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Sample records for phospho protein phosphatase

  1. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    PubMed

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  2. A Specific Form of Phospho Protein Phosphatase 2 Regulates Anaphase-promoting Complex/Cyclosome Association with Spindle Poles

    PubMed Central

    Ban, Kenneth H.

    2010-01-01

    In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation. PMID:20089842

  3. Chromosomal localization of the chicken and mammalian orthologues of the orphan phosphatase PHOSPHO1 gene.

    PubMed

    Houston, B; Paton, I R; Burt, D W; Farquharson, C

    2002-12-01

    PHOSPHO1 is a recently identified phosphatase expressed at high levels in the chicken growth plate and which may be involved in generating inorganic phosphate for skeletal matrix mineralization. Using a degenerate RT-PCR approach a fragment of human PHOSPHO1 was cloned. This enabled the identification of the human orthologue on HSA17q21, and the mouse orthologue on a region of MMU11 that exhibits conservation of synteny with HSA17q21. Chicken PHOSPHO1 was mapped by SSCP analysis to position 44 cM on GGA27, adjacent to the HOXB@ (44 cM) and COL1A1 (36 cM) loci. Comparison of genes on GGA27 with their orthologues on the preliminary draft of the human genome identifies regions of conserved synteny equivalent to 25 Mb on HSA17q21.2-23.3 and approximately 20 Mb on GGA27 in which the gene order appears to be conserved. Mapping of the PHOSPHO1 genes to regions of HSA17q21.3, MMU11 and GGA27 that exhibit conservation of synteny provides strong evidence that they are orthologous. PMID:12464021

  4. 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

  5. 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.

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

    PubMed Central

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

    2014-01-01

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

  7. A Theileria parva type 1 protein phosphatase activity.

    PubMed

    Cayla, X; Garcia, A; Baumgartner, M; Ozon, R; Langsley, G

    2000-09-01

    The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1. PMID:10989153

  8. Loss of Skeletal Mineralization by the Simultaneous Ablation of PHOSPHO1 and Alkaline Phosphatase Function: A Unified Model of the Mechanisms of Initiation of Skeletal Calcification

    PubMed Central

    Yadav, Manisha C; Simão, Ana Maria Sper; Narisawa, Sonoko; Huesa, Carmen; McKee, Marc D; Farquharson, Colin; Millán, José Luis

    2011-01-01

    Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PPi). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1−/− mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1−/− tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1−/− mice show reduced levels of TNAP and elevated plasma PPi concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1−/− mice despite normalization of their plasma PPi levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research. PMID:20684022

  9. 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

  10. Phospho-tyrosine dependent protein–protein interaction network

    PubMed Central

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  11. 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

  12. 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

  13. Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

    PubMed Central

    Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

    2013-01-01

    Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

  14. 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

  15. Role of PHOSPHO1 in Periodontal Development and Function.

    PubMed

    Zweifler, L E; Ao, M; Yadav, M; Kuss, P; Narisawa, S; Kolli, T N; Wimer, H F; Farquharson, C; Somerman, M J; Millán, J L; Foster, B L

    2016-07-01

    The tooth root and periodontal apparatus, including the acellular and cellular cementum, periodontal ligament (PDL), and alveolar bone, are critical for tooth function. Cementum and bone mineralization is regulated by factors including enzymes and extracellular matrix proteins that promote or inhibit hydroxyapatite crystal growth. Orphan Phosphatase 1 (Phospho1, PHOSPHO1) is a phosphatase expressed by chondrocytes, osteoblasts, and odontoblasts that functions in skeletal and dentin mineralization by initiating deposition of hydroxyapatite inside membrane-limited matrix vesicles. The role of PHOSPHO1 in periodontal formation remains unknown and we aimed to determine its functional importance in these tissues. We hypothesized that the enzyme would regulate proper mineralization of the periodontal apparatus. Spatiotemporal expression of PHOSPHO1 was mapped during periodontal development, and Phospho1(-/-) mice were analyzed using histology, immunohistochemistry, in situ hybridization, radiography, and micro-computed tomography. The Phospho1 gene and PHOSPHO1 protein were expressed by active alveolar bone osteoblasts and cementoblasts during cellular cementum formation. In Phospho1(-/-) mice, acellular cementum formation and mineralization were unaffected, whereas cellular cementum deposition increased although it displayed delayed mineralization and cementoid. Phospho1(-/-) mice featured disturbances in alveolar bone mineralization, shown by accumulation of unmineralized osteoid matrix and interglobular patterns of protein deposition. Parallel to other skeletal sites, deposition of mineral-regulating protein osteopontin (OPN) was increased in alveolar bone in Phospho1(-/-) mice. In contrast to the skeleton, genetic ablation of Spp1, the gene encoding OPN, did not ameliorate dentoalveolar defects in Phospho1(-/-) mice. Despite alveolar bone mineralization defects, periodontal attachment and function appeared undisturbed in Phospho1(-/-) mice, with normal PDL

  16. Essential Phosphatases and a Phospho-Degron Are Critical for Regulation of SRC-3/AIB1 Coactivator Function and Turnover

    PubMed Central

    Li, Chao; Liang, Yao-Yun; Feng, Xin-Hua; Tsai, Sophia Y.; Tsai, Ming-Jer; O’Malley, Bert W.

    2008-01-01

    SRC-3/AIB1 is a master growth coactivator and oncogene, and phosphorylation activates it into a powerful coregulator. Dephosphorylation is a potential regulatory mechanism for SRC-3 function but the identity of such phosphatases remains unexplored. Herein, we report that using functional genomic screening of human Ser/Thr phosphatases targeting SRC-3’s known phosphorylation sites, the phosphatases PDXP, PP1 and PP2A were identified to be key negative regulators of SRC-3 transcriptional coregulatory activity in steroid receptor signalings. PDXP and PP2A dephosphorylate SRC-3 and inhibit its ligand-dependent association with estrogen receptor. PP1 stabilizes SRC-3 protein by blocking its proteasome-dependent turnover through dephosphorylation of two previously unidentified phosphorylation sites (Ser101 and S102) required for activity. These two sites are located within a degron of SRC-3, and are primary determinants of SRC-3 turnover. Moreover, PP1 regulates the oncogenic cell proliferation and invasion functions of SRC-3 in breast cancer cells. PMID:18922467

  17. 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

  18. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    PubMed

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  19. 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

  20. The Arabidopsis thaliana response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

    PubMed Central

    Horák, Jakub; Grefen, Christopher; Berendzen, Kenneth W; Hahn, Achim; Stierhof, York-Dieter; Stadelhofer, Bettina; Stahl, Mark; Koncz, Csaba; Harter, Klaus

    2008-01-01

    Background The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood. Results We present evidence that ARR22 is a preferentially cytoplasmic protein, exclusively expressed in the chalaza of developing seeds. ARR22 specifically interacts with AHP2, AHP3 and AHP5 in yeast and living plant cells. Two new loss-of-function alleles, arr22-2 and arr22-3, were isolated and characterized. With respect to their morphology and metabolite status, no significant difference in the developing seeds of the arr22 mutants was observed compared to wild type. The genetic complementation of the arr22 mutants with a genomic ARR22 fragment resulted in plants (arr22/gARR22) with a pleiotropic phenotype of different penetrance. This phenotype was not observed when the phosphorylatable Asp74 of ARR22 was changed to either a dominant-active Glu or a dominant-inactive Asn. The phenotype of the arr22/gARR22 plants was comparable to that of multiple ahk, ahp and B-type arr mutants. Conclusion Our results favor the model that ARR22 acts as a phospho-histidine phosphatase on specific AHPs in the cytoplasm of Arabidopsis chalaza cells. The lack of any aberrant morphological and metabolite phenotype in the seeds of the arr22 mutants indicates that ARR22 is probably primarily responsible for the fine tuning of specific branches of chalaza-based TCS signalling. Even when slightly mis-expressed, ARR22 interferes with hormone homeostasis in non

  1. Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

    PubMed Central

    2010-01-01

    Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples. PMID:21092202

  2. Protein phosphatases and their regulation in the control of mitosis

    PubMed Central

    Mochida, Satoru; Hunt, Tim

    2012-01-01

    Cell cycle transitions depend on protein phosphorylation and dephosphorylation. The discovery of cyclin-dependent kinases (CDKs) and their mode of activation by their cyclin partners explained many important aspects of cell cycle control. As the cell cycle is basically a series of recurrences of a defined set of events, protein phosphatases must obviously be as important as kinases. However, our knowledge about phosphatases lags well behind that of kinases. We still do not know which phosphatase(s) is/are truly responsible for dephosphorylating CDK substrates, and we know very little about whether and how protein phosphatases are regulated. Here, we summarize our present understanding of the phosphatases that are important in the control of the cell cycle and pose the questions that need to be answered as regards the regulation of protein phosphatases. PMID:22482124

  3. Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

    SciTech Connect

    Agarwal,R.; Burley, S.; Swaminathan, S.

    2008-01-01

    Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-{beta} and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 angstrom resolution. The polypeptide chain adopts the typical a{beta}a PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.

  4. Protein Phosphatase 1 β Paralogs Encode the Zebrafish Myosin Phosphatase Catalytic Subunit

    PubMed Central

    Jayashankar, Vaishali; Nguyen, Michael J.; Carr, Brandon W.; Zheng, Dale C.; Rosales, Joseph B.; Rosales, Joshua B.; Weiser, Douglas C.

    2013-01-01

    Background The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. Methodology/Principal Findings We observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. Conclusions/Significance Our work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required. PMID:24040418

  5. Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons.

    PubMed Central

    Desdouits, F; Siciliano, J C; Nairn, A C; Greengard, P; Girault, J A

    1998-01-01

    DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1. PMID:9461512

  6. 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. PMID:26275498

  7. Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase.

    PubMed

    Pato, M D; Sutherland, C; Winder, S J; Walsh, M P

    1993-07-01

    Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase

  8. 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

  9. Phospho-proteins patial gradients in a cell of spheroidal shape

    NASA Astrophysics Data System (ADS)

    Sosa, Gerardo; Ramirez-Santiago, Guillermo

    2010-03-01

    Many signalling proteins undergo phosphorilated-dephosphorilated cycles at different locations inside the cell. These cycles give rise to spatial gradients of phosphoproteins. In this work we solve the reaction-difussion equation in a spheroidal geometry and investigate the diffusion of the phosphorilated form of the proteins to evaluate the size of the spatial gradients. This is done in terms of diffusion coefficients as well as protein kinase and phosphatase activities. Previous estimations of these gradients have been done for two geometries [1]: (i) a spherical cell and (ii) for a kinase and a protein each one located on two parallel planar membranes. This type of quantitative analyzes may have important implications in the cellular signaling processes [2].[4pt] [1] G.C. Brown, B.N. Kholodenko, FEBS Letters, vol. 457, p. 452-454[0pt] [2] B.N. Kholodenko, G.C. Brown, J.B. Hoek, Biochem. J. vol. 350, p. 901-907.

  10. A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling.

    PubMed

    Khatlani, Tanvir; Pradhan, Subhashree; Da, Qi; Shaw, Tanner; Buchman, Vladimir L; Cruz, Miguel A; Vijayan, K Vinod

    2016-08-12

    The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity. PMID:27334924

  11. Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer

    PubMed Central

    Lui, Vivian Wai Yan; Peyser, Noah D.; Ng, Patrick Kwok-Shing; Hritz, Jozef; Zeng, Yan; Lu, Yiling; Li, Hua; Wang, Lin; Gilbert, Breean R.; General, Ignacio J.; Bahar, Ivet; Ju, Zhenlin; Wang, Zhenghe; Pendleton, Kelsey P.; Xiao, Xiao; Du, Yu; Vries, John K.; Hammerman, Peter S.; Garraway, Levi A.; Mills, Gordon B.; Johnson, Daniel E.; Grandis, Jennifer R.

    2014-01-01

    The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a “driver” phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine–substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents. PMID:24395800

  12. Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

    PubMed Central

    Márk, Ágnes; Hajdu, Melinda; Kenessey, István; Sticz, Tamás; Nagy, Eszter; Barna, Gábor; Váradi, Zsófia; Kovács, Gábor; Kopper, László; Csóka, Monika

    2013-01-01

    Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments. PMID:23573198

  13. [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

  14. 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

  15. Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways.

    PubMed

    Dimchev, Georgi A; Al-Shanti, Nasser; Stewart, Claire E

    2013-05-01

    Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration. PMID:23553034

  16. The extended human PTPome: a growing tyrosine phosphatase family.

    PubMed

    Alonso, Andrés; Pulido, Rafael

    2016-04-01

    Tyr phosphatases are, by definition, enzymes that dephosphorylate phospho-Tyr (pTyr) from proteins. This activity is found in several structurally diverse protein families, including the protein Tyr phosphatase (PTP), arsenate reductase, rhodanese, haloacid dehalogenase (HAD) and His phosphatase (HP) families. Most of these families include members with substrate specificity for non-pTyr substrates, such as phospho-Ser/phospho-Thr, phosphoinositides, phosphorylated carbohydrates, mRNAs, or inorganic moieties. A Cys is essential for catalysis in PTPs, rhodanese and arsenate reductase enzymes, whereas this work is performed by an Asp in HAD phosphatases and by a His in HPs, via a catalytic mechanism shared by all of the different families. The category that contains most Tyr phosphatases is the PTP family, which, although it received its name from this activity, includes Ser, Thr, inositide, carbohydrate and RNA phosphatases, as well as some inactive pseudophosphatase proteins. Here, we propose an extended collection of human Tyr phosphatases, which we call the extended human PTPome. The addition of new members (SACs, paladin, INPP4s, TMEM55s, SSU72, and acid phosphatases) to the currently categorized PTP group of enzymes means that the extended human PTPome contains up to 125 proteins, of which ~ 40 are selective for pTyr. We set criteria to ascribe proteins to the extended PTPome, and summarize the more important features of the new PTPome members in the context of their phosphatase activity and their relationship with human disease. PMID:26573778

  17. Characterization of the PEST family protein tyrosine phosphatase BDP1.

    PubMed

    Kim, Y W; Wang, H; Sures, I; Lammers, R; Martell, K J; Ullrich, A

    1996-11-21

    Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues. PMID:8950995

  18. 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.

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

    PubMed Central

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

    2014-01-01

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

  20. Displacement affinity chromatography of protein phosphatase one (PP1) complexes

    PubMed Central

    Moorhead, Greg BG; Trinkle-Mulcahy, Laura; Nimick, Mhairi; De Wever, Veerle; Campbell, David G; Gourlay, Robert; Lam, Yun Wah; Lamond, Angus I

    2008-01-01

    Background Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes. PMID:19000314

  1. PARP1 and phospho-p65 protein expression is increased in human HER2-positive breast cancers

    PubMed Central

    Stanley, Jennifer; Klepczyk, Lisa; Keene, Kimberly; Wei, Shi; Li, Yufeng; Forero, Andres; Grizzle, William; Wielgos, Monica; Brazelton, Jason; LoBuglio, Albert F.; Yang, Eddy S.

