase beyond the normal physiologic amount with subsequent return to baseline after delivery. Here we present a similar case of extreme elevation of alkaline phosphatase in a pregnancy complicated by gestational diabetes and subsequently by neonatal alloimmune thrombocytopenia (NAIT). PMID:27610256

  15. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  16. Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase.

    PubMed Central

    Spencer, D B; Chen, C P; Hulett, F M

    1981-01-01

    The effect of CO2+ on the synthesis and activation of Bacillus licheniformis MC14 alkaline phosphatase has been shown by the development of a defined minimal salts medium in which this organism produces 35 times more (assayable) alkaline phosphatase than when grown in a low-phosphate complex medium or in the defined medium without cobalt. Stimulation of enzyme activity with cobalt is dependent on a low phosphate concentration in the medium (below 0.075 mM) and continued protein synthesis. Cobalt stimulation resulted in alkaline phosphate production being a major portion of total protein synthesized during late-logarithmic and early-stationary-phase culture growth. Cells cultured in the defined medium minus cobalt, or purified enzyme partially inactivated with a chelating agent, showed a 2.5-fold increase in activity when assayed in the presence of cobalt. Atomic spectral analysis indicated the presence of 3.65 +/- 0.45 g-atoms of cobalt associated with each mole of purified active alkaline phosphatase. A biochemical localization as a function of culture age in this medium showed that alkaline phosphatase was associated with the cytoplasmic membrane and was also found as a soluble enzyme in the periplasmic region and secreted into the growth medium. PMID:7462163

  17. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    PubMed

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. PMID:24548880

  18. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    PubMed

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. PMID:26071590

  19. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. PMID:27043172

  20. [Biological profile of tartrate-resistant acid phosphatase as a marker of bone resorption].

    PubMed

    Rico, H; Iritia, M; Arribas, I; Revilla, M

    1990-12-01

    Tartrate-resistant serum acid phosphatase was measured in 123 subjects, 80 of which were normal and the rest pathologic, in order to define the profile and value of this parameter as a biological marker of osteoclastic activity. Normal subjects were divided into age groups based on the period where skeletal growth ends (under 20 years), at the age of menopause in women (50 years, between 20 and 50 years) and those over 50 years. There was an increase in tartrate-resistant serum acid phosphatase coinciding with puberty and no sex differences were observed after the 50 year mark, when women showed higher values than men (p less than 0.001). Such tartrate-resistant serum acid phosphatase increase, is reflected as higher values in the 50 year group than in the 20 to 50 year group (p less than 0.001), the only age limit where a negative significant correlation between tartrate-resistant serum acid phosphatase values and age could be observed (p less than 0.05). Values were higher up to the age of 20 years (p less than 0.001) than in any other older age group. Levels increased significantly (p less than 0.001 for both groups) in post-menopausal osteoporosis (n = 20) and in Paget's disease of bone (n = 15), and decreased significantly (p less than 0.05) in imperfect osteogenesis (n = 8), thus revealing its value as a biological marker of osteoclastic activity. PMID:2099535

  1. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition.

  2. Heat stable alkaline phosphatase from thermophiles. Final report, March-October 1993

    SciTech Connect

    Combie, J.D.; Runnion, K.N.; Williamson, M.L.

    1994-07-01

    Alkaline phosphatase has been the most widely used enzyme for colorimetric immunoassays. The current potential for this enzyme lies in biosensors, fieldable assay kits, biotechnology applications, degradation of certain nerve agents and pesticides and detoxification of heavy metal waste streams. While the commercial source of this enzyme is predominantly from mammalian tissues, expanded commercial application is restricted by the enzyme's instability at elevated temperatures. Although alkaline phosphatases are ubiquitous in nature, two isolates out of 44 alkaline phosphatase producing isolates occurring in habitats at 50 deg C and above have been isolated possessing extremely stable enzymes. One enzyme retained 98% of original activity following boiling for 1 hr. The secretion of the enzyme by the organism is an added benefit promoting efficient and economical production capability. Procedures for the screening, isolation, and optimal growth and fermentation of organisms acquired from geothermal sources located in Yellowstone National Park, WY are described. Purification was most effectively achieved using size exclusion chromatography where 101% of the activity and 33% of the crude mother liquor protein were recovered. Although the presence of manganese in the assay buffer was observed to significantly elevate the enzyme's catalytic activity, a precipitate incompatibility with calcium chloride, a requirement for high temperature stability, prohibits its use. Bacteria, Fermentation, Alkaline phosphatase, Biosensors, Biotechnology, Heat stable enzymes, Biochemistry, Bioremediation, Thermophilic microorganisms.

  3. Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

    PubMed

    Kumar, Mukesh; Khan, Imran; Sinha, Subrata

    2015-01-01

    Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

  4. Purification and characterization of a protein phosphatase that dephosphorylates pyruvate kinase in an anoxia tolerant animal.

    PubMed

    Brooks, S P; Storey, K B

    1996-05-01

    A protein phosphatase that dephosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia tolerant gastropod mollusc Busycon canaliculatum. Purification involved three steps: negative chromatography through Blue Dextran and CM Sephadex, affinity chromatography on DEAE Sephadex and gel exclusion chromatography on Sephacryl S-400. Pyruvate kinase phosphatase (PK-Pase) activity was monitored by following changes in PK I50 values for L-alanine that had previously been linked to changes in the degree of PK phosphorylation. The purified PK-Pase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 41 +/- 1 kdaltons. Isoelectric focusing analysis showed that the PK-Pase had an isoelectric point of 4.2 +/- 0.1. Kinetic analysis showed that the enzyme was a Type 2C protein phosphatase with a pH optimum of 6.5. Maximal activity required the presence of magnesium ions (KM = 7.9 +/- 0.6 microM) although high concentrations of Mg2+ were inhibitory (I50 = 2.3 +/- 0.4 mM). The protein phosphatase activity was not affected by either spermine, cAMP, cGMP, potassium phosphate, tartrate, NaF, HgCl2, citrate or concentrations of CaCl2 less than 10 mM. The enzyme could also use ATP, ADP, and GTP as substrates. PMID:8739044

  5. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    EPA Science Inventory

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  6. ISOLATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE ACTIVITY FROM SPIRODELA OLIGORHIZA

    EPA Science Inventory

    An acid phosphatase activity from the aquatic plant Spirodela oligorhiza (duckweed) was isolated and partially characterized. S. oligorhiza was grown in a hydroponic growth medium, harvested, and ground up in liquid nitrogen. The ground plant material was added to a biological ...

  7. UIS2: A Unique Phosphatase Required for the Development of Plasmodium Liver Stages

    PubMed Central

    Zhang, Min; Mishra, Satish; Sakthivel, Ramanavelan; Fontoura, Beatriz M. A.; Nussenzweig, Victor

    2016-01-01

    Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection. PMID:26735921

  8. Characterization of New Substrates Targeted By Yersinia Tyrosine Phosphatase YopH

    PubMed Central

    de la Puerta, María Luisa; Trinidad, Antonio G.; del Carmen Rodríguez, María; Bogetz, Jori; Sánchez Crespo, Mariano; Mustelin, Tomas; Alonso, Andrés; Bayón, Yolanda

    2009-01-01

    YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates. PMID:19221593

  9. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus

    PubMed Central

    Winkelströter, Lizziane K.; Dolan, Stephen K.; Fernanda dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W.; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H.

    2015-01-01

    Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen. PMID:25943523

  10. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase

    PubMed Central

    Linford, Alicia S.; Jiang, Nona M.; Edwards, Thomas E.; Sherman, Nicholas E.; Van Voorhis, Wesley C.; Stewart, Lance J.; Myler, Peter J.; Staker, Bart L.; Petri, William A.

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. PMID:24548880

  11. Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch.

    PubMed

    Qian, Junbin; Beullens, Monique; Huang, Jin; De Munter, Sofie; Lesage, Bart; Bollen, Mathieu

    2015-01-01

    RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates. PMID:26674376

  12. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  13. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition. PMID:20934434

  14. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparat...

  15. [Effect of phosphorus deficiency on activity of acid phosphatase exuded by wheat roots].

    PubMed

    Sun, Haiguo; Zhang, Fusuo

    2002-03-01

    The activity of acid phosphatase exuded by roots, the tissue location of the enzyme, and the relationship between the enzyme activity and phosphorus efficiency of wheat were studied. The results showed that the activity of acid phosphatase exuded by wheat 81(85)5-3-3-3 and NC37 under P-sufficiency treat were lower than those under P-deficiency, and the enzyme activity of the former variety was significantly higher than that of the latter. There was a significant difference in the enzyme activity among 12 wheat genotypes grown under P-deficiency treat. Acid phosphatase was exuded by epidermis cell of root, especially by epidermal cell of root apex. Thus, there was a linear relationship between the enzyme activity and the surface area of root or the number of root apexes. It implied that the enzyme activity was markedly related to the size of root system. The linear relationship between relative grain yield and acid phosphatase activity was significant. It indicates that the enzyme activity could be used as an early indicator to select P-efficient wheat genotypes.

  16. Receptor protein tyrosine phosphatases are novel components of a polycystin complex

    PubMed Central

    Boucher, Catherine A.; Ward, Heather H.; Case, Ruth L.; Thurston, Katie S.; Li, Xiaohong; Needham, Andrew; Romero, Elsa; Hyink, Deborah; Qamar, Seema; Roitbak, Tamara; Powell, Samantha; Ward, Christopher; Wilson, Patricia D.; Wandinger-Ness, Angela; Sandford, Richard N.

    2011-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, but the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. PMID:21126580

  17. Seleninate in Place of Phosphate: Irreversible Inhibition of Protein Tyrosine Phosphatases

    SciTech Connect

    Abdo, Mohannad; Liu, Sijiu; Zhou, Bo; Walls, Chad D.; Wu, Li; Knapp, Spencer; Zhang, Zhong-Yin

    2009-02-16

    A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization.

  18. Possible protein phosphatase inhibition by bis(hydroxyethyl) sulfide, a hydrolysis product of mustard gas

    SciTech Connect

    Brimfield, A.A.

    1995-12-31

    Recently, the natural vesicant cantharidin was shown to bind exclusively to and inhibit protein phosphatase 2A (PP2A) in mouse tissue extracts (Li and Casida (1992) Proc. Nati. Acad. Sci. USA 89, 11867-11870). To explore the generality of this effect in vesicant action, we measured the protein serinelthreonine phosphatase activity in mouse liver cytosol (in the form of the okadaic acid inhibitable increment of p-nitrophenyl phosphate (p-NPP) phosphatase activity) in the presence of aqueous sulfur mustard or its hydrolysis product, bis(hydroxyethyl)sulfide (TDG). Sulfur mustard inhibited p-NPP hydrolysis. However, inhibition correlated with the time elapsed between thawing and the addition of mustard to the enzyme preparation, not with concentration. TDG exhibited a direct, concentration-related inhibition of p-NPP hydrolysis between 30 and 300 1LM. We conclude that sulfur mustard also has an inhibitory effect on protein serinelthreonine phosphatases. However, the inhibition is an effect of its non-alkykating hydrolysis product TDG, not of sulfur mustard itself.