    2015-01-01

    Purpose Previous studies have shown that basal breast cancers, which may have an inherent “BRCAness” phenotype and sensitivity to inhibitors of poly (ADP-Ribose) polymerase (PARP), express elevated levels of PARP1. Our lab recently reported that HER2+ breast cancers also exhibit sensitivity to PARP inhibitors (PARPi) by attenuating the NF-kB pathway. In this study, we assessed PARP1 and phospho-p65, a marker of activated NF-kB levels in human breast cancer tissues. Methods PARP1 and PARP2 copy number, mRNA, and protein expression was assessed by interrogating the PAM-50 defined breast cancer patient set from the TCGA using the cBioPortal. PARP1 and phospho-p65 immunohistochemistry and correlation to clinical parameters was conducted using 307 primary breast cancer specimens (132 basal, 82 luminal, 93 HER2+) through univariate and multivariate analyses. Results In the PAM50 breast cancer data set, PARP1 and 2 expression was altered in 24/58 (41%) HER2+, 32/81 (40%) basal, and 75/324 (23%) luminal A/B breast cancer patients. This correlated with a statistically significant increase in PARP1 protein levels in HER2+ and basal but not luminal breast cancers (p=0.003, p=0.027, p=0.289, respectively). No change in PARP2 protein level was observed. Interestingly, using breast cancer specimens from 307 patients, HER2 positivity correlated with elevated PARP1 expression (p<0.0001) and was three times more likely than HER2 negative breast cancers to exhibit high PARP1 levels. No significant differences were noted between race, ER status, or PR status for PARP1 expression. Additionally, we found a significant correlation between HER2 status and phospho-p65 expression (p<0.0001). Lastly, a direct correlation between PARP1 and phospho-p65 (p<0.0001) was noted. Conclusions These results indicate a potential connection between HER2, PARP1, and phospho-p65. Furthermore, these data suggest that the PARPi sensitivity we previously observed in HER2+ breast cancer cells may be due

  2. Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse*

    PubMed Central

    Corradini, Eleonora; Vallur, Raghavan; Raaijmakers, Linsey M.; Feil, Susanne; Feil, Robert; Heck, Albert J. R.; Scholten, Arjen

    2014-01-01

    The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. PMID:24925903

  3. 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

  4. Enzymatic and Functional Analysis of a Protein Phosphatase, Pph3, from Myxococcus xanthus ▿

    PubMed Central

    Kimura, Yoshio; Mori, Yumi; Ina, Youhei; Takegawa, Kaoru

    2011-01-01

    A protein phosphatase, designated Pph3, from Myxococcus xanthus showed the enzymatic characteristics of PP2C-type serine/threonine protein phosphatases, which are metal ion-dependent, okadaic acid-insensitive protein phosphatases. The pph3 mutant under starvation conditions formed immature fruiting bodies and reduced sporulation. PMID:21398555

  5. Role of protein phosphatase-1 inhibitor-1 in cardiac physiology and pathophysiology

    PubMed Central

    Nicolaou, Persoulla; Hajjar, Roger J.; Kranias, Evangelia G.

    2009-01-01

    The type 1 protein phosphatase (PP1) is a critical negative regulator of Ca2+ cycling and contractility in the cardiomyocyte. In particular, it mediates restoration of cardiac function to basal levels, after β-adrenergic stimulation, by dephosphorylating key phospho-proteins. PP1 is a holoenzyme comprised of its catalytic and auxiliary subunits. These regulatory proteins dictate PP1's subcellular localization, substrate specificity and activity. Amongst them, inhibitor-1 is of particular importance since it has been implicated as an integrator of multiple neurohormonal pathways, which finely regulate PP1 activity, at the level of the sarcoplasmic reticulum (SR). In fact, perturbations in the regulation of PP1 by inhibitor-1 have been implicated in the pathogenesis of heart failure, suggesting that inhibitor-1-based therapeutic interventions may ameliorate cardiac dysfunction and remodeling in the failing heart. This review will discuss the current views on the role of inhibitor-1 in cardiac physiology, its possible contribution to cardiac disease and its potential as a novel therapeutic strategy. PMID:19481088

  6. Centromeric binding and activity of Protein Phosphatase 4

    PubMed Central

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

    2015-01-01

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

  7. 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.

  8. 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

  9. Searching for the role of protein phosphatases in eukaryotic microorganisms.

    PubMed

    da-Silva, A M; Zapella, P D; Andrioli, L P; Campanhã, R B; Fiorini, L C; Etchebehere, L C; da-Costa-Maia, J C; Terenzi, H F

    1999-07-01

    Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism. PMID:10454741

  10. Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

    PubMed

    Ramírez-Tapia, Ana Lilia; Baylón-Pacheco, Lidia; Espíritu-Gordillo, Patricia; Rosales-Encina, José Luis

    2015-12-01

    Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism. PMID:26431820

  11. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    PubMed Central

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  12. 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.

  13. Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

    PubMed Central

    McGovern, K; Ehrmann, M; Beckwith, J

    1991-01-01

    We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them. Images PMID:1915262

  14. Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome†

    PubMed Central

    Phatnani, Hemali P.; Jones, Janice C.; Greenleaf, Arno L.

    2009-01-01

    CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO4 residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957–24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus. PMID:15595826

  15. Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

    PubMed Central

    Venerando, Andrea; Franchin, Cinzia; Cant, Natasha; Cozza, Giorgio; Pagano, Mario A.; Tosoni, Kendra; Al-Zahrani, Ateeq; Arrigoni, Giorgio; Ford, Robert C.; Mehta, Anil; Pinna, Lorenzo A.

    2013-01-01

    By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein. PMID:24058532

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

    PubMed

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

    2015-07-21

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

  17. Protein Phosphatase-1α Interacts with and Dephosphorylates Polycystin-1

    PubMed Central

    Parnell, Stephen C.; Puri, Sanjeev; Wallace, Darren P.; Calvet, James P.

    2012-01-01

    Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme. PMID:22675472

  18. 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). PMID:26636645

  19. The receptor protein tyrosine phosphatase LAR promotes R7 photoreceptor axon targeting by a phosphatase-independent signaling mechanism

    PubMed Central

    Hofmeyer, Kerstin; Treisman, Jessica E.

    2009-01-01

    Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors. PMID:19889974

  20. New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression.

    PubMed

    Galinier, A; Kravanja, M; Engelmann, R; Hengstenberg, W; Kilhoffer, M C; Deutscher, J; Haiech, J

    1998-02-17

    Carbon catabolite repression (CCR) is the prototype of a signal transduction mechanism. In enteric bacteria, cAMP was considered to be the second messenger in CCR by playing a role reminiscent of its actions in eukaryotic cells. However, recent results suggest that CCR in Escherichia coli is mediated mainly by an inducer exclusion mechanism. In many Gram-positive bacteria, CCR is triggered by fructose-1,6-bisphosphate, which activates HPr kinase, presumed to be one of the most ancient serine protein kinases. We here report cloning of the Bacillus subtilis hprK and hprP genes and characterization of the encoded HPr kinase and P-Ser-HPr phosphatase. P-Ser-HPr phosphatase forms a new family of phosphatases together with bacterial phosphoglycolate phosphatase, yeast glycerol-3-phosphatase, and 2-deoxyglucose-6-phosphate phosphatase whereas HPr kinase represents a new family of protein kinases on its own. It does not contain the domain structure typical for eukaryotic protein kinases. Although up to now the HPr modifying/demodifying enzymes were thought to exist only in Gram-positive bacteria, a sequence comparison revealed that they also are present in several Gram-negative pathogenic bacteria. PMID:9465101

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

    PubMed Central

    Sontag, Jean-Marie; Sontag, Estelle

    2014-01-01

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

  2. New functional aspects of the atypical protein tyrosine phosphatase VHZ

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.

    2013-01-01

    LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date, and was originally misclassified as an atypical dual specificity phosphatase (DSP). Kinetic isotope effects with steady state and pre-steady state kinetics of VHZ and mutants with para-nitrophenol phosphate (pNPP) have revealed several unusual properties. VHZ is significantly more active than previously reported, but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations to Q-loop residues affect this phosphotransferase activity, mutations on the IPD-loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer. PMID:24073992

  3. 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.

  4. Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1.

    PubMed

    Czikora, István; Kim, Kyung-mi; Kása, Anita; Bécsi, Bálint; Verin, Alexander D; Gergely, Pál; Erdodi, Ferenc; Csortos, Csilla

    2011-07-01

    TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (K(a) = 1.80 × 10(6) M(-1)) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (~60% inhibition), mono- (~50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level. PMID:21466834

  5. Phospho-specific recognition by 14-3-3 proteins and antibodies monitored by a high throughput label-free optical biosensor.

    PubMed

    Wu, Meng; Coblitz, Brian; Shikano, Sojin; Long, Shunyou; Spieker, Matt; Frutos, Anthony G; Mukhopadhyay, Sunil; Li, Min

    2006-10-16

    Label-free detection of molecular interactions has considerable potential in facilitating assay development. When combined with high throughput capability, it may be applied to small molecule screens for drug candidates. Phosphorylation is a key posttranslational process that confers diverse regulation in biological systems involving specific protein-protein interactions recognizing the phosphorylated motifs. Using a resonant waveguide grating biosensor, the Epic mark System, we have developed a generic assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with a solution-based fluorescence anisotropy assay, our results support that the high throughput resonant waveguide grating biosensor system has favorable technical profiles in detecting protein-protein interactions that recognize phosphorylated motifs. Hence it provides a new generic HTS platform for phospho-detection. PMID:17011553

  6. A chronoamperometric screen printed carbon biosensor based on alkaline phosphatase inhibition for W(IV) determination in water, using 2-phospho-L-ascorbic acid trisodium salt as a substrate.

    PubMed

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver-Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  7. A Chronoamperometric Screen Printed Carbon Biosensor Based on Alkaline Phosphatase Inhibition for W(VI) Determination in Water, Using 2-Phospho-l-Ascorbic Acid Trisodium Salt as a Substrate

    PubMed Central

    Alvarado-Gámez, Ana Lorena; Alonso-Lomillo, María Asunción; Domínguez-Renedo, Olga; Arcos-Martínez, María Julia

    2015-01-01

    This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 μM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 μM. This study was performed by means of a Lineweaver–Burk plot, showing a mixed kinetic inhibition. PMID:25621602

  8. 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

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

    PubMed

    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

  10. 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

  11. 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

  12. 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

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

    PubMed Central

    Janssens, V; Goris, J

    2001-01-01

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

  14. Viewing serine/threonine protein phosphatases through the eyes of drug designers

    PubMed Central

    Zhang, Mengmeng; Yogesha, S. D.; Mayfield, Joshua E.; Gill, Gordon N.; Zhang, Yan

    2015-01-01

    Protein phosphatases, as the counterpart to protein kinases, are essential for homeostatic balance of cell signaling. Small chemical compounds that modulate the specific activity of phosphatases can be powerful tools to elucidate the biological functions of these enzymes. More importantly, many phosphatases are central players in the development of pathological pathways where inactivation can reverse or delay the onset of human diseases. Therefore, potent inhibitors for such phosphatases can be of great therapeutic benefit. In contrast to the seemingly identical enzymatic mechanism and structural characterization of eukaryotic protein kinases, protein phosphatases evolved from diverse ancestors, resulting in different domain architectures, reaction mechanisms and active site properties. In this review, we will discuss for each family of serine/threonine protein phosphatases, their involvement in biological process and corresponding strategies for small chemical intervention. Recent advances in modern drug discovery technologies have markedly facilitated the identification of selective inhibitors for some members of the phosphatase family. Furthermore, the rapid growth in knowledge about structure-activity relationships related to possible new drug targets has aided the discovery of natural product inhibitors for phosphatase family. This review summarizes the current state of investigation of the small molecules that regulate the function of serine/threonine phosphatases, the challenges presented and also strategies to overcome these obstacles. PMID:23937612

  15. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  16. 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.

  17. 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

  18. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    PubMed

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  19. Redox and zinc signalling pathways converging on protein tyrosine phosphatases.

    PubMed

    Bellomo, Elisa; Hogstrand, Christer; Maret, Wolfgang

    2014-10-01

    Zinc ions, though redox-inert, have either pro-antioxidant or pro-oxidant functions at critical junctures in redox metabolism and redox signalling. They are released from cells and in cells, e.g. from metallothionein, a protein that transduces redox signals into zinc signals (1). The released zinc ions inhibit enzymes such as protein tyrosine phosphatases (PTPs), key regulatory enzymes of cellular phosphorylation signalling. The Ki(Zn) value for inhibition of receptor PTPB is 21pM (2). The binding is about as tight as the binding of zinc to zinc metalloenzymes and suggests tonic zinc inhibition. PTP1-B (PTPN1), an enzyme regulating the insulin and leptin receptors and involved in cancer and diabetes pathobiochemistry, has a Ki(Zn) value of about 5nM (3). Zinc ions bind to the enzyme in the closed conformation when additional metal-binding ligands are brought into the vicinity of the active site. In contrast, redox reactions target cysteines in the active sites of PTPs in the open conformation. This work provides a molecular basis how hydrogen peroxide and free zinc ions generated by growth factor signalling stimulate phosphorylation signalling differentially. (Supported by the Biotechnology and Biological Sciences Research Council UK, grant BB/K001442/1.). PMID:26461422

  20. Protein Phosphatase-1 regulates Rift Valley fever virus replication.

    PubMed

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

    2016-03-01

    Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target. PMID:26801627

  1. Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho-protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

    PubMed Central

    2013-01-01

    Background The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. Methods We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. Results The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. Conclusions We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell

  2. 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.

  3. Okadaic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sustained hypoxia.

    PubMed

    Wilkerson, Julia E R; Satriotomo, Irawan; Baker-Herman, Tracy L; Watters, Jyoti J; Mitchell, Gordon S

    2008-03-12

    Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined. PMID:18337426

  4. Isolation of Human Mitotic Protein Phosphatase Complexes: Identification of a Complex between Protein Phosphatase 1 and the RNA Helicase Ddx21

    PubMed Central

    De Wever, Veerle; Lloyd, David C.; Nasa, Isha; Nimick, Mhairi; Trinkle-Mulcahy, Laura; Gourlay, Robert; Morrice, Nick; Moorhead, Greg B. G.