  19. Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis.

    PubMed

    De Leon, Gabriel; Sherry, Tara C; Krucher, Nancy A

    2008-06-01

    There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies. PMID:18360108

  20. Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch

    PubMed Central

    Qian, Junbin; Beullens, Monique; Huang, Jin; De Munter, Sofie; Lesage, Bart; Bollen, Mathieu

    2015-01-01

    RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates. PMID:26674376

  1. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    PubMed

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  2. Vanadate inhibition of fungal phyA and bacterial appA2 histidine acid phosphatases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal PhyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could also serve as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading ac...

  3. A potent and selective inhibitor for the UBLCP1 proteasome phosphatase

    PubMed Central

    He, Yantao; Guo, Xing; Yu, Zhi-Hong; Wu, Li; Gunawan, Andrea M.; Zhang, Yan; Dixon, Jack E.; Zhang, Zhong-Yin

    2015-01-01

    The ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) has been implicated as a negative regulator of the proteasome, a key mediator in the ubiquitin-dependent protein degradation. Small molecule inhibitors that block UBLCP1 activity would be valuable as research tools and potential therapeutics for human diseases caused by the cellular accumulation of misfold/damaged proteins. We report a salicylic acid fragment-based library approach aimed at targeting both the phosphatase active site and its adjacent binding pocket for enhanced affinity and selectivity. Screening of the focused libraries led to the identification of the first potent and selective UBLCP1 inhibitor 13. Compound 13 exhibits an IC50 of 1.0 μM for UBLCP1 and greater than 5-fold selectivity against a large panel of protein phosphatases from several distinct families. Importantly, the inhibitor possesses efficacious cellular activity and is capable of inhibiting UBLCP1 function in cells, which in turn up-regulates nuclear proteasome activity. These studies set the groundwork for further developing compound 13 into chemical probes or potential therapeutic agents targeting the UBLCP1 phosphatase. PMID:25907364

  4. Prostatic acid phosphatase: structural aspects of inhibition by L-(+)-tartrate ions.

    PubMed

    Lovelace, L; Lewiński, K; Jakob, C G; Kuciel, R; Ostrowski, W; Lebioda, L

    1997-01-01

    The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.

  5. Highly sensitive detection of alkaline phosphatase using molecular beacon probes based on enzymatic polymerization.

    PubMed

    Ma, Changbei

    2012-06-01

    We have developed a new methodology for highly sensitive alkaline phosphatase assay using molecular beacon probes. No incubation step is needed to obtain a limit of detection for ALP of 2×10(-16) M. Furthermore, ALP inhibition by the inhibitor okadaic acid is shown, demonstrating the potential for high-throughput screening for inhibitors.

  6. Ligand Binding to the Androgen Receptor Induces Conformational Changes That Regulate Phosphatase Interactions▿

    PubMed Central

    Yang, Chun-Song; Xin, Hong-Wu; Kelley, Joshua B.; Spencer, Adam; Brautigan, David L.; Paschal, Bryce M.

    2007-01-01

    We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation. PMID:17325038

  7. Serine/threonine protein phosphatases: multi-purpose enzymes in control of defense mechanisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine/threonine protein phosphatases are a group of enzymes involved in the regulation of defense mechanisms in plants. This paper describes the effects of an inhibitor of these enzymes on the expression of all of the genes associated with these defense mechanisms. The results suggest that inhibi...

  8. Therapeutic strategies for anchored kinases and phosphatases: exploiting short linear motifs and intrinsic disorder

    PubMed Central

    Nygren, Patrick J.; Scott, John D.

    2015-01-01

    Phosphorylation events that occur in response to the second messenger cAMP are controlled spatially and temporally by protein kinase A (PKA) interacting with A-kinase anchoring proteins (AKAPs). Recent advances in understanding the structural basis for this interaction have reinforced the hypothesis that AKAPs create spatially constrained signaling microdomains. This has led to the realization that the PKA/AKAP interface is a potential drug target for modulating a plethora of cell-signaling events. Pharmacological disruption of kinase–AKAP interactions has previously been explored for disease treatment and remains an interesting area of research. However, disrupting or enhancing the association of phosphatases with AKAPs is a therapeutic concept of equal promise, particularly since they oppose the actions of many anchored kinases. Accordingly, numerous AKAPs bind phosphatases such as protein phosphatase 1 (PP1), calcineurin (PP2B), and PP2A. These multimodal signaling hubs are equally able to control the addition of phosphate groups onto target substrates, as well as the removal of these phosphate groups. In this review, we describe recent advances in structural analysis of kinase and phosphatase interactions with AKAPs, and suggest future possibilities for targeting these interactions for therapeutic benefit. PMID:26283967

  9. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  12. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  13. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  14. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  15. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  16. 21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alkaline phosphatase or isoenzymes test system. 862.1050 Section 862.1050 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  17. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.1020 - Acid phosphatase (total or prostatic) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Acid phosphatase (total or prostatic) test system. 862.1020 Section 862.1020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. The Structure of Fcp1, an Essential RNA Polymerase II CTD Phosphatase

    SciTech Connect

    Ghosh, A.; Shuman, S.; Lima, C.D.