    2012-01-01

    Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis. PMID:22761809

  5. Counteracting Protein Kinase Activity in the Heart: The Multiple Roles of Protein Phosphatases

    PubMed Central

    Weber, Silvio; Meyer-Roxlau, Stefanie; Wagner, Michael; Dobrev, Dobromir; El-Armouche, Ali

    2015-01-01

    Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. PMID:26617522

  6. 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.

  7. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    PubMed

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  8. 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

  9. 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

  10. 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. PMID:17615669

  11. Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

    PubMed Central

    Stanford, Stephanie M.; Ahmed, Vanessa

    2014-01-01

    Abstract Significance: The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and influence many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. Critical Issues: Of particular importance are the elucidation of the biological substrates of individual PTPs and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. Recent Advances: Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. Future Directions: Further development of approaches to investigate the intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways. Antioxid. Redox Signal. 20, 2160–2178. PMID:24294920

  12. 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.

  13. Identification of the Interaction Sites of Inhibitor-3 for Protein Phosphatase-1

    PubMed Central

    Zhang, Lifang; Qi, Zhiqing; Gao, Yan; Lee, Ernest Y.C.

    2008-01-01

    Inhibitor-3 is a potent inhibitor of protein phosphatase-1, with an IC50 in the nanomolar range for the inhibition of the dephosphorylation of phosphorylase a. Human Inhibitor-3 possesses a putative protein phosphatase-1 binding motif, 39KKVEW43. We provide direct evidence that this sequence is involved in PP1 interaction by examining the effects of site-directed mutations of Inhibitor-3 on its ability to inhibit protein phosphatase-1. A second interaction site whose deletion led to loss of inhibitory potency was identified between residues 65–77. The existence of two interaction sites is consistent with the high inhibitory potency of Inhibitor-3, and with current models for other inhibitor and targeting proteins that interact with protein phosphatase-1 with high affinity. PMID:18951879

  14. Hypothermic Preconditioning Reverses Tau Ontogenesis in Human Cortical Neurons and is Mimicked by Protein Phosphatase 2A Inhibition

    PubMed Central

    Rzechorzek, Nina M.; Connick, Peter; Livesey, Matthew R.; Borooah, Shyamanga; Patani, Rickie; Burr, Karen; Story, David; Wyllie, David J.A.; Hardingham, Giles E.; Chandran, Siddharthan

    2015-01-01

    Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure. Changes in neuronal tau protein are of interest, since tau becomes hyperphosphorylated in injury-resistant, hypothermic brains. Noting inter-species differences in tau isoforms, we have used functional cortical neurons differentiated from human pluripotent stem cells (hCNs) to interrogate tau modulation during hypothermic preconditioning at clinically-relevant temperatures. Key tau developmental transitions (phosphorylation status and splicing shift) are recapitulated during hCN differentiation and subsequently reversed by mild (32 °C) to moderate (28 °C) cooling — conditions which reduce oxidative and excitotoxic stress-mediated injury in hCNs. Blocking a major tau kinase decreases hCN tau phosphorylation and abrogates hypothermic neuroprotection, whilst inhibition of protein phosphatase 2A mimics cooling-induced tau hyperphosphorylation and protects normothermic hCNs from oxidative stress. These findings indicate a possible role for phospho-tau in hypothermic preconditioning, and suggest that cooling drives human tau towards an earlier ontogenic phenotype whilst increasing neuronal resilience to common neurotoxic insults. This work provides a critical step forward in understanding how we might exploit the neuroprotective benefits of cooling without cooling patients. PMID:26870825

  15. Protein phosphatase magnesium dependent 1A governs the wound healing-inflammation-angiogenesis cross talk on injury.

    PubMed

    Dvashi, Zeev; Sar Shalom, Hadas; Shohat, Meytal; Ben-Meir, Daniella; Ferber, Shiran; Satchi-Fainaro, Ronit; Ashery-Padan, Ruth; Rosner, Mordechai; Solomon, Arieh S; Lavi, Sara

    2014-11-01

    Protein phosphatase magnesium dependent 1A (PPM1A) has been implicated in fibrosis and skin wounding. We generated PPM1A knockout mice to study the role of PPM1A in the wound healing-inflammation-angiogenesis cross talk. The role of PPM1A in these processes was studied using the ocular alkali burn model system. In the injured cornea the absence of PPM1A led to enhanced inflammatory response, stromal keratocyte transactivation, fibrosis, increased p38 mitogen-activated protein kinase phosphorylation, elevated expression of transforming growth factor-β-related genes (including Acta2, TGF-β, Col1, MMP9, and VEGF) and subsequently to neovascularization. Augmented angiogenesis in the absence of PPM1A is a general process occurring in vivo in PPM1A knockout mice upon subcutaneous Matrigel injection and ex vivo in aortic ring Matrigel cultures. Using primary keratocyte cultures and various experimental approaches, we found that phospho-p38 is a favored PPM1A substrate and that by its dephosphorylation PPM1A participates in the regulation of the transforming growth factor-β signaling cascade, the hallmark of inflammation and the angiogenic process. On the whole, the studies presented here position PPM1A as a new player in the wound healing-inflammation-angiogenesis axis in mouse, reveal its crucial role in homeostasis on injury, and highlight its potential as a therapeutic mediator in pathologic conditions, such as inflammation and angiogenesis disorders, including cancer. PMID:25196308

  16. Hypothermic Preconditioning Reverses Tau Ontogenesis in Human Cortical Neurons and is Mimicked by Protein Phosphatase 2A Inhibition.

    PubMed

    Rzechorzek, Nina M; Connick, Peter; Livesey, Matthew R; Borooah, Shyamanga; Patani, Rickie; Burr, Karen; Story, David; Wyllie, David J A; Hardingham, Giles E; Chandran, Siddharthan

    2016-01-01

    Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure. Changes in neuronal tau protein are of interest, since tau becomes hyperphosphorylated in injury-resistant, hypothermic brains. Noting inter-species differences in tau isoforms, we have used functional cortical neurons differentiated from human pluripotent stem cells (hCNs) to interrogate tau modulation during hypothermic preconditioning at clinically-relevant temperatures. Key tau developmental transitions (phosphorylation status and splicing shift) are recapitulated during hCN differentiation and subsequently reversed by mild (32 °C) to moderate (28 °C) cooling--conditions which reduce oxidative and excitotoxic stress-mediated injury in hCNs. Blocking a major tau kinase decreases hCN tau phosphorylation and abrogates hypothermic neuroprotection, whilst inhibition of protein phosphatase 2A mimics cooling-induced tau hyperphosphorylation and protects normothermic hCNs from oxidative stress. These findings indicate a possible role for phospho-tau in hypothermic preconditioning, and suggest that cooling drives human tau towards an earlier ontogenic phenotype whilst increasing neuronal resilience to common neurotoxic insults. This work provides a critical step forward in understanding how we might exploit the neuroprotective benefits of cooling without cooling patients. PMID:26870825

  17. Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein.

    PubMed Central

    Burchell, A; Burchell, B; Monaco, M; Walls, H E; Arion, W J

    1985-01-01

    Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2996501

  18. Inhibition of CDC25B Phosphatase Through Disruption of Protein-Protein Interaction

    SciTech Connect

    Lund, George; Dudkin, Sergii; Borkin, Dmitry; Ni, Wendi; Grembecka, Jolanta; Cierpicki, Tomasz

    2015-04-29

    CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

  19. Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

    PubMed Central

    Hadler, Kieran S; Huber, Thomas; Cassady, A Ian; Weber, Jane; Robinson, Jodie; Burrows, Allan; Kelly, Gregory; Guddat, Luke W; Hume, David A; Schenk, Gerhard; Flanagan, Jack U

    2008-01-01

    Background Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. Findings A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. Conlusion The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism. PMID:18771593

  20. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    PubMed

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice. PMID:27166156

  1. 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

  2. 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.

  3. 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. PMID:27208174

  4. The endogenous inhibitor of protein kinase-C in the rat ovary is a protein phosphatase.

    PubMed

    Eyster, K M; Waller, M S; Miller, T L; Miller, C J; Johnson, M J; Persing, J S

    1993-09-01

    Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase. PMID:7689949

  5. Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

    PubMed

    Etchebehere, L C; Simon, M N; Campanhã, R B; Zapella, P D; Véron, M; Maia, J C

    1993-08-01

    Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases. PMID:8394312

  6. Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase.

    PubMed

    Inaba, Kazuo

    2002-10-01

    Sperm flagellar movements are regulated by cAMP-dependent protein phosphorylation. Tctex2-related light chain of outer arm dynein is a well-defined phosphorylated protein that is phosphorylated at activation of sperm motility. Here, the protein phosphatase that dephosphorylates Tctex2-related dynein light chain (LC2) has been characterized in salmonid fish sperm. Most of the phosphatase activity against LC2 is found in Triton-soluble fraction of flagella but trace extent of the activity is retained in the axoneme. The dephosphorylation of LC2 is inhibited by okadaic acid at more than 1nM, whereas that of dynein alpha heavy chain is inhibited at more than 10nM. The addition of Ca(2+) gives no direct effect on LC2 dephosphorylation, but it accelerates the dephosphorylation of the regulatory subunit of cAMP-dependent protein kinase, resulting in the decrease of LC2 phosphorylation. The activity to dephosphorylate the LC2 is separated by MonoQ ion-exchange column chromatography along with the immunoreactivity to the antibody against the catalytic subunit of type 2A protein phosphatase. These results suggest that LC2 is dephosphorylated by type 2A protein phosphatase and that dynein alpha heavy chain and the regulatory subunit of cAMP-dependent protein kinase are dephosphorylated by other types of protein phosphatases. PMID:12359223

  7. 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

  8. 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

  9. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    PubMed Central

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  10. Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry.

    PubMed Central

    Kennelly, Peter J

    2003-01-01

    Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'. PMID:12444920

  11. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    SciTech Connect

    Eto, Masumi; Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  12. Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii.

    PubMed

    Gentry, Matthew S; Romá-Mateo, Carlos; Sanz, Pascual

    2013-01-01

    Lafora disease (LD) is a rare, fatal neurodegenerative disorder characterized by the accumulation of glycogen-like inclusions in the cytoplasm of cells from most tissues of affected patients. One hundred years after the first description of these inclusions, the molecular bases underlying the processes involved in LD physiopathology are finally being elucidated. The main cause of the disease is related to the activity of two proteins, the dual-specificity phosphatase laforin and the E3-ubiquitin ligase malin, which form a functional complex. Laforin is unique in humans, as it is composed of a carbohydrate-binding module attached to a cysteine-based catalytic dual-specificity phosphatase domain. Laforin directly dephosphorylates glycogen, but other proteinaceous substrates, if they exist, have remained elusive. Recently, an emerging set of laforin-binding partners apart from malin have been described, suggestive of laforin roles unrelated to its catalytic activity. Further investigations based on different transgenic mouse models have shown that the laforin-malin complex is also involved in other cellular processes, such as response to endoplasmic reticulum stress and misfolded protein clearance by the lysosomal pathway. However, controversial data and some missing links still make it difficult to assess the concrete relationship between glycogen deregulation and neuronal damage leading to the fatal symptoms observed in LD patients, such as myoclonic seizures and epilepsy. Consequently, clinical treatments are far from being achieved. In the present review, we focus on the knowledge of laforin biology, not only as a glucan phosphatase, but also as an adaptor protein involved in several physiological pathways. PMID:22364389

  13. 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.

  14. Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases.

    PubMed

    Wehenkel, Annemarie; Bellinzoni, Marco; Schaeffer, Francis; Villarino, Andrea; Alzari, Pedro M

    2007-12-01

    Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the alpha isoform of human PP2C (PP2Calpha), which folds into an alpha/beta domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn(2+)-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions. PMID:17961594

  15. Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

    PubMed

    Yamaguchi, Fuminori; Tsuchiya, Mitsumasa; Shimamoto, Seiko; Fujimoto, Tomohito; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2016-01-01

    Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis. PMID:27600583

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

    PubMed Central

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

    2013-01-01

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

  17. Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases

    PubMed Central

    Ishida, A; Sueyoshi, N; Shigeri, Y; Kameshita, I

    2008-01-01

    Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their ‘switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their ‘switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases. PMID:18454172

  18. Dephosphorylation of Tyrosine 393 in Argonaute 2 by Protein Tyrosine Phosphatase 1B Regulates Gene Silencing in Oncogenic RAS-Induced Senescence

    PubMed Central

    Yang, Ming; Haase, Astrid D.; Huang, Fang-Ke; Coulis, Gérald; Rivera, Keith D.; Dickinson, Bryan C.; Chang, Christopher J.; Pappin, Darryl J.; Neubert, Thomas A.; Hannon, Gregory J.; Boivin, Benoit; Tonks, Nicholas K.

    2014-01-01

    SUMMARY Oncogenic RAS (H-RASV12) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RASV12-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RASV12-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RASV12-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNA) and thus miRNA-mediated gene silencing, which counteracted the function of H-RASV12-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RASV12-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing. PMID:25175024

  19. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  20. Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis.