    2009-03-27

    Kinases and phosphatases regulate mRNA synthesis and processing by phosphorylating and dephosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Fcp1 is an essential CTD phosphatase that preferentially hydrolyzes Ser2-PO{sub 4} of the tandem YSPTSPS CTD heptad array. Fcp1 crystal structures were captured at two stages of the reaction pathway: a Mg-BeF{sub 3} complex that mimics the aspartylphosphate intermediate and a Mg-AlF{sub 4}{sup -} complex that mimics the transition state of the hydrolysis step. Fcp1 is a Y-shaped protein composed of an acylphosphatase domain located at the base of a deep canyon formed by flanking modules that are missing from the small CTD phosphatase (SCP) clade: an Fcp1-specific helical domain and a C-terminal BRCA1 C-terminal (BRCT) domain. The structure and mutational analysis reveals that Fcp1 and Scp1 (a Ser5-selective phosphatase) adopt different CTD-binding modes; we surmise the CTD threads through the Fcp1 canyon to access the active site.

  20. ZN2+ INDUCES COX-2 EXPRESSION THROUGH DOWNREGULATION OF LIPID PHOSPHATASE PTEN

    EPA Science Inventory

    Zn2+ Induces COX-2 Expression through Downregulation of Lipid Phosphatase PTEN
    Weidong Wu*, James M. Samet, Philip A. Bromberg*?, Young E. Whang?, and Lee M. Graves* ?
    *CEMALB, ?Department of Medicine, and ?Department of Pharmacology, UNC-Chapel Hill, NC27599; Human Studie...

  1. Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

    SciTech Connect

    Nelson, R.L.; Branton, P.E.

    1984-06-01

    Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using /sup 32/P-labeled immunoglobulin G. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII) were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn/sup 2 +/, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F/sup -/ under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

  2. Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.

    PubMed

    Rininsland, Frauke; Xia, Wensheng; Wittenburg, Shannon; Shi, Xiaobo; Stankewicz, Casey; Achyuthan, Komandoor; McBranch, Duncan; Whitten, David

    2004-10-26

    An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation. PMID:15494445

  3. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-01-01

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade. PMID:26304753

  4. Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase.

    PubMed

    Hou, Hsin-Han; Liao, Yi-Jen; Hsiao, Sheng-Huang; Shyue, Song-Kun; Lee, Tzong-Shyuan

    2015-08-25

    Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor-c-Src-increased Tyr phosphorylation of sEH and formation of an sEH-Akt-AMPK-eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt-AMPK-eNOS signaling cascade.

  5. Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods.

    PubMed

    Albillos, S M; Reddy, R; Salter, R

    2011-07-01

    Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.

  6. Isolation, transcription, and inactivation of the gene for an atypical alkaline phosphatase of Synechococcus sp. strain PCC 7942.

    PubMed Central

    Ray, J M; Bhaya, D; Block, M A; Grossman, A R

    1991-01-01

    The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake. Images PMID:1712356

  7. Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.

    PubMed

    Romá-Mateo, Carlos; Sacristán-Reviriego, Almudena; Beresford, Nicola J; Caparrós-Martín, José Antonio; Culiáñez-Macià, Francisco A; Martín, Humberto; Molina, María; Tabernero, Lydia; Pulido, Rafael

    2011-04-01

    Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including

  8. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    SciTech Connect

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-05-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca/sup 2 +/. One mechanism by which Ca/sup 2 +/ may trigger neutrophil activation is through Ca/sup 2 +//calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca/sup 2 +//CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and /sup 125/I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner. One predominant region of /sup 125/I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca/sup 2 +/-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 ..mu..g/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca/sup 2 +/-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca/sup 2 +//calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase.

  9. Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases

    PubMed Central

    Forrest, Alistair RR; Taylor, Darrin F; Crowe, Mark L; Chalk, Alistair M; Waddell, Nic J; Kolle, Gabriel; Faulkner, Geoffrey J; Kodzius, Rimantas; Katayama, Shintaro; Wells, Christine; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Grimmond, Sean M

    2006-01-01

    Background Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse . Results By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. Conclusion These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways. PMID:16507138

  10. Structural basis of the inhibition of class C acid phosphatases by adenosine 5;#8242;-phosphorothioate

    SciTech Connect

    Singh, Harkewal; Reilly, Thomas J.; Tanner, John J.

    2012-01-20

    The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-{angstrom} resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64 {yields} Asn mutant enzyme was also determined at 1.35-{angstrom} resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64 {yields} Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.

  11. Myxococcus xanthus Pph2 Is a Manganese-dependent Protein Phosphatase Involved in Energy Metabolism*

    PubMed Central

    García-Hernández, Raquel; Moraleda-Muñoz, Aurelio; Castañeda-García, Alfredo; Pérez, Juana; Muñoz-Dorado, José