    PubMed

    Amand, Mathieu; Erpicum, Charlotte; Gilles, Christine; Noël, Agnès; Rahmouni, Souad

    2016-01-01

    Therapeutic perspectives targeting angiogenesis in cancer stimulated an intense investigation of the mechanisms triggering and governing angiogenic processes. Several publications have highlighted the importance of typical dual-specificity phosphatases (DSPs) or MKPs in endothelial cells and their role in controlling different biological functions implicated in angiogenesis such as migration, proliferation, apoptosis, tubulogenesis, and cell adhesion. However, among atypical DSPs, the only one investigated in angiogenesis was DUSP3. We recently identified this DSP as a new key player in endothelial cells and angiogenesis. In this chapter we provide with detailed protocols and models used to investigate the role of DUSP3 in endothelial cells and angiogenesis. We start the chapter with an overview of the role of several DSPs in angiogenesis. We continue with providing a full description of a highly efficient transfection protocol to deplete DUSP3 using small interfering RNA (siRNA) in the primary human umbilical vein endothelial cells (HUVEC). We next describe the major assays used to investigate different processes involved in angiogenesis such as tube formation assay, proliferation assay and spheroids sprouting assay. We finish the chapter by validating our results in DUSP3-knockout mice using in vivo angiogenesis assays such as Matrigel plug and Lewis lung carcinoma cell subcutaneous xenograft model followed by anti-CD31 immunofluorescence and ex vivo aortic ring assay. All methods described can be adapted to other phosphatases and signaling molecules. PMID:27514814

  1. An immunochemical approach to detect oxidized protein tyrosine phosphatases using a selective C-nucleophile tag.

    PubMed

    Garcia, Francisco J; Carroll, Kate S

    2016-05-24

    Protein tyrosine phosphatases are crucial regulators of signal transduction and function as antagonists towards protein tyrosine kinases to control reversible tyrosine phosphorylation, thereby regulating fundamental physiological processes. Growing evidence has supported the notion that reversible oxidative inactivation of the catalytic cysteine residue in protein tyrosine phosphatases serves as an oxidative post-translational modification that regulates its activity to influence downstream signaling by promoting phosphorylation and induction of the signaling cascade. The oxidation of cysteine to the sulfenic acid is often transient and difficult to detect, thus making it problematic in understanding the role that this oxidative post-translational modification plays in redox-biology and pathogenesis. Several methods to detect cysteine oxidation in biological systems have been developed, though targeted approaches to directly detect oxidized phosphatases are still lacking. Herein we describe the development of a novel immunochemical approach to directly profile oxidized phosphatases. This immunochemical approach consists of an antibody designed to recognize the conserved sequence of the PTP active site (VHCDMDSAG) harboring the catalytic cysteine modified with dimedone (CDMD), a nucleophile that chemoselectively reacts with cysteine sulfenic acids to form a stable thioether adduct. Additionally, we provide biochemical and mass spectrometry workflows to be used in conjugation with this newly developed immunochemical approach to assist in the identification and quantification of basal and oxidized phosphatases. PMID:26757830

  2. Tailoring a low-molecular weight protein tyrosine phosphatase into an efficient reporting protein

    SciTech Connect

    Liu, Xiao-Yan; Li, Lan-Fen; Su, Xiao-Dong; Shenzhen Graduate School of Peking University, Shenzhen 518055

    2009-05-15

    Fusion reporter methods are important tools for biology and biotechnology. An ideal reporter protein in a fusion system should have little effects on its fusion partner and provide an easy and accurate readout. Therefore, a small monomeric protein with high activity for detection assays often has advantages as a reporter protein. For this purpose, we have tailored the human B-form low-molecular-weight phosphotyrosyl phosphatase (HPTP-B) to increase its general applicability as a potent reporter protein. With the aim to eliminate interference from cysteine residues in the native HPTP-B, combined with a systematic survey of N- and C-terminal truncated variants, a series of cysteine to serine mutations were introduced, which allowed isolation of an engineered soluble protein with suitable biophysical properties. When we deleted both the first six residues and the last two residues, we still obtained a soluble mutant protein with correct folding and similar activity with wild-type protein. This mutant with two cysteine to serine mutations, HPTP-B{sup N{sub {Delta}}6-C{sub {Delta}}2-C90S-C109S}, has good potential as an optimal reporter.

  3. Molecular Cloning and Functional Expression of a Protein-Serine/Threonine Phosphatase from the Hyperthermophilic Archaeon Pyrodictium abyssi TAG11

    PubMed Central

    Mai, Bianca; Frey, Gerhard; Swanson, Ronald V.; Mathur, Eric J.; Stetter, K. O.

    1998-01-01

    An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo. PMID:9696747

  4. Analysis of Smad Phosphatase Activity In Vitro.

    PubMed

    Shen, Tao; Qin, Lan; Lin, Xia

    2016-01-01

    Phosphorylation of Smad1/5/8 at the C-terminal SXS motif by BMP type I receptors is one of the most critical events in BMP signaling. Conversely, protein phosphatases that dephosphorylate phospho-Smad1/5/8 can consequently prevent or terminate BMP signaling. PPM1H is an undercharacterized phosphatase in the PPM family. We recently demonstrated that PPM1H can dephosphorylate Smad1 in the cytoplasm and block BMP signaling responses in cellular assays. Here we describe in vitro method showing that PPM1H is a bona fide phosphatase for Smad1/5/8. PPM1H is produced as GST fusion protein in E. coli, and purified against glutathione sepharose beads. Bacterially purified recombinant PPM1H possesses phosphatase activity toward artificial substrate para-nitrophenyl phosphate (pNPP). Recombinant PPM1H also dephosphorylates immuno-purified phosphorylated Smad1 in test tubes. These direct in vitro phosphatase assays provide convincing evidence demonstrating the role of PPM1H as a specific phosphatase for P-Smad1. PMID:26520120

  5. Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis.

    PubMed

    Li, Xue; Oghi, Kenneth A; Zhang, Jie; Krones, Anna; Bush, Kevin T; Glass, Christopher K; Nigam, Sanjay K; Aggarwal, Aneel K; Maas, Richard; Rose, David W; Rosenfeld, Michael G

    2003-11-20

    The precise mechanistic relationship between gene activation and repression events is a central question in mammalian organogenesis, as exemplified by the evolutionarily conserved sine oculis (Six), eyes absent (Eya) and dachshund (Dach) network of genetically interacting proteins. Here, we report that Six1 is required for the development of murine kidney, muscle and inner ear, and that it exhibits synergistic genetic interactions with Eya factors. We demonstrate that the Eya family has a protein phosphatase function, and that its enzymatic activity is required for regulating genes encoding growth control and signalling molecules, modulating precursor cell proliferation. The phosphatase function of Eya switches the function of Six1-Dach from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival in mammalian organogenesis. PMID:14628042

  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. Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase, TOPP4

    PubMed Central

    Qin, Qianqian; Wang, Wei; Guo, Xiaola; Yue, Jing; Huang, Yan; Xu, Xiufei; Li, Jia; Hou, Suiwen

    2014-01-01

    Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway. PMID:25010794

  8. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    PubMed Central

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

    PubMed Central

    Bozaquel-Morais, Bruno L.; Madeira, Juliana B.; Maya-Monteiro, Clarissa M.; Masuda, Claudio A.; Montero-Lomeli, Mónica

    2010-01-01

    In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis. PMID:21060891

  11. The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

    PubMed Central

    Santos, Mariana; Rebelo, Sandra; Van Kleeff, Paula J. M.; Kim, Connie E.; Dauer, William T.; Fardilha, Margarida; da Cruz e Silva, Odete A.; da Cruz e Silva, Edgar F.

    2013-01-01

    Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases. PMID:24116158

  12. Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin.

    PubMed Central

    Reardon, D B; Wood, S L; Brautigan, D L; Bell, G I; Dent, P; Sturgill, T W

    1996-01-01

    Human somatostatin receptor 3 ('hsstr3') was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro. PMID:8670047

  13. 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 ...

  14. 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...

  15. 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

  16. REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

    PubMed Central

    Tu, J; Carlson, M

    1995-01-01

    Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway. Images PMID:8846786

  17. PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity.

    PubMed Central

    Iwanicki, Adam; Herman-Antosiewicz, Anna; Pierechod, Marcin; Séror, Simone J; Obuchowski, Michał

    2002-01-01

    Bacillus subtilis is a Gram-positive bacterium with a relatively large number of protein phosphatases. Previous studies have shown that some Ser/Thr phosphatases play an important role in the life cycle of this bacterium [Losick and Stragier (1992) Nature (London) 355, 601-604; Yang, Kang, Brody and Price (1996) Genes Dev. 10, 2265-2275]. In this paper, we report the biochemical properties of a putative, previously uncharacterized phosphatase, PrpE, belonging to the PPP family. This enzyme shares homology with other PPP phosphatases as well as with symmetrical diadenosine tetraphosphatases related to ApaH (symmetrical Ap(4)A hydrolase) from Escherichia coli. A His-tagged recombinant PrpE was purified from E. coli and shown to have Ni(2+)-dependent and okadaic acid-resistant phosphatase activity against a synthetic phosphorylated peptide and hydrolase activity against diadenosine 5',5"'-tetraphosphate. Unexpectedly, PrpE was able to remove phosphate from phosphotyrosine, but not from phosphothreonine or phosphoserine. PMID:12059787

  18. Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Travis, Sue M.; Berger, Herbert A.; Welsh, Michael J.

    1997-01-01

    cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target. PMID:9380758

  19. 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

  20. 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.

  1. 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

  2. Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.

    PubMed

    Mommen, Geert P M; van de Waterbeemd, Bas; Meiring, Hugo D; Kersten, Gideon; Heck, Albert J R; de Jong, Ad P J M

    2012-09-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO(2)) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO(2), keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products. PMID:22729381

  3. 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

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

    PubMed Central

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

    2004-01-01

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

  5. EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases.

    PubMed

    Wang, Yongbo; Liu, Zexian; Cheng, Han; Gao, Tianshun; Pan, Zhicheng; Yang, Qing; Guo, Anyuan; Xue, Yu

    2014-01-01

    We present here EKPD (http://ekpd.biocuckoo.org), a hierarchical database of eukaryotic protein kinases (PKs) and protein phosphatases (PPs), the key molecules responsible for the reversible phosphorylation of proteins that are involved in almost all aspects of biological processes. As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50,000 PKs and >10,000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript. PMID:24214991

  6. The protein phosphatase inhibitor calyculin A stimulates chemokine production by human synovial cells.

    PubMed Central

    Jordan, N J; Watson, M L; Westwick, J

    1995-01-01

    Cultured human synovial fibroblasts express mRNA for the chemotactic cytokines (chemokines) interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and presumably secreted (RANTES), when stimulated with IL-1 or tumour necrosis factor alpha (TNF alpha). Calyculin A, a potent type 1/2A protein serine/threonine phosphatase inhibitor, was used to examine the role of protein phosphatases in the regulation of chemokine gene expression. Calyculin A (1 nM) mimicked IL-1 by inducing IL-8 and MCP-1 mRNA expression in synovial cells. IL-8 mRNA was induced over a similar time period (1-6 h) in response to IL-1 or calyculin A, whereas MCP-1 mRNA was induced more rapidly (1-2 h) by calyculin A than by IL-1 (4-6 h). Expression of RANTES mRNA occurred in response to TNF alpha, but could not be induced by stimulation with calyculin A alone. These results suggest that inhibition of protein phosphatase type 1/2A may have a differential role in the regulation of the expression of each of the chemokine genes. Synovial fibroblasts also secreted IL-8 and IL-6 peptide when stimulated with either IL-1/TNF alpha or calyculin A. The amount of IL-8 and IL-6 peptide produced in response to calyculin A was significantly increased above that produced by untreated synovial cells, though it was much less than the amount induced by IL-1 or TNF alpha. Calyculin A also acted synergistically with IL-1 or TNF alpha to cause a 2-fold potentiation of IL-1- or TNF alpha-induced IL-8 mRNA and peptide and RANTES mRNA expression. These results suggest that although inhibition of a protein phosphatase may be able to regulate the magnitude of IL-1-induced chemokine gene expression, the IL-1 signal transduction pathway involves components in addition to phosphatase inhibition, possibly including the activation of a protein kinase, the action of which may be opposed by a protein phosphatase inhibited by calyculin A. Images Figure 1 Figure 2 Figure 3

  7. Kinetic Characterization of O-Phospho-L-Tyrosine Phosphohydrolase Activity of Two Fungal Phytases.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal phytases belonging to 'Histidine Acid Phosphatase' or HAP class of phosphomonoesterase that catalyzes the hydrolysis of phytic acid could also hydrolyze O-phospho-tyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phospho-tyrosine hydrola...

  8. Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential.

    PubMed

    Wehenkel, Annemarie; Bellinzoni, Marco; Graña, Martin; Duran, Rosario; Villarino, Andrea; Fernandez, Pablo; Andre-Leroux, Gwénaëlle; England, Patrick; Takiff, Howard; Cerveñansky, Carlos; Cole, Stewart T; Alzari, Pedro M

    2008-01-01

    Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds. PMID:17869195

  9. Reactivity of Cdc25 phosphatase at low pH and with thiophosphorylated protein substrate.

    PubMed

    Rudolph, Johannes

    2005-08-01

    Cdc25s, dual-specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases, are important regulators of the eukaryotic cell cycle. Herein, we probe the protonation state of the phosphate on the protein substrate of Cdc25 by pH-dependent studies and thiosubstitution. We have extended the useable range of pH for this enzyme substrate pair by using high concentrations of glycerol under acidic conditions. Using the protein substrate, we find a slope of 2 for the acidic side of the bell-shaped pH-rate profile, as found with other protein tyrosine phosphatases. Using thiophosphorylated protein substrate, we find no change in the basic side of the pH-rate profile, despite a large reduction in activity as measured by kcat/Km (0.18%) or kcat (0. 11%). In contrast, the acidic side of the profile changes shows a slope of 1, consistent with the 1.5 pH unit shift associated with thiosubstitution. Thus, Cdc25, like other protein phosphatases, uses a dianionic phosphorylated substrate. PMID:16023486

  10. Deletion of conserved protein phosphatases reverses defects associated with mitochondrial DNA damage in Saccharomyces cerevisiae.

    PubMed

    Garipler, Görkem; Mutlu, Nebibe; Lack, Nathan A; Dunn, Cory D

    2014-01-28

    Mitochondrial biogenesis is regulated by signaling pathways sensitive to extracellular conditions and to the internal environment of the cell. Therefore, treatments for disease caused by mutation of mtDNA may emerge from studies of how signal transduction pathways command mitochondrial function. We have examined the role of phosphatases under the control of the conserved α4/Tap42 protein in cells lacking a mitochondrial genome. We found that deletion of protein phosphatase 2A (PP2A) or of protein phosphatase 6 (PP6) protects cells from the reduced proliferation, mitochondrial protein import defects, lower mitochondrial electrochemical potential, and nuclear transcriptional response associated with mtDNA damage. Moreover, PP2A or PP6 deletion allows viability of a sensitized yeast strain after mtDNA loss. Interestingly, the Saccharomyces cerevisiae ortholog of the mammalian AMP-activated protein kinase was required for the full benefits of PP6 deletion and also for proliferation of otherwise wild-type cells lacking mtDNA. Our work highlights the important role that nutrient-responsive signaling pathways can play in determining the response to mitochondrial dysfunction. PMID:24474773

  11. A cluster of protein kinases and phosphatases modulated in fetal Down syndrome (trisomy 21) brain.

    PubMed

    Weitzdoerfer, Rachel; Toran, Nuria; Subramaniyan, Saraswathi; Pollak, Arnold; Dierssen, Mara; Lubec, Gert

    2015-06-01

    Down syndrome (DS; trisomy 21) is the most frequent cause of mental retardation with major cognitive and behavioral deficits. Although a series of aberrant biochemical pathways has been reported, work on signaling proteins is limited. It was, therefore, the aim of the study to test a selection of protein kinases and phosphatases known to be essential for memory and learning mechanisms in fetal DS brain. 12 frontal cortices from DS brain were compared to 12 frontal cortices from controls obtained at legal abortions. Proteins were extracted from brains and western blotting with specific antibodies was carried out. Primary results were used for networking (IntAct Molecular Interaction Database) and individual predicted pathway components were subsequently quantified by western blotting. Levels of calcium-calmodulin kinase II alpha, transforming growth factor beta-activated kinase 1 as well as phosphatase and tensin homolog (PTEN) were reduced in cortex of DS subjects and network generation pointed to interaction between PTEN and the dendritic spine protein drebrin that was subsequently determined and reduced levels were observed. The findings of reduced levels of cognitive-function-related protein kinases and the phosphatase may be relevant for interpretation of previous work and may be useful for the design of future studies on signaling in DS brain. Moreover, decreased drebrin levels may point to dendritic spine abnormalities. PMID:25740605

  12. Integrative Transcriptome Profiling of Cognitive Aging and Its Preservation through Ser/Thr Protein Phosphatase Regulation

    PubMed Central

    Park, C. Sehwan; Valomon, Amandine; Welzl, Hans

    2015-01-01

    Environmental enrichment has been reported to delay or restore age-related cognitive deficits, however, a mechanism to account for the cause and progression of normal cognitive decline and its preservation by environmental enrichment is lacking. Using genome-wide SAGE-Seq, we provide a global assessment of differentially expressed genes altered with age and environmental enrichment in the hippocampus. Qualitative and quantitative proteomics in naïve young and aged mice was used to further identify phosphorylated proteins differentially expressed with age. We found that increased expression of endogenous protein phosphatase-1 inhibitors in aged mice may be characteristic of long-term environmental enrichment and improved cognitive status. As such, hippocampus-dependent performances in spatial, recognition, and associative memories, which are sensitive to aging, were preserved by environmental enrichment and accompanied by decreased protein phosphatase activity. Age-associated phosphorylated proteins were also found to correspond to the functional categories of age-associated genes identified through transcriptome analysis. Together, this study provides a comprehensive map of the transcriptome and proteome in the aging brain, and elucidates endogenous protein phosphatase-1 inhibition as a potential means through which environmental enrichment may ameliorate age-related cognitive deficits. PMID:26102285

  13. Chasing Phosphoarginine Proteins: Development of a Selective Enrichment Method Using a Phosphatase Trap*

    PubMed Central

    Trentini, Débora Broch; Fuhrmann, Jakob; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging post-translational protein modification implicated in the bacterial stress response. Although early reports suggested that arginine phosphorylation also occurs in higher eukaryotes, its overall prevalence was never studied using modern mass spectrometry methods, owing to technical difficulties arising from the acid lability of phosphoarginine. As shown recently, the McsB and YwlE proteins from Bacillus subtilis function as a highly specific protein arginine kinase and phosphatase couple, shaping the phosphoarginine proteome. Using a B. subtilis ΔywlE strain as a source for arginine-phosphorylated proteins, we were able to adapt mass spectrometry (MS) protocols to the special chemical properties of the arginine modification. Despite this progress, the analysis of protein arginine phosphorylation in eukaryotes is still challenging, given the great abundance of serine/threonine phosphorylations that would compete with phosphoarginine during the phosphopeptide enrichment procedure, as well as during data-dependent MS acquisition. We thus set out to establish a method for the selective enrichment of arginine-phosphorylated proteins as an initial step in the phosphoproteomic analysis. For this purpose, we developed a substrate-trapping mutant of the YwlE phosphatase that retains binding affinity toward arginine-phosphorylated proteins but cannot hydrolyze the captured substrates. By testing a number of active site substitutions, we identified a YwlE mutant (C9A) that stably binds to arginine-phosphorylated proteins. We further improved the substrate-trapping efficiency by impeding the oligomerization of the phosphatase mutant. The engineered YwlE trap efficiently captured arginine-phosphorylated proteins from complex B. subtilis ΔywlE cell extracts, thus facilitating identification of phosphoarginine sites in the large pool of cellular protein modifications. In conclusion, we present a novel tool for the selective enrichment and

  14. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    SciTech Connect

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

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

    PubMed

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

    2016-01-01

    Protein phosphatase 2A (PP2A) is one of the most abundant serine-threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias. PMID:27611305

  16. A subset of RAB proteins modulates PP2A phosphatase activity

    PubMed Central

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

    2016-01-01

    Protein phosphatase 2A (PP2A) is one of the most abundant serine–threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias. PMID:27611305

  17. Circadian Regulation of Sucrose Phosphate Synthase Activity in Tomato by Protein Phosphatase Activity.

    PubMed Central

    Jones, T. L.; Ort, D. R.

    1997-01-01

    Sucrose phosphate synthase (SPS), a key enzyme in sucrose biosynthesis, is regulated by protein phosphorylation and shows a circadian pattern of activity in tomato. SPS is most active in its dephosphorylated state, which normally coincides with daytime. Applying okadaic acid, a potent protein phosphatase inhibitor, prevents SPS activation. More interesting is that a brief treatment with cycloheximide, a cytoplasmic translation inhibitor, also prevents the light activation of SPS without any effect on the amount of SPS protein. Cordycepin, an inhibitor of transcript synthesis and processing, has the same effect. Both of these inhibitors also prevent the activation phase of the circadian rhythm in SPS activity. Conversely, cycloheximide and cordycepin do not prevent the decline in circadian SPS activity that normally occurs at night. These observations indicate that SPS phosphatase activity but not SPS kinase activity is controlled, directly or indirectly, at the level of gene expression. Taken together, these data imply that there is a circadian rhythm controlling the transcription of a protein phosphatase that subsequently dictates the circadian rhythm in SPS activity via effects on this enzyme's phosphorylation state. PMID:12223667

  18. Positive Regulation of TRAF6-Dependent Innate Immune Responses by Protein Phosphatase PP1-γ

    PubMed Central

    Chiang, Chih-yuan; Nguyen, Quy T.; Maestre, Ana M.; Mulder, Lubbertus C. F.; Secundino, Ismael; De Jesus, Paul D.; König, Renate; Simon, Viviana; Nizet, Victor; MacLeod, Graham; Varmuza, Susannah; Fernandez-Sesma, Ana; Chanda, Sumit K.

    2014-01-01

    Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses. PMID:24586659

  19. 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.

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  1. 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

  2. Disruption of striatal-enriched protein tyrosine phosphatase (STEP) function in neuropsychiatric disorders

    PubMed Central

    Karasawa, Takatoshi; Lombroso, Paul J.

    2014-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer’s disease, schizophrenia, fragile X syndrome, Huntington’s disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses. PMID:25218562

  3. Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

    PubMed Central

    2015-01-01

    CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes. PMID:25423142

  4. 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

  5. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus.

    PubMed

    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

    2015-07-01

    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

  6. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability

    PubMed Central

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-01

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ−/− mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ−/− mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ−/− mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper–tyrosine-phosphorylated in the PTPσ−/− mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ−/− mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD. PMID:24385580

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. Activation of SPS from darkened spinach leaves by an endogenous protein phosphatase

    SciTech Connect

    Huber, S.C.; Huber, J.L. )

    1990-05-01

    Sucrose-phosphate synthase from darkened spinach leaves has a low activation state but can undergo a time-dependent activation in desalted leaf extracts that is inhibited by Pi, molybdate, okadaic acid and vanadate, but stimulated by fluoride. SPS labeled in vivo with ({sup 32}P)Pi in excised leaves in the dark loses incorporated {sup 32}P with time when extracts are incubated at 25{degree}C. This loss is largely prevented by vanadate, suggesting that an endogenous protein phosphatase can use SPS as substrate. Changes in phosphorylation state are closely paralleled by changes in SPS activation state. The spontaneous activation achieved in the extracts can be reversed by addition of 2 mM MgATP. Feeding okadaic acid to darkened leaves prevents light activation of SPS suggesting that the endogenous protein phosphatase is similar to the type-1 enzyme of animal tissues. Overall, the results are consistent with the notion that light activation of SPS involves dephosphorylation of inhibitory phosphorylation site(s). Regulation of the protein phosphatase by Pi may be of physiological significance.

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

    PubMed Central

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

    2016-01-01

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

  11. Is Protein Phosphatase Inhibition Responsible for the Toxic Effects of Okadaic Acid in Animals?

    PubMed Central

    Munday, Rex

    2013-01-01

    Okadaic acid (OA) and its derivatives, which are produced by dinoflagellates of the genera Prorocentrum and Dinophysis, are responsible for diarrhetic shellfish poisoning in humans. In laboratory animals, these toxins cause epithelial damage and fluid accumulation in the gastrointestinal tract, and at high doses, they cause death. These substances have also been shown to be tumour promoters, and when injected into the brains of rodents, OA induces neuronal damage reminiscent of that seen in Alzheimer’s disease. OA and certain of its derivatives are potent inhibitors of protein phosphatases, which play many roles in cellular metabolism. In 1990, it was suggested that inhibition of these enzymes was responsible for the diarrhetic effect of these toxins. It is now repeatedly stated in the literature that protein phosphatase inhibition is not only responsible for the intestinal effects of OA and derivatives, but also for their acute toxic effects, their tumour promoting activity and their neuronal toxicity. In the present review, the evidence for the involvement of protein phosphatase inhibition in the induction of the toxic effects of OA and its derivatives is examined, with the conclusion that the mechanism of toxicity of these substances requires re-evaluation. PMID:23381142

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

    PubMed Central

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

    2015-01-01

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

  13. Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿

    PubMed Central

    Cornell, Timothy T.; Rodenhouse, Paul; Cai, Qing; Sun, Lei; Shanley, Thomas P.

    2010-01-01

    Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production. PMID:20351138

  14. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  15. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  16. 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.

  17. 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

  18. Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina.

    PubMed

    Thoen, C; Van Hove, L; Cohen, P; Slegers, H

    1985-08-30

    In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP-Y) can be accomplished by ethanol treatment of the native enzymes, or freeze-thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins. PMID:2994667

  19. 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

  20. Structural Diversity in Free and Bound States of Intrinsically Disordered Protein Phosphatase 1 Regulators

    SciTech Connect

    Marsh, J.A.; Allaire, M.; Dancheck, B.; Ragusa, M.J.; Forman-Kay, J.D.; Peti, Wolfgang

    2010-09-08

    Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three nonhomologous, intrinsically disordered proteins: I-2, spinophilin, and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, preformed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex.

  1. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

    PubMed Central

    van Zyl, W; Huang, W; Sneddon, A A; Stark, M; Camier, S; Werner, M; Marck, C; Sentenac, A; Broach, J R

    1992-01-01

    We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase. Images PMID:1328868

  2. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

    PubMed Central

    Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

    2012-01-01

    This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

  3. Analysis of Protein Phosphatase-1 and Aurora Protein Kinase Suppressors Reveals New Aspects of Regulatory Protein Function in Saccharomyces cerevisiae

    PubMed Central

    Ghosh, Anuprita; Cannon, John F.

    2013-01-01

    Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates. PMID:23894419

  4. Mannose 6 Dephosphorylation of Lysosomal Proteins Mediated by Acid Phosphatases Acp2 and Acp5

    PubMed Central

    Makrypidi, Georgia; Damme, Markus; Müller-Loennies, Sven; Trusch, Maria; Schmidt, Bernhard; Schlüter, Hartmut; Heeren, Joerg; Lübke, Torben; Saftig, Paul

    2012-01-01

    Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5−/−) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5−/− mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. PMID:22158965

  5. Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

    PubMed

    Verrastro, Ivan; Tveen-Jensen, Karina; Woscholski, Rudiger; Spickett, Corinne M; Pitt, Andrew R

    2016-01-01

    Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured. PMID:26561776

  6. Protein Phosphatase 1 (PP1) Is a Post-Translational Regulator of the Mammalian Circadian Clock

    PubMed Central

    Schmutz, Isabelle; Wendt, Sabrina; Schnell, Anna; Kramer, Achim; Mansuy, Isabelle M.; Albrecht, Urs

    2011-01-01

    Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock. PMID:21712997

  7. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  8. Carboxyl-Terminal Receptor Domains Control the Differential Dephosphorylation of Somatostatin Receptors by Protein Phosphatase 1 Isoforms

    PubMed Central

    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. PMID:24637622

  9. Functional diversity of protein phosphatase-1, a cellular economizer and reset button.

    PubMed

    Ceulemans, Hugo; Bollen, Mathieu

    2004-01-01

    The protein serine/threonine phosphatase protein phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that regulates a variety of cellular processes through the dephosphorylation of dozens of substrates. This multifunctionality of PP1 relies on its association with a host of function-specific targetting and substrate-specifying proteins. In this review we discuss how PP1 affects the biochemistry and physiology of eukaryotic cells. The picture of PP1 that emerges from this analysis is that of a "green" enzyme that promotes the rational use of energy, the recycling of protein factors, and a reversal of the cell to a basal and/or energy-conserving state. Thus PP1 promotes a shift to the more energy-efficient fuels when nutrients are abundant and stimulates the storage of energy in the form of glycogen. PP1 also enables the relaxation of actomyosin fibers, the return to basal patterns of protein synthesis, and the recycling of transcription and splicing factors. In addition, PP1 plays a key role in the recovery from stress but promotes apoptosis when cells are damaged beyond repair. Furthermore, PP1 downregulates ion pumps and transporters in various tissues and ion channels that are involved in the excitation of neurons. Finally, PP1 promotes the exit from mitosis and maintains cells in the G1 or G2 phases of the cell cycle. PMID:14715909

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

    SciTech Connect

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

    2008-01-01

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

  11. Protein Phosphatase 1ß Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

    PubMed Central

    Yamamoto, Shinya; Bayat, Vafa; Bellen, Hugo J.; Tan, Change

    2013-01-01

    Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well. PMID:23936219

  12. A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes.

    PubMed

    Smith, Miles C; Box, Andrew C; Haug, Jeffrey S; Lane, William S; Davido, David J

    2014-01-01

    Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML's activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1. PMID:25513827

  13. A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

    PubMed Central

    Smith, Miles C.; Box, Andrew C.; Haug, Jeffrey S.; Lane, William S.; Davido, David J.

    2014-01-01

    Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML’s activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1. PMID:25513827

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

    SciTech Connect

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

    2008-12-26

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

  15. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  16. New aspects of the phosphatase VHZ revealed by a high-resolution structure with vanadate and substrate screening

    PubMed Central

    Kuznetsov, Vyacheslav I.; Hengge, Alvan C.; Johnson, Sean J.