    2009-01-01

    The multicellular behavior of the myxobacterium Myxococcus xanthus requires the participation of an elevated number of signal-transduction mechanisms to coordinate the cell movements and the sequential changes in gene expression patterns that lead to the morphogenetic and differentiation events. These signal-transduction mechanisms are mainly based on two-component systems and on the reversible phosphorylation of protein targets mediated by eukaryotic-like protein kinases and phosphatases. Among all these factors, protein phosphatases are the elements that remain less characterized. Hence, we have studied in this work the physiological role and biochemical activity of the protein phosphatase of the family PPP (phosphoprotein phosphatases) designated as Pph2, which is forming part of the same operon as the two-component system phoPR1. We have demonstrated that this operon is induced upon starvation in response to the depletion of the cell energy levels. The increase in the expression of the operon contributes to an efficient use of the scarce energy resources available for developing cells to ensure the completion of the life cycle. In fact, a Δpph2 mutant is defective in aggregation, sporulation yield, morphology of the myxospores, and germination efficiency. The yeast two-hybrid technology has shown that Pph2 interacts with the gene products of MXAN_1875 and 5630, which encode a hypothetical protein and a glutamine synthetase, respectively. Because Pph2 exhibits Ser/Thr, and to some extent Tyr, Mn2+-dependent protein phosphatase activity, it is expected that this function is accomplished by dephosphorylation of the specific protein substrates. PMID:19706604

  12. LEPS2, a Phosphorus Starvation-Induced Novel Acid Phosphatase from Tomato1

    PubMed Central

    Baldwin, James C.; Karthikeyan, Athikkattuvalasu S.; Raghothama, Kashchandra G.

    2001-01-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase. PMID:11161030

  13. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    PubMed

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

  14. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-01

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  15. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    PubMed

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family. PMID:26537753

  16. LEPS2, a phosphorus starvation-induced novel acid phosphatase from tomato.

    PubMed

    Baldwin, J C; Karthikeyan, A S; Raghothama, K G

    2001-02-01

    Phosphate (Pi) is one of the least available plant nutrients found in the soil. A significant amount of phosphate is bound in organic forms in the rhizosphere. Phosphatases produced by plants and microbes are presumed to convert organic phosphorus into available Pi, which is absorbed by plants. In this study we describe the isolation and characterization of a novel tomato (Lycopersicon esculentum) phosphate starvation-induced gene (LePS2) representing an acid phosphatase. LePS2 is a member of a small gene family in tomato. The cDNA is 942 bp long and contains an open reading frame encoding a 269-amino acid polypeptide. The amino acid sequence of LePS2 has a significant similarity with a phosphatase from chicken. Distinct regions of the peptide also share significant identity with the members of HAD and DDDD super families of phosphohydrolases. Many plant homologs of LePS2 are found in the databases. The LePS2 transcripts are induced rapidly in tomato plant and cell culture in the absence of Pi. However, the induction is repressible in the presence of Pi. Divided root studies indicate that internal Pi levels regulate the expression of LePS2. The enhanced expression of LePS2 is a specific response to Pi starvation, and it is not affected by starvation of other nutrients or abiotic stresses. The bacterially (Escherichia coli) expressed protein exhibits phosphatase activity against the synthetic substrate p-nitrophenyl phosphate. The pH optimum of the enzyme activity suggests that LePS2 is an acid phosphatase.

  17. Evaluating transition state structures of vanadium-phosphatase protein complexes using shape analysis.

    PubMed

    Sánchez-Lombardo, Irma; Alvarez, Santiago; McLauchlan, Craig C; Crans, Debbie C

    2015-06-01

    Shape analysis of coordination complexes is well-suited to evaluate the subtle distortions in the trigonal bipyramidal (TBPY-5) geometry of vanadium coordinated in the active site of phosphatases and characterized by X-ray crystallography. Recent studies using the tau (τ) analysis support the assertion that vanadium is best described as a trigonal bipyramid, because this geometry is the ideal transition state geometry of the phosphate ester substrate hydrolysis (C.C. McLauchlan, B.J. Peters, G.R. Willsky, D.C. Crans, Coord. Chem. Rev. http://dx.doi.org/10.1016/j.ccr.2014.12.012 ; D.C. Crans, M.L. Tarlton, C.C. McLauchlan, Eur. J. Inorg. Chem. 2014, 4450-4468). Here we use continuous shape measures (CShM) analysis to investigate the structural space of the five-coordinate vanadium-phosphatase complexes associated with mechanistic transformations between the tetrahedral geometry and the five-coordinate high energy TBPY-5 geometry was discussed focusing on the protein tyrosine phosphatase 1B (PTP1B) enzyme. No evidence for square pyramidal geometries was observed in any vanadium-protein complexes. The shape analysis positioned the metal ion and the ligands in the active site reflecting the mechanism of the cleavage of the organic phosphate in a phosphatase. We identified the umbrella distortions to be directly on the reaction path between tetrahedral phosphate and the TBPY-5-types of high-energy species. The umbrella distortions of the trigonal bipyramid are therefore identified as being the most relevant types of transition state structures for the phosphoryl group transfer reactions for phosphatases and this may be related to the possibility that vanadium is an inhibitor for enzymes that support both exploded and five-coordinate transition states.