    2013-01-01

    The recently discovered 150-residue human VHZ (VH1 related protein, Z member) is one of the smallest protein tyrosine phosphatases (PTPs) known, and contains only the minimal structural elements common to all PTPs. We report a substrate screening analysis and a crystal structure of the VHZ complex with vanadate at 1.1 Å resolution, with a detailed structural comparison with other members of the protein tyrosine phosphatase family, including classical tyrosine-specific protein tyrosine phosphatases (PTPs) and dual specific phosphatases (DSPs). A screen with 360 phosphorylated peptides shows VHZ efficiently catalyzes the hydrolysis of phospho-tyrosine(pY)-containing peptides, but exhibits no activity toward phospho-serine (pS) or phospho-threonine (pT) peptides. The new structure reveals a deep and narrow active site more typical of the classical tyrosine specific PTPs. Despite the high structural and sequence similarities between VHZ and classical PTPs, its general acid IPD-loop is most likely conformationally rigid, in contrast to the flexible WPD counterpart of classical PTPs. VHZ also lacks substrate recognition domains and other domains typically found on classical PTPs. It is therefore proposed that VHZ is more properly classified as an atypical PTP rather than an atypical DSP, as has been suggested. PMID:23145819

  17. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  18. Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells1[W

    PubMed Central

    Kaida, Rumi; Serada, Satoshi; Norioka, Naoko; Norioka, Shigemi; Neumetzler, Lutz; Pauly, Markus; Sampedro, Javier; Zarra, Ignacio; Hayashi, Takahisa; Kaneko, Takako S.

    2010-01-01

    It is not yet known whether dephosphorylation of proteins catalyzed by phosphatases occurs in the apoplastic space. In this study, we found that tobacco (Nicotiana tabacum) purple acid phosphatase could dephosphorylate the phosphoryl residues of three apoplastic proteins, two of which were identified as α-xylosidase and β-glucosidase. The dephosphorylation and phosphorylation of recombinant α-xylosidase resulted in a decrease and an increase in its activity, respectively, when xyloglucan heptasaccharide was used as a substrate. Attempted overexpression of the tobacco purple acid phosphatase NtPAP12 in tobacco cells not only decreased the activity levels of the glycosidases but also increased levels of xyloglucan oligosaccharides and cello-oligosaccharides in the apoplast during the exponential phase. We suggest that purple acid phosphatase controls the activity of α-xylosidase and β-glucosidase, which are responsible for the degradation of xyloglucan oligosaccharides and cello-oligosaccharides in the cell walls. PMID:20357138

  19. The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro.

    SciTech Connect

    Li, Ruiliang; Potters, M B.; Shi, Liang; Kennelly, Peter J.

    2005-09-01

    The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp(608) by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg(2+) or Mn(2+), for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

  20. Structure of thermotoga maritima stationary phase survival protein SurE : a novel acid phosphatase.

    SciTech Connect

    Zhang, R.-G; Skarina, T.; Katz, J. E.; Khachatryan, A; Vyas, S.; Arrowsmith, C. H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.; Biosciences Division; Univ. of Toronto; Univ. of California; Clinical Genomics Centre /Proteomics, Univ. Health Network

    2001-11-01

    Background: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase {sigma} subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results: The structure of SurE from Thermotoga maritima was determined at 2.0 Angstroms. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. Conclusions: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

  1. Phosphacan and Receptor Protein Tyrosine Phosphatase β Expression Mediates Deafferentation-Induced Synaptogenesis

    PubMed Central

    Harris, Janna L.; Reeves, Thomas M.; Phillips, Linda L.

    2009-01-01

    This study documents the spatial and temporal expression of three structurally related chondroitin sulfated proteoglycans (CSPGs) during synaptic regeneration induced by brain injury. Using the unilateral entorhinal cortex lesion model of adaptive synaptogenesis, we documented mRNA and protein profiles of phosphacan and its two splice variants, full length receptor protein tyrosine phosphatase β (RPTPβ) and the short transmembrane receptor form (sRPTPβ), at 2, 7, and 15 d postlesion. We report that whole hippocampal sRPTPβ protein and mRNA are persistently elevated over the first two weeks after UEC. As predicted, this transmembrane family member was localized adjacent to synaptic sites in the deafferented neuropil and showed increased distribution over that zone following lesion. By contrast, whole hippocampal phosphacan protein was not elevated with deafferentation, however, its mRNA was increased during the period of sprouting and synapse formation (7d). When the zone of synaptic reorganization was sampled using molecular layer/granule cell (ML/GCL) enriched dissections, we observed an increase in phosphacan protein at 7d, concurrent with the observed hippocampal mRNA elevation. Immunohistochemistry also showed a shift in phosphacan distribution from granule cell bodies to the deafferented ML at 2 and 7d postlesion. Phosphacan and sRPTPβ were not co-localized with glial fibrillary acid protein (GFAP), suggesting that reactive astrocytes were not a major source of either proteoglycan. While transcript for the developmentally prominent full length RPTPβ was also increased at 2 and 15d, its protein was not detected in our adult samples. These results indicate that phosphacan and RPTPβ splice variants participate in both the acute degenerative and long-term regenerative phases of reactive synaptogenesis. These results suggest that increase in the transmembrane sRPTPβ tyrosine phosphatase activity is critical to this plasticity, and that local elevation of

  2. Discovery and optimization of sulfonyl acrylonitriles as selective, covalent inhibitors of protein phosphatase methylesterase-1.

    PubMed

    Bachovchin, Daniel A; Zuhl, Andrea M; Speers, Anna E; Wolfe, Monique R; Weerapana, Eranthie; Brown, Steven J; Rosen, Hugh; Cravatt, Benjamin F

    2011-07-28

    The serine hydrolase protein phosphatase methylesterase-1 (PME-1) regulates the methylesterification state of protein phosphatase 2A (PP2A) and has been implicated in cancer and Alzheimer's disease. We recently reported a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) high-throughput screen for PME-1 that uncovered a remarkably potent and selective class of aza-β-lactam (ABL) PME-1 inhibitors. Here, we describe a distinct set of sulfonyl acrylonitrile inhibitors that also emerged from this screen. The optimized compound, 28 (AMZ30), selectively inactivates PME-1 and reduces the demethylated form of PP2A in living cells. Considering that 28 is structurally unrelated to ABL inhibitors of PME-1, these agents, together, provide a valuable set of pharmacological probes to study the role of methylation in regulating PP2A function. We furthermore observed that several serine hydrolases were sensitive to analogues of 28, suggesting that more extensive structural exploration of the sulfonyl acrylonitrile chemotype may result in useful inhibitors for other members of this large enzyme class. PMID:21639134

  3. PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

    PubMed Central

    Patsoukis, Nikolaos; Li, Lequn; Sari, Duygu; Petkova, Victoria

    2013-01-01

    Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis. PMID:23732914

  4. Structural and Mechanistic Characterization of L-Histidinol Phosphate Phosphatase from the PHP Family of Proteins

    PubMed Central

    Ghodge, Swapnil V.; Fedorov, Alexander A.; Fedorov, Elena V.; Hillerich, Brandan; Seidel, Ronald; Almo, Steven C.; Raushel, Frank M.

    2013-01-01

    l-Histidinol phosphate phosphatase (HPP) catalyzes the hydrolysis of L-histidinol phosphate to L-histidinol and inorganic phosphate, the penultimate step in the biosynthesis of L-histidine. HPP from the polymerase and histidinol phosphatase (PHP) family of proteins possesses a trinuclear active site and a distorted (β/α)7-barrel protein fold. This group of enzymes is closely related to the amidohydrolase superfamily of enzymes. The mechanism of phosphomonoester bond hydrolysis by the PHP family of HPP enzymes was addressed. Recombinant HPP from Lactococcus lactis subsp. lactis that was expressed in Escherichia coli contained a mixture of iron and zinc in the active site and had a catalytic efficiency of ~103 M−1 s−1. Expression of the protein under iron-free conditions resulted in the production of enzyme with a two orders of magnitude improvement in catalytic efficiency and a mixture of zinc and manganese in the active site. Solvent isotope and viscosity effects demonstrated that proton transfer steps and product dissociation steps are not rate-limiting. X-ray structures of HPP were determined with sulfate, L-histidinol/phosphate, and a complex of L-histidinol and arsenate bound in the active site. These crystal structures and the catalytic properties of variants were used to identify the structural elements required for catalysis and substrate recognition by the HPP family of enzymes within the amidohydrolase superfamily. PMID:23327428

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

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

  6. Discovery and Optimization of Sulfonyl Acrylonitriles as Selective, Covalent Inhibitors of Protein Phosphatase Methylesterase-1

    PubMed Central

    Bachovchin, Daniel A.; Zuhl, Andrea M.; Speers, Anna E.; Wolfe, Monique R.; Weerapana, Eranthie; Brown, Steven J.; Rosen, Hugh; Cravatt, Benjamin F.

    2011-01-01

    The serine hydrolase protein phosphatase methylesterase-1 (PME-1) regulates the methylesterification state of protein phosphatase 2A (PP2A) and has been implicated in cancer and Alzheimer's disease. We recently reported a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) high-throughput screen for PME-1 that uncovered a remarkably potent and selective class of aza-β-lactam (ABL) PME-1 inhibitors. Here, we describe a distinct set of sulfonyl acrylonitrile inhibitors that also emerged from this screen. The optimized compound, 28 (AMZ30), selectively inactivates PME-1 and reduces the demethylated form of PP2A in living cells. Considering that 28 is structurally unrelated to ABL inhibitors of PME-1, these agents, together, provide a valuable set of pharmacological probes to study the role of methylation in regulating PP2A function. We furthermore observed that several serine hydrolases were sensitive to analogs of 28, suggesting that more extensive structural exploration of the sulfonyl acrylonitrile chemotype may result in useful inhibitors for other members of this large enzyme class. PMID:21639134

  7. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    PubMed

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  8. Kinetic isotope effects in the characterization of catalysis by protein tyrosine phosphatases.

    PubMed

    Hengge, Alvan C

    2015-11-01

    Although thermodynamically favorable, the uncatalyzed hydrolysis of phosphate monoesters is extraordinarily slow, making phosphatases among the most catalytically efficient enzymes known. Protein-tyrosine phosphatases (PTPs) are ubiquitous in biology, and kinetic isotope effects were one of the key mechanistic tools used to discern molecular details of their catalytic mechanism and the transition state for phosphoryl transfer. Later, the unique level of detail KIEs provided led to deeper questions about the potential role of protein motions in PTP catalysis. The recent discovery that such motions are responsible for different catalytic rates between PTPs arose from questions originating from KIE data showing that the transition states and chemical mechanisms are identical, combined with structural data demonstrating superimposable active sites. KIEs also reveal perturbations to the transition state as mutations are made to residues directly involved in chemistry, and to residues that affect protein motions essential for catalysis. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. PMID:25840000

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

    PubMed

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

    2015-04-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study. PMID:25645927

  10. HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

    PubMed Central

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

    2015-01-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study. PMID:25645927

  11. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

    SciTech Connect

    Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih

    1995-08-01

    Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.

  12. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast.

    PubMed Central

    Jiang, Y; Broach, J R

    1999-01-01

    Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22. Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway. PMID:10329624

  13. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

    PubMed

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F; Mori Sequeiros García, M Mercedes; Maloberti, Paula M; Orlando, Ulises D; Mele, Pablo G; Poderoso, Cecilia; Podesta, Ernesto J

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed. PMID:27375556

  14. Sodium selenate retards epileptogenesis in acquired epilepsy models reversing changes in protein phosphatase 2A and hyperphosphorylated tau.

    PubMed

    Liu, Shi-Jie; Zheng, Ping; Wright, David K; Dezsi, Gabi; Braine, Emma; Nguyen, Thanh; Corcoran, Niall M; Johnston, Leigh A; Hovens, Christopher M; Mayo, Jamie N; Hudson, Matthew; Shultz, Sandy R; Jones, Nigel C; O'Brien, Terence J

    2016-07-01

    There are no treatments in clinical practice known to mitigate the neurobiological processes that convert a healthy brain into an epileptic one, a phenomenon known as epileptogenesis. Downregulation of protein phosphatase 2A, a protein that causes the hyperphosphorylation of tau, is implicated in neurodegenerative diseases commonly associated with epilepsy, such as Alzheimer's disease and traumatic brain injury. Here we used the protein phosphatase 2A activator sodium selenate to investigate the role of protein phosphatase 2A in three different rat models of epileptogenesis: amygdala kindling, post-kainic acid status epilepticus, and post-traumatic epilepsy. Protein phosphatase 2A activity was decreased, and tau phosphorylation increased, in epileptogenic brain regions in all three models. Continuous sodium selenate treatment mitigated epileptogenesis and prevented the biochemical abnormalities, effects which persisted after drug withdrawal. Our studies indicate that limbic epileptogenesis is associated with downregulation of protein phosphatase 2A and the hyperphosphorylation of tau, and that targeting this mechanism with sodium selenate is a potential anti-epileptogenic therapy. PMID:27289302

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

    PubMed Central

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

    2015-01-01

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

  16. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    PubMed Central

    Chen, Xi’en; Lü, Shumin; Zhang, Yalin

    2013-01-01

    Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin. PMID:24351830

  17. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

    NASA Technical Reports Server (NTRS)

    Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

    2001-01-01

    Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

  18. Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum.

    PubMed

    Noguchi, Munenori; Sasaki, Jun-Ya; Kamachi, Hiroyuki; Inoue, Hiroshi

    2003-02-01

    Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP. PMID:12529856

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization*

    PubMed Central

    Liao, Fang-Hsuean; Chan, Yi-Chiao; Huang, Ching-Yu

    2015-01-01

    Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability. PMID:26710253

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  4. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    PubMed

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  5. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  6. A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation.

    PubMed

    Sokolov, Lubomir N; Dominguez-Solis, Jose R; Allary, Anne-Laure; Buchanan, Bob B; Luan, Sheng

    2006-06-20

    Starch is the ultimate storage molecule formed in the photosynthetic fixation of carbon dioxide by chloroplasts. Starch accumulates during the day and is degraded at night to intermediates that are exported to heterotrophic organs. The mechanism by which diurnal cycles control the transitory biosynthesis and degradation of chloroplast starch has long remained a mystery. We now report evidence that a dual-specificity protein phosphatase, DSP4, binds to starch granules during the day and dissociates at night. Disruption of the DSP4 gene resulted in a dramatic increase in the level of starch in mutant Arabidopsis plants. Moreover, although composition was apparently unchanged, the morphology of the starch granule was significantly altered compared to the wild type counterpart. Two regulatory factors linked to light (i.e., pH and redox status) changed both the activity and the starch-binding capacity of DSP4. The results further revealed that DSP4 represents a major fraction of granule-bound phosphatase activity during the day but not at night. Our study suggests that DSP4 acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation. PMID:16772378

  7. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  8. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B

    PubMed Central

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  9. Alterations in the Interactome of Serine/Threonine Protein Phosphatase Type-1 in Atrial Fibrillation Patients

    PubMed Central

    Chiang, David Y.; Lebesgue, Nicolas; Beavers, David L.; Alsina, Katherina M.; A. Damen, J. Mirjam; Voigt, Niels; Dobrev, Dobromir; Wehrens, Xander H.T.; Scholten, Arjen

    2015-01-01

    BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown. OBJECTIVES The purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits. METHODS Cardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation. RESULTS A total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone. CONCLUSIONS Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug

  10. A RP-UFLC Assay for Protein Tyrosine Phosphatases: Focus on Protein Tyrosine Phosphatase Non-Receptor Type 2 (PTPN2).

    PubMed

    Duval, Romain; Bui, Linh-Chi; Berthelet, Jérémy; Dairou, Julien; Mathieu, Cécile; Guidez, Fabien; Dupret, Jean-Marie; Cools, Jan; Chomienne, Christine; Rodrigues-Lima, Fernando

    2015-01-01

    Protein tyrosine phosphatases (PTPs) are involved in numerous signaling pathways and dysfunctions of certain of these enzymes have been linked to several human diseases including cancer and autoimmune diseases. PTPN2 is a PTP mainly expressed in hematopoietic cells and involved in growth factor and JAK/STAT signaling pathways. Loss of function analyses in patients with mutation/deletion of the PTPN2 gene and knock-out mouse models indicate that PTPN2 acts as a tumor suppressor in T-cell malignancies and as a regulator of inflammation and immunity. The use of sensitive and quantitative assays is of prime importance to better characterize the biochemical properties of PTPN2 and its biological roles. We report a highly sensitive non-radioactive assay that allows the measurement of the activity of purified PTPN2 and of endogenous PTPN2 immunoprecipitated on agarose beads. The assay relies on separation and quantitation by reverse-phase ultra fast liquid chromatography (RP-UFLC) of a fluorescein-labeled phosphotyrosine peptide substrate derived from the sequence of STAT1. The applicability and reliability of this approach is supported by kinetic and mechanistic studies using PTP inhibitors. More broadly, our PTPN2 assay provides the basis for the design of flexible methods for the measurement of other PTPs. PMID:26040922

  11. Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

    PubMed

    Amador, Fátima Camões; Henriques, Ana Gabriela; da Cruz E Silva, Odete A B; da Cruz E Silva, Edgar F

    2004-01-01

    Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future. PMID:15113600

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

    PubMed Central

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

    2014-01-01

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

  13. Protein tyrosine phosphatase variants in human hereditary disorders and disease susceptibilities.

    PubMed

    Hendriks, Wiljan J A J; Pulido, Rafael

    2013-10-01

    Reversible tyrosine phosphorylation of proteins is a key regulatory mechanism to steer normal development and physiological functioning of multicellular organisms. Phosphotyrosine dephosphorylation is exerted by members of the super-family of protein tyrosine phosphatase (PTP) enzymes and many play such essential roles that a wide variety of hereditary disorders and disease susceptibilities in man are caused by PTP alleles. More than two decades of PTP research has resulted in a collection of PTP genetic variants with corresponding consequences at the molecular, cellular and physiological level. Here we present a comprehensive overview of these PTP gene variants that have been linked to disease states in man. Although the findings have direct bearing for disease diagnostics and for research on disease etiology, more work is necessary to translate this into therapies that alleviate the burden of these hereditary disorders and disease susceptibilities in man. PMID:23707412

  14. Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B.

    PubMed

    Li, Xun; Köhn, Maja

    2016-08-01

    Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B. PMID:27025565

  15. Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity*

    PubMed Central

    Kok, Bernard P. C.; Skene-Arnold, Tamara D.; Ling, Ji; Benesch, Matthew G. K.; Dewald, Jay; Harris, Thurl E.; Holmes, Charles F. B.; Brindley, David N.

    2014-01-01

    Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg2+ or Mn2+ and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ. PMID:24558042

  16. Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

    PubMed

    Azkona, Garikoitz; Saavedra, Ana; Aira, Zigor; Aluja, David; Xifró, Xavier; Baguley, Tyler; Alberch, Jordi; Ellman, Jonathan A; Lombroso, Paul J; Azkue, Jon J; Pérez-Navarro, Esther

    2016-02-01

    The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception. PMID:26270590

  17. Networks of protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Mucic, Goran; Sase, Sunetra; Stork, Oliver; Lubec, Gert; Li, Lin

    2015-03-01

    Although protein kinases and phosphatases have been reported to be involved in fear memory, information about these signalling molecules in the individual phases of contextual fear conditioning (cFC) is limited. C57BL/6J mice were tested in cFC, sacrificed and hippocampi were used for screening of approximately 800 protein kinases and phosphatases by protein microarrays with subsequent Western blot confirmation of threefold higher or lower hippocampal levels as compared to foot shock controls. Immunoblotting of the protein kinases and phosphatases screened out was carried out by Western blotting. A network of protein kinases and phosphatases was generated (STRING 9.1). Animals learned the task in the paradigm and protein kinase and phosphatase levels were determined in the individual phases acquisition, consolidation and retrieval and compared to foot shock controls. Protein kinases discoidin containing receptor 2 (DDR2), mitogen activated protein kinase kinase kinase 7 (TAK1), protein phosphatases dual specificity protein phosphatase (PTEN) and protein phosphatase 2a (PP2A) were modulated in the individual phases of cFC. Phosphatidyl-inositol-3,4,5-triphosphate 3-phosphatase and phosphatidylinositol-3 kinase (PI3K) that is interacting with PTEN were modulated as well. Freezing time was correlating with PP2A levels in the retrieval phase of cFC. The abovementioned protein kinases, phosphatases and inositol-signalling enzymes were not reported so far in cFC and the results are relevant for interpretation of previous and design of future studies in cFC or fear memory. Protein phosphatase PP2A was, however, the only signalling compound tested that was directly linked to retrieval in the cFC. PMID:25461266

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

    PubMed

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

    2008-03-01

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

  19. Protein tyrosine phosphatase SHP2 promotes invadopodia formation through suppression of Rho signaling

    PubMed Central

    Tsai, Wan-Chen; Chen, Chien-Lin; Chen, Hong-Chen

    2015-01-01

    Invadopodia are actin-enriched membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Src homolog domain-containing phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, has been shown to play an important role in promoting cancer metastasis, but the underlying mechanism is unclear. In this study, we found that depletion of SHP2 by short-hairpin RNA suppressed invadopodia formation in several cancer cell lines, particularly in the SAS head and neck squamous cell line. In contrast, overexpression of SHP2 promoted invadopodia formation in the CAL27 head and neck squamous cell line, which expresses low levels of endogenous SHP2. The depletion of SHP2 in SAS cells significantly decreased their invasive motility. The suppression of invadopodia formation by SHP2 depletion was restored by the Clostridium botulinum C3 exoenzyme (a Rho GTPase inhibitor) or Y27632 (a specific inhibitor for Rho-associated kinase). Together, our results suggest that SHP2 may promote invadopodia formation through inhibition of Rho signaling in cancer cells. PMID:26204488

  20. Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

    PubMed Central

    Hellmuth, Klaus; Grosskopf, Stefanie; Lum, Ching Tung; Würtele, Martin; Röder, Nadine; von Kries, Jens Peter; Rosario, Marta; Rademann, Jörg; Birchmeier, Walter

    2008-01-01

    The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases. PMID:18480264

  1. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

    PubMed

    Bunin, Anna; Sisirak, Vanja; Ghosh, Hiyaa S; Grajkowska, Lucja T; Hou, Z Esther; Miron, Michelle; Yang, Cliff; Ceribelli, Michele; Uetani, Noriko; Chaperot, Laurence; Plumas, Joel; Hendriks, Wiljan; Tremblay, Michel L; Häcker, Hans; Staudt, Louis M; Green, Peter H; Bhagat, Govind; Reizis, Boris

    2015-08-18

    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation. PMID:26231120

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

    SciTech Connect

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

    2006-01-01

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

  3. Protein Tyrosine Phosphatase α in the Dorsomedial Striatum Promotes Excessive Ethanol-Drinking Behaviors

    PubMed Central

    Ben Hamida, Sami; Darcq, Emmanuel; Wang, Jun; Wu, Su; Phamluong, Khanhky; Kharazia, Viktor

    2013-01-01

    We previously found that excessive ethanol drinking activates Fyn in the dorsomedial striatum (DMS) (Wang et al., 2010; Gibb et al., 2011). Ethanol-mediated Fyn activation in the DMS leads to the phosphorylation of the GluN2B subunit of the NMDA receptor, to the enhancement of the channel's activity, and to the development and/or maintenance of ethanol drinking behaviors (Wang et al., 2007, 2010). Protein tyrosine phosphatase α (PTPα) is essential for Fyn kinase activation (Bhandari et al., 1998), and we showed that ethanol-mediated Fyn activation is facilitated by the recruitment of PTPα to synaptic membranes, the compartment where Fyn resides (Gibb et al., 2011). Here we tested the hypothesis that PTPα in the DMS is part of the Fyn/GluN2B pathway and is thus a major contributor to the neuroadaptations underlying excessive ethanol intake behaviors. We found that RNA interference (RNAi)-mediated PTPα knockdown in the DMS reduces excessive ethanol intake and preference in rodents. Importantly, no alterations in water, saccharine/sucrose, or quinine intake were observed. Furthermore, downregulation of PTPα in the DMS of mice significantly reduces ethanol-mediated Fyn activation, GluN2B phosphorylation, and ethanol withdrawal-induced long-term facilitation of NMDAR activity without altering the intrinsic features of DMS neurons. Together, these results position PTPα upstream of Fyn within the DMS and demonstrate the important contribution of the phosphatase to the maladaptive synaptic changes that lead to excessive ethanol intake. PMID:24005290

  4. An Affinity-Based Fluorescence Polarization Assay for Protein Tyrosine Phosphatases

    PubMed Central

    Zhang, Sheng; Chen, Lan; Kumar, Sanjai; Wu, Li; Lawrence, David S.; Zhang, Zhong-Yin

    2007-01-01

    Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate “false” positives due to modification of the active site Cys that destroy the phosphatase activity. PMID:17532513

  5. Essential, Overlapping and Redundant Roles of the Drosophila Protein Phosphatase 1α and 1β Genes

    PubMed Central

    Kirchner, Jasmin; Gross, Sascha; Bennett, Daimark; Alphey, Luke

    2007-01-01

    Protein serine/threonine phosphatase type 1 (PP1) has been found in all eukaryotes examined to date and is involved in the regulation of many cellular functions, including glycogen metabolism, muscle contraction, and mitosis. In Drosophila, four genes code for the catalytic subunit of PP1 (PP1c), three of which belong to the PP1α subtype. PP1β9C (flapwing) encodes the fourth PP1c gene and has a specific and nonredundant function as a nonmuscle myosin phosphatase. PP1α87B is the major form and contributes ∼80% of the total PP1 activity. We describe the first mutant alleles of PP1α96A and show that PP1α96A is not an essential gene, but seems to have a function in the regulation of nonmuscle myosin. We show that overexpression of the PP1α isozymes does not rescue semilethal PP1β9C mutants, whereas overexpression of either PP1α96A or PP1β9C does rescue a lethal PP1α87B mutant combination, showing that the lethality is due to a quantitative reduction in the level of PP1c. Overexpression of PP1β9C does not rescue a PP1α87B, PP1α96A double mutant, suggesting an essential PP1α-specific function in Drosophila. PMID:17513890

  6. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    PubMed

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone. PMID:26373709

  7. Protein tyrosine phosphatase SHP2 promotes invadopodia formation through suppression of Rho signaling.

    PubMed

    Tsai, Wan-Chen; Chen, Chien-Lin; Chen, Hong-Chen

    2015-09-15

    Invadopodia are actin-enriched membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Src homolog domain-containing phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, has been shown to play an important role in promoting cancer metastasis, but the underlying mechanism is unclear. In this study, we found that depletion of SHP2 by short-hairpin RNA suppressed invadopodia formation in several cancer cell lines, particularly in the SAS head and neck squamous cell line. In contrast, overexpression of SHP2 promoted invadopodia formation in the CAL27 head and neck squamous cell line, which expresses low levels of endogenous SHP2. The depletion of SHP2 in SAS cells significantly decreased their invasive motility. The suppression of invadopodia formation by SHP2 depletion was restored by the Clostridium botulinum C3 exoenzyme (a Rho GTPase inhibitor) or Y27632 (a specific inhibitor for Rho-associated kinase). Together, our results suggest that SHP2 may promote invadopodia formation through inhibition of Rho signaling in cancer cells. PMID:26204488

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

    PubMed Central

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

    1996-01-01

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

  9. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    PubMed Central

    Primo, María E.; Jakoncic, Jean; Noguera, Martín E.; Risso, Valeria A.; Sosa, Laura; Sica, Mauricio P.; Solimena, Michele; Poskus, Edgardo; Ermácora, Mario R.

    2011-01-01

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo. PMID:21935384

  10. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    SciTech Connect

    Primo M. E.; Jakoncic J.; Noguera M.E.; Risso V.A.; Sosa L.; Sica M.P.; Solimena M.; Poskus E. and Ermacora M.

    2011-09-15

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  11. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    SciTech Connect

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-07

    Research highlights: {yields} Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. {yields} However, it is not clear exactly how PP5 participates in this process. {yields} Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  12. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations. PMID:2119547

  13. 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.

  14. Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

    PubMed

    Kleppe, Rune; Herfindal, Lars; Døskeland, Stein Ove

    2015-10-01

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity. PMID:26506362

  15. Cell Death Inducing Microbial Protein Phosphatase Inhibitors—Mechanisms of Action

    PubMed Central

    Kleppe, Rune; Herfindal, Lars; Døskeland, Stein Ove

    2015-01-01

    Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca2+/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity. PMID:26506362

  16. INHIBITORY POTENTIAL OF POLYHYDROXYLATED FULLERENES AGAINST PROTEIN TYROSINE PHOSPHATASE 1B.

    PubMed

    Kobzar, O L; Trush, V V; Tanchuk, V Yu; Vovk, A I

    2015-01-01

    Inhibition of PTP1B by polyhydroxylated fullerenes was studied in silico and in vitro. The enzyme kinetics in the presence of polyhydroxy small gap fullerenes showed that reciprocal value of maximum velocity non-linearly increases with increasing the inhibitor concentration. Analysis of the dose-dependent curve of PTP1B inhibition suggests an apparent positive cooperativity with involvement of at least two binding sites for the hydroxylated fullerene cages. Molecular docking calculations indicated that highly hydroxylated fullerene C60 may occupy the active site and additional allosteric binding site with similar affinity. In silico analysis of a number of fullerenols with 6, 12, 18, 24, 30, and 36 hydroxyl groups showed that the inhibitory activity may depend on the degree of hydroxylation of the nanoparticles surface. These data provide some understanding of the mechanisms of inhibitory action of fullerenols on activity of protein tyrosine phosphatases. PMID:26547960

  17. Implication of protein tyrosine phosphatase SHP-1 in cancer-related signaling pathways.

    PubMed

    Sharma, Yadhu; Ahmad, Altaf; Bashir, Samina; Elahi, Asif; Khan, Farah

    2016-05-01

    The altered expression of SHP-1 (SH2 domain-containing protein tyrosine phosphatase) as a consequence of promoter hypermethylation or mutations has evidently been linked to cancer development. The notion of being a cancer drug target is conceivable as SHP-1 negatively regulates cell cycle and inflammatory pathways which are an inevitable part of oncogenic transformation. In the present review, we try to critically analyze the role of SHP-1 in cancer progression via regulating the above mentioned pathways with the major emphasis on cell cycle components and JAK/STAT pathway, commencing with the SHP-1 biology in immune cell signaling. Lastly, we have provided the future directions for researchers to encourage SHP-1 as a prognostic marker and curative target for this debilitating disease called as cancer. PMID:26987952

  18. Protein phosphatase PHLPP1 controls the light-induced resetting of the circadian clock

    PubMed Central

    Masubuchi, Satoru; Gao, Tianyan; O'Neill, Audrey; Eckel-Mahan, Kristin; Newton, Alexandra C.; Sassone-Corsi, Paolo

    2010-01-01

    The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) differentially attenuates Akt, PKC, and ERK1/2 signaling, thereby controlling the duration and amplitude of responses evoked by these kinases. PHLPP1 is expressed in the mammalian central clock, the suprachiasmatic nucleus, where it oscillates in a circadian fashion. To explore the role of PHLPP1 in vivo, we have generated mice with a targeted deletion of the PHLPP1 gene. Here we show that PHLPP1-null mice, although displaying normal circadian rhythmicity, have a drastically impaired capacity to stabilize the circadian period after light-induced resetting, producing a large phase shift after light resetting. Our findings reveal that PHLPP1 exerts a previously unappreciated role in circadian control, governing the consolidation of circadian periodicity after resetting. PMID:20080691

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2008-01-01

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

  1. The Trypanosoma brucei protein phosphatase gene: polycistronic transcription with the RNA polymerase II largest subunit gene.

    PubMed Central

    Evers, R; Cornelissen, A W

    1990-01-01

    We have previously described the trypanosomal gene encoding the largest subunit of RNA polymerase II (RNAP II) and found that two almost identical genes are encoded within the Trypanosoma brucei genome. Here we show by Southern analyses that the 5' breakpoint between both loci is located approximately 7.5 kb upstream of the RNAP II genes. Northern analyses revealed that the 5' duplicated segment contains at least four other genes, which are transcribed in both bloodstream and procyclic trypanosomes. The gene located immediately upstream of the RNAP II gene in both loci was characterized by sequence analyses. The deduced amino acid sequences show a high degree of similarity to the catalytic subunit of protein phosphatase class 1 (PP1) genes. S1 mapping provided strong evidence in support of the fact that the PP1 and RNAP II genes belong to a single transcription unit. Images PMID:2169604

  2. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    SciTech Connect

    Streuli, M.; Krueger, N.X.; Saito, H. ); Tsai, A.Y.M. )

    1989-11-01

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a {sup 32}P-labeled (Tyr(P))peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity.

  3. Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory

    PubMed Central

    Abdul Rahman, Nor Zaihana; Greenwood, Sam M.; Brett, Ros R.; Tossell, Kyoko; Ungless, Mark A.; Plevin, Robin

    2016-01-01

    Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2−/− mice), we show that long-term potentiation is impaired in MKP-2−/− mice compared with MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2−/− mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2−/− mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. SIGNIFICANCE STATEMENT Recently, there has been significant focus on proteins that control mitogen-activated protein kinases' (MAPKs) function, namely the mitogen-activated protein kinase phosphatases (MKPs). Recent studies have revealed novel

  4. Involvement of protein phosphatases in the destabilization of methamphetamine-associated contextual memory.

    PubMed

    Yu, Yang-Jung; Huang, Chien-Hsuan; Chang, Chih-Hua; Gean, Po-Wu

    2016-09-01

    Destabilization refers to a memory that becomes unstable when reactivated and is susceptible to disruption by amnestic agents. Here we delineated the cellular mechanism underlying the destabilization of drug memory. Mice were conditioned with methamphetamine (MeAM) for 3 d, and drug memory was assessed with a conditioned place preference (CPP) protocol. Anisomycin (ANI) was administered 60 min after the CPP retrieval to disrupt reconsolidation. We found that destabilization of MeAM CPP after the application of ANI was blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 and the NR2B antagonist ifenprodil (IFN) but not by the NR2A antagonist NVP-AAM077 (NVP). In addition, decrease in the phosphorylation of GluR1 at Serine845 (p-GluR1-Ser845), decrease in spine density, and a reduction in the AMPAR/NMDAR ratio in the basolateral amygdala (BLA) were reversed after the MK-801 treatment. The effect of ANI on destabilization was prevented by the protein phosphatase 2B (calcineurin, CaN) inhibitors cyclosporine A (CsA) and FK-506 and the protein phosphatase 1 (PP1) inhibitors calyculin A (CA) and okadaic acid (OA). These results suggest that memory destabilization involves the activation of NR2B-containing NMDARs, which in turn allows the influx of Ca(2+) Increased intracellular Ca(2+) stimulates CaN, leading to the dephosphorylation and inactivation of inhibitor 1 and the activation of PP1. PP1 then dephosphorylates p-GluR1-Ser845 to elicit AMPA receptor (AMPAR) endocytosis and destabilization of the drug memory. PMID:27531839

  5. The Potent Inhibitors of Protein Tyrosine Phosphatase 1B from the Fruits of Melaleuca leucadendron

    PubMed Central

    Saifudin, Azis; Lallo, Subehan Ab; Tezuka, Yasuhiro

    2016-01-01

    Background: Melaleuca leucadendron (Myrtaceae) is a kind of fruit used as Indonesian medicinal component and recorded in Jamu (tonic made of medical herbs) prescription records for the diabetes treatment. Its methanol extract exhibited a strong inhibitory activity with the half maximal inhibitory concentration (IC50) value of 2.05 μg/mL, while it is the same value with positive control RK-682. Objective: To isolate the chemical constituents of M. leucadendron and to evaluate their activity against protein tyrosine phosphatase 1B (PTP1B). Further, determine their toxicity potential against T-cell protein tyrosine phosphatase (TCPTP). Materials and Methods: Methanol extract was fractionated using silica column chromatography, and the obtained fraction was purified using Sephadex 20-LH. The structure of isolated compounds was identified based on 1H and 13Nuclear Magnetic Resonance Spectrometry. Furthermore, the compounds were examined against PTP1B and TCPTP. Results: Methanol extract of M. leucadendron (Myrtaceae) afforded two triterpenes: Betulinic acid and ursolic acid in high quantities. Both compounds exhibited a strong inhibitory activity against PTP1B inhibition with IC50 value of 1.5 and 2.3 μg/mL, respectively (positive control RK-682, IC50 = 2.05 μg/mL). Their activity toward TCPTP, on the other hand, were at 2.4 and 3.1 μg/mL, respectively. Based on this purification work, betulinic acid and ursolic acid presented 7.6% and 2.4%, respectively, as markedly M. leucadendron most potential for betulinic acid source among Indonesian plants. The result should have demonstrated that the antidiabetes of M. dendron could be through the inhibition of PTP1B. SUMMARY Melaleuca leucadendron is a good source for ursolic acid.Confirming traditional use for type II diabetes via PTP1B inhibition. PMID:27114690

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

    PubMed Central

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

    2010-01-01

    Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rβ, but not IL-2Rγ. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rβ. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rβ, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rβ by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rγ in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease. PMID:19923221

  7. Cellular Casein Kinase 2 and Protein Phosphatase 2A Modulate Replication Site Assembly of Bluetongue Virus*

    PubMed Central

    Mohl, Bjorn-Patrick; Roy, Polly

    2016-01-01

    A number of cytoplasmic replicating viruses produce cytoplasmic inclusion bodies or protein aggregates; however, a hallmark of viruses of the Reoviridae family is that they utilize these sites for purposes of replication and capsid assembly, functioning as viral assembly factories. Here we have used bluetongue virus (BTV) as a model system for this broad family of important viruses to understand the mechanisms regulating inclusion body assembly. Newly synthesized viral proteins interact with sequestered viral RNA molecules prior to capsid assembly and double-stranded RNA synthesis within viral inclusion bodies (VIBs). VIBs are predominantly comprised of a BTV-encoded non-structural protein 2 (NS2). Previous in vitro studies indicated that casein kinase 2 (CK2) mediated the phosphorylation of NS2, which regulated the propensity of NS2 to form larger aggregates. Using targeted pharmacological reagents, specific mutation in the viral genome by reverse genetics and confocal microscopy, here we demonstrate that CK2 activity is important for BTV replication. Furthermore, we show that a novel host cell factor, protein phosphatase 2A, is involved in NS2 dephosphorylation and that, together with CK2, it regulates VIB morphology and virus replication. Thus, these two host enzymes influence the dynamic nature of VIB assembly/disassembly, and these concerted activities may be relevant to the assembly and the release of these cores from VIBs. PMID:27226558

  8. Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase.

    PubMed Central

    Pei, D; Neel, B G; Walsh, C T

    1993-01-01

    A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides. Images PMID:8430079

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

    PubMed

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

    2015-01-01

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

  10. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    NASA Technical Reports Server (NTRS)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  11. Inhibition of PHOSPHO1 activity results in impaired skeletal mineralization during limb development of the chick

    PubMed Central

    MacRae, Vicky E.; Davey, Megan G.; McTeir, Lynn; Narisawa, Sonoko; Yadav, Manisha C.; Millan, Jose Luis; Farquharson, Colin.

    2010-01-01

    PHOSPHO1 is a bone specific phosphatase implicated in the initiation of inorganic phosphate generation for matrix mineralization. The control of mineralization is attributed to the actions of tissue-non specific alkaline phosphatase (TNAP). However, matrix vesicles (MVs) containing apatite crystals are present in patients with hypophosphatasia as well as TNAP null (Akp2-/-) mice. It is therefore likely that other phosphatases work with TNAP to regulate matrix mineralization. Although PHOSPHO1 and TNAP expression is associated with MVs, it is not known if PHOSPHO1 and TNAP are co-expressed during the early stages of limb development. Furthermore the functional in-vivo role of PHOSPHO1 in matrix mineralization has yet to be established. Here, we studied the temporal expression and functional role of PHOSPHO1 within chick limb bud mesenchymal micromass cultures and also in wild-type and talpid3 chick mutants. These mutants are characterized by defective hedgehog signalling and the absence of endochondral mineralization. The ability of in-vitro micromass cultures to differentiate and mineralize their matrix was temporally associated with increased expression of PHOSPHO1 and TNAP. Comparable changes in expression were noted in developing embryonic legs (developmental stages 23–36HH). Micromass cultures treated with lansoprazole, a small-molecule inhibitor of PHOSPHO1 activity, or FGF2, an inhibitor of chondrocyte differentiation, resulted in reduced alizarin red staining (P<0.05). FGF2 treatment also caused a reduction in PHOSPHO1 (P<0.001) and TNAP (P<0.001) expression. Expression analysis by whole mount RNA in-situ hybridization, correlated with qPCR micromass data and demonstrated the existence of a tightly regulated pattern of Phospho1 and Tnap expression which precedes mineralization. Treatment of developing embryos for 5-days with lansoprazole completely inhibited mineralization of all leg and wing long bones as assessed by alcian blue/alizarin red staining

  12. Inhibition of Phospho-S6 Kinase, a Protein Involved in the Compensatory Adaptive Response, Increases the Efficacy of Paclitaxel in Reducing the Viability of Matrix-Attached Ovarian Cancer Cells

    PubMed Central

    Choi, Jeong In; Park, Sang Hi; Lee, Hee-Jin; Lee, Dae Woo; Lee, Hae Nam

    2016-01-01

    Objective To identify the proteins involved the compensatory adaptive response to paclitaxel in ovarian cancer cells and to determine whether inhibition of the compensatory adaptive response increases the efficacy of paclitaxel in decreasing the viability of cancer cells. Methods We used a reverse-phase protein array and western blot analysis to identify the proteins involved in the compensatory mechanism induced by paclitaxel in HeyA8 and SKOV3 ovarian cancer cells. We used a cell viability assay to examine whether inhibition of the proteins involved in the compensatory adaptive response influenced the effects of paclitaxel on cancer cell viability. All experiments were performed in three-dimensional cell cultures. Results Paclitaxel induced the upregulation of pS6 (S240/S244) and pS6 (S235/S236) in HeyA8 and SKOV3 cells, and pPRAS40 (T246) in HeyA8 cells. BX795 and CCT128930 were chosen as inhibitors of pS6 (S240/S244), pS6 (S235/S236), and pPRAS40 (T246). BX795 and CCT128930 decreased pS6 (S240/S244) and pS6 (S235/S236) expression in HeyA8 and SKOV3 cells. However, pPRAS40 (T246) expression was inhibited only by BX795 and not by CCT128930 in HeyA8 cells. Compared with paclitaxel alone, addition of BX795 or CCT128930 to paclitaxel was more effective in decreasing the viability of HeyA8 and SKOV3 cells. Conclusion Addition of BX795 or CCT128930 to inhibit pS6 (S240/S244) or pS6 (S235/S236) restricted the compensatory adaptive response to paclitaxel in HeyA8 and SKOV3 cells. These inhibitors increased the efficacy of paclitaxel in reducing cancer cell viability. PMID:27148873

  13. Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice.

    PubMed

    Yadav, Manisha C; Bottini, Massimo; Cory, Esther; Bhattacharya, Kunal; Kuss, Pia; Narisawa, Sonoko; Sah, Robert L; Beck, Laurent; Fadeel, Bengt; Farquharson, Colin; Millán, José Luis

    2016-06-01

    We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ∼80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ∼50% for the Phospho1(-/-) MVs and ∼25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and

  14. Protein phosphatase methylesterase-1 (PME-1) expression predicts a favorable clinical outcome in colorectal cancer.

    PubMed

    Kaur, Amanpreet; Elzagheid, Adam; Birkman, Eva-Maria; Avoranta, Tuulia; Kytölä, Ville; Korkeila, Eija; Syrjänen, Kari; Westermarck, Jukka; Sundström, Jari

    2015-12-01

    Colorectal cancer (CRC) accounts for high mortality. So far, there is lack of markers capable of predicting which patients are at risk of aggressive course of the disease. Protein phosphatase-2A (PP2A) inhibitor proteins have recently gained interest as markers of more aggressive disease in certain cancers. Here, we report the role of PP2A inhibitor PME-1 in CRC. PME-1 expression was assessed from a rectal cancer patient cohort by immunohistochemistry, and correlations were performed for various clinicopathological variables and patient survival