  18. Effects of phosphoprotein phosphatase inhibitors (phenylarsine oxide and cantharidin) on Tetrahymena.

    PubMed

    Kovács, P; Pintér, M

    2001-09-01

    The effects of phenylarsine oxide (PAO) (phosphotyrosine phosphatase inhibitor) and cantharidin (serine/threonine phosphatase [PP2A] inhibitor) treatments were analysed on the synthesis of phospholipids and glycolipids, and on the cytoskeletal elements (F-actin and tubulin containing structures) of Tetrahymena pyriformis. Both phosphatase inhibitors reduced the amount of incorporated 32P of the whole phospholipid content, but the ratio of phosphatidylserine (PS) and phosphatidylcholine (PC) to the total phospholipid content increased. Both treatments influenced the phosphatidylinositol (PI) system. These inhibitors also influenced the incorporation of palmitic acid into the phospholipids: in general PAO decreased, whereas cantharidin increased the amount of incorporated palmitic acid; 1 microM cantharidin significantly increased the labelling of PE and PA. The incorporation of mannose and glucosamine was influenced differently by PAO and cantharidin treatments: the latter elevated, while PAO decreased the labelling of glycolipids with these sugars. The effects of these treatments were visible also in the case of confocal scanning laser microscopic (CSLM) images: after treatments with both inhibitors, the F-actin containing cortical elements were destroyed, but the tubulin containing ones (longitudinal and transversal microtubules, oral apparatus and deep fibres) did not display significant alterations. The different effects of phosphatase inhibitors were visible also on the scanning electron microscopic (sEM) images: cantharidin treatments (1 microM) decreased the amount of dissolved membrane lipids after chemical dehydration of the cells with 2, 2-dimethoxy propane (DMP), but in the case of treatments with 10 microM, the surface pattern of cells was similar to the controls. On the other hand, after PAO treatments the surface pattern of Tetrahymena showed significant alterations. Both phosphatase inhibitors inhibited the phagocytotic activity of the cells. On the

  19. Histochemical Phosphatases and Metachromasia in Murine Tumours Induced by Bone Seeking Radionuclides

    PubMed Central

    Bland, M. R.; Loutit, J. F.; Sansom, Janet M.

    1974-01-01

    Tumours induced in mice, either CBA normal and chimaerical, or C3H, by 90Sr or 226Ra or plutonium have been examined histochemically with (1) diazotate fast red violet LB salt in naphthol AS-MX phosphate buffer at pH 8·6 and 5·2, (2) 1: 9 dimethyl methylene blue (Taylor). It is concluded: (a) The diagnosis of osteosarcoma is facilitated with Taylor's Blue which stains osteoid metachromatically. Cells of osteosarcoma, like normal osteoblasts, contain alkaline phosphatase but this may be lost by mutation either in the original tumour or subsequently on passage of the tumour serially to compatible hosts. (b) Osteosarcomata may contain giant-cells of two forms, bizarre tumour cells and osteoclasts; the latter contain acid phosphatase. Osteosarcomata which retain their osteoid on serial passage have few cells containing acid phosphatases. (c) Primitive mesenchymal cell tumours of angiomatous form may occur, if the bone marrow is irradiated, e.g. by 90Sr-90Y and Pu. These tumours lack osteoid and cells interpretable as osteoblasts or osteoclasts (though they destroy bone). (d) Tumours classifiable as fibrosarcomata occur rarely, and may be truly of fibroblastic origin or be mutated osteosarcomata. (e) Lymphomata also occur when the marrow is irradiated (90Sr-90Y and Pu). They may be generalized, when their cells may contain alkaline phosphatase or lack it. They may be localized to abdominal viscera, the reticulo-sarcomatous form, in which case the cells lack alkaline phosphatase. ImagesFig. 1Fig. 3Fig. 5Fig. 8Fig. 10Fig. 11Fig. 2Fig. 4Fig. 6Fig. 7Fig. 9Fig. 12 PMID:4133784

  20. Phosphorylation of Protein Phosphatase Inhibitor-1 by Protein Kinase C*s

    PubMed Central

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D.; Nairn, Angus C.; Bibb, James A.

    2015-01-01

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr35. Moreover, Ser67 of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser67 inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser65 in vitro. In contrast, Ser67 phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser65. Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser65 and Ser67, but not Ser65 alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser65 inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser67 protects phospho-Ser65 inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser65/Ser67 inhibitor-1 in this tissue. In contrast, the activation of N-methyl-D-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser65/Ser67 inhibitor-1 levels. Phosphomimetic mutation of Ser65 and/or Ser67 did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser65/Ser67 inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser67 and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation. PMID:16772299

  1. Global Analysis of Serine/Threonine and Tyrosine Protein Phosphatase Catalytic Subunit Genes in Neurospora crassa Reveals Interplay Between Phosphatases and the p38 Mitogen-Activated Protein Kinase

    PubMed Central

    Ghosh, Arit; Servin, Jacqueline A.; Park, Gyungsoon; Borkovich, Katherine A.

    2013-01-01

    Protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. The genome of the filamentous fungus and model organism Neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. In this study, we have characterized 24 viable N. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. We found that 91% of the mutants had defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock. PMID:24347630

  2. Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

    PubMed

    Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

    2013-03-15

    The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

  3. Comparative Evaluation of Efficacy of Three Different Herbal Toothpastes on Salivary Alkaline Phosphatase and Salivary Acid Phosphatase - A Randomized Controlled Trial

    PubMed Central

    Dodamani, Arun; Karibasappa, G. N.; Deshmukh, Manjiri; Naik, Rahul

    2016-01-01

    Introduction Very few researches in the past have tried to evaluate the effect of herbal toothpaste on saliva and salivary constituents like alkaline phosphatase and acid phosphatase which play an important role in maintaining oral health. Aim To evaluate and compare the effect of three different herbal toothpastes on Salivary Alkaline Phosphatase (ALP) and salivary Acid Phosphatase (ACP). Material and Methods The present study was a preliminary study conducted among 45 dental students (15 subjects in each group) in the age group of 19-21 years. Subjects in each group were randomly intervened with three different herbal toothpastes respectively (Group A – Patanjali Dant Kanti, Group B - Himalaya Complete Care and Group C – Vicco Vajradanti). Unstimulated saliva sample were collected before and after brushing and salivary ACP and salivary ALP levels were assessed at an interval of one week each for a period of four weeks starting from day one. Compiled data was analyzed using chi square test, paired t-test and ANOVA based on the nature of the obtained data. Results All the three toothpastes showed significant (p<0.001) reduction in ACP and ALP levels at each interval. For patanjali toothpaste, the mean reduction was in the range of 2.55 – 2.62 IU/L for ACP and 2.94 – 2.99 IU/L for ALP. For Himalaya toothpaste, the mean reduction was in the range of 1.39 – 1.47 IU/L for ACP and 1.55 – 1.61 IU/L for ALP. For Vicco toothpaste, the mean reduction was in the range of 2.46 – 2.50 IU/L for ACP and 2.64 – 2.77 IU/L for ALP. Patanjali and Vicco toothpaste were significantly effective in reducing the levels of salivary ACP and ALP more than Himalaya toothpaste (p<0.05). Conclusion Herbal toothpastes, especially Dant Kanti and Vicco Vajradanti, showed significant reduction in levels of ACP and ALP resulting in overall improvement towards the oral health. PMID:27790584

  4. Crystal Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family

    PubMed Central

    2016-01-01

    The inositol polyphosphate 5-phosphatase INPP5B hydrolyzes the 5-phosphate group from water- and lipid-soluble signaling messengers. Two synthetic benzene and biphenyl polyphosphates (BzP/BiPhPs), simplified surrogates of inositol phosphates and phospholipid headgroups, were identified by thermodynamic studies as potent INPP5B ligands. The X-ray structure of the complex between INPP5B and biphenyl 3,3′,4,4′,5,5′-hexakisphosphate [BiPh(3,3′,4,4′,5,5′)P6, IC50 5.5 μM] was determined at 2.89 Å resolution. One inhibitor pole locates in the phospholipid headgroup binding site and the second solvent-exposed ring binds to the His-Tag of another INPP5B molecule, while a molecule of inorganic phosphate is also present in the active site. Benzene 1,2,3-trisphosphate [Bz(1,2,3)P3] [one ring of BiPh(3,3′,4,4′,5,5′)P6] inhibits INPP5B ca. 6-fold less potently. Co-crystallization with benzene 1,2,4,5-tetrakisphosphate [Bz(1,2,4,5)P4, IC50 = 6.3 μM] yielded a structure refined at 2.9 Å resolution. Conserved residues among the 5-phosphatase family mediate interactions with Bz(1,2,4,5)P4 and BiPh(3,3′,4,4′,5,5′)P6 similar to those with the polar groups present in positions 1, 4, 5, and 6 on the inositol ring of the substrate. 5-Phosphatase specificity most likely resides in the variable zone located close to the 2- and 3-positions of the inositol ring, offering insights to inhibitor design. We propose that the inorganic phosphate present in the INPP5B–BiPh(3,3′,4,4′,5,5′)P6 complex mimics the postcleavage substrate 5-phosphate released by INPP5B in the catalytic site, allowing elucidation of two new key features in the catalytic mechanism proposed for the family of phosphoinositide 5-phosphatases: first, the involvement of the conserved Arg-451 in the interaction with the 5-phosphate and second, identification of the water molecule that initiates 5-phosphate hydrolysis. Our model also has implications for the proposed “moving metal” mechanism

  5. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

    PubMed Central

    Sneddon, A A; Cohen, P T; Stark, M J

    1990-01-01

    Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell. Images Fig. 5. PMID:2176150

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

    PubMed

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

    2016-09-01

    Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages. PMID:27220404

  7. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

    PubMed

    Jaumot, M; Hancock, J F

    2001-07-01

    Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

  8. Carboxyl-terminal receptor domains control the differential dephosphorylation of somatostatin receptors by protein phosphatase 1 isoforms.

    PubMed

    Lehmann, Andreas; Kliewer, Andrea; Märtens, Jan Carlo; Nagel, Falko; Schulz, Stefan

    2014-01-01

    We have recently identified protein phosphatase 1β (PP1β) as G protein-coupled receptor (GPCR) phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ) as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

  9. A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine.

    PubMed

    Bruder, S P; Caplan, A I

    1990-01-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.

  10. Crystal Structures of Protein Phosphatase-1 Bound to Nodularin-R and Tautomycin: A Novel Scaffold for Structure Based Drug Design of Serine/threonine Phosphatase Inhibitors

    SciTech Connect

    Kelker, M.; Page, R; Peti, W

    2009-01-01

    Protein phosphatase 1 occurs in all tissues and regulates many pathways, ranging from cell-cycle progression to carbohydrate metabolism. Many naturally occurring, molecular toxins modulate PP1 activity, though the exact mechanism of this differential regulation is not understood. A detailed elucidation of these interactions is crucial for understanding the cellular basis of phosphatase function and signaling pathways but, more importantly, they can serve as the basis for highly specific therapeutics, e.g. against cancer. We report the crystal structures of PP1 in complex with nodularin-R at 1.63 {angstrom} and tautomycin at 1.70 {angstrom} resolution. The PP1:nodularin-R complex was used to demonstrate the utility of our improved PP1 production technique, which produces highly active, soluble PP1. Tautomycin is one of the few toxins that reportedly preferentially binds PP1> PP2A. Therefore, the PP1:tautomycin structure is the first complex structure with a toxin with preferred PP1 specificity. Furthermore, since tautomycin is a linear non-peptide-based toxin, our reported structure will aid the design of lead compounds for novel PP1-specific pharmaceuticals.

  11. Small Molecule Receptor Protein Tyrosine Phosphatase γ (RPTPγ) Ligands That Inhibit Phosphatase Activity via Perturbation of the Tryptophan-Proline-Aspartate (WPD) Loop

    SciTech Connect

    Sheriff, Steven; Beno, Brett R; Zhai, Weixu; Kostich, Walter A; McDonnell, Patricia A; Kish, Kevin; Goldfarb, Valentina; Gao, Mian; Kiefer, Susan E; Yanchunas, Joseph; Huang, Yanling; Shi, Shuhao; Zhu, Shirong; Dzierba, Carolyn; Bronson, Joanne; Macor, John E; Appiah, Kingsley K; Westphal, Ryan S; O’Connell, Jonathan; Gerritz, Samuel W

    2012-11-09

    Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an 'open' conformation or a 'closed' conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced 'superopen' conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.

  12. Bistability of a coupled Aurora B kinase-phosphatase system in cell division.

    PubMed

    Zaytsev, Anatoly V; Segura-Peña, Dario; Godzi, Maxim; Calderon, Abram; Ballister, Edward R; Stamatov, Rumen; Mayo, Alyssa M; Peterson, Laura; Black, Ben E; Ataullakhanov, Fazly I; Lampson, Michael A; Grishchuk, Ekaterina L

    2016-01-01

    Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division. PMID:26765564

  13. Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

    PubMed

    Jiang, Huan; Sui, Yuan; Cui, Yue; Lin, Peng; Li, Wannan; Xing, Shu; Wang, Deli; Hu, Min; Fu, Xueqi

    2015-03-01

    Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0. PMID:25462809

  14. Insect freeze tolerance: Roles of protein phosphatases and protein kinase A.

    PubMed

    Pfister, Thomas D; Storey, Kenneth B

    2006-01-01

    Freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis Fitch, show multiple metabolic adaptations for subzero survival including the autumn synthesis of high concentrations of polyols. The induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. The present study evaluates changes in the activities of cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) over the course of the winter season and also in insects exposed to -4, -20 degrees C, or anoxic conditions in the laboratory. The increased PKA and decreased PP1 over the winter season and/or at subzero temperature support a regulatory role for these enzymes in cryoprotectant polyol synthesis. PTP activities were also strongly increased under these conditions and may act to antagonize tyrosine kinase mediated cell growth and proliferation responses and, thereby, contribute to hypometabolism and diapause over the winter.

  15. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

  16. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP. PMID:10556574

  17. Phosphatase activity in Antarctica soil samples as a biosignature of extant life

    NASA Astrophysics Data System (ADS)

    Sato, Shuji; Itoh, Yuki; Takano, Yoshinori; Fukui, Manabu; Kaneko, Takeo; Kobayashi, Kensei

    Microbial activities have been detected in such extreme terrestrial environments as deep lithosphere, a submarine hydrothermal systems, stratosphere, and Antarctica. Microorganisms have adapted to such harsh environments by evolving their biomolecules. Some of these biomolecules such as enzymes might have different characteristics from those of organisms in ordinary environments. Many biosignatures (or biomarkers) have been proposed to detect microbial activities in such extreme environments. A number of techniques are proposed to evaluate biological activities in extreme environments including cultivation methods, assay of metabolism, and analysis of bioorganic compounds like amino acids and DNA. Enzyme activities are useful signature of extant life in extreme environments. Among many enzymes, phosphatase could be a good indicator of biological activities, since phosphate esters are essential for all the living terrestrial organisms. In addition, alkaline phosphatase is known as a typical zinc-containing metalloenzyme and quite stable in environments. We analyzed phosphatase activities in Antarctica soil samples to see whether they can be used as biosignatures for extant life. In addition, we characterized phosphatases extracted from the Antarctica soil samples, and compared with those obtained from other types of environments. Antarctica surface environments are quite severe environments for life since it is extremely cold and dry and exposed to strong UV and cosmic rays. We tried to evaluate biological activities in Antarctica by measuring phosphatase activities. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) are measured spectrophotometrically after mixing the powdered sample and p-nitrophenyl phosphate solution (pH 6.5 for ACP, pH 8.0 for ALP). ALP was characterized after extraction from soils with

  18. Receptor tyrosine phosphatase CLR-1 acts in skin cells to promote sensory dendrite outgrowth.

    PubMed

    Liu, Xianzhuang; Wang, Xiangming; Shen, Kang

    2016-05-01

    Sensory dendrite morphogenesis is directed by intrinsic and extrinsic factors. The extracellular environment plays instructive roles in patterning dendrite growth and branching. However, the molecular mechanism is not well understood. In Caenorhabditis elegans, the proprioceptive neuron PVD forms highly branched sensory dendrites adjacent to the hypodermis. We report that receptor tyrosine phosphatase CLR-1 functions in the hypodermis to pattern the PVD dendritic branches. Mutations in clr-1 lead to loss of quaternary branches, reduced secondary branches and increased ectopic branches. CLR-1 is necessary for the dendrite extension but not for the initial filopodia formation. Its role is dependent on the intracellular phosphatase domain but not the extracellular adhesion domain, indicating that it functions through dephosphorylating downstream factors but not through direct adhesion with neurons. Genetic analysis reveals that clr-1 also functions in parallel with SAX-7/DMA-1 pathway to control PVD primary dendrite development. We provide evidence of a new environmental factor for PVD dendrite morphogenesis. PMID:26968353

  19. Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps.

    PubMed

    Henry, Y; Steer, M W

    1985-04-01

    The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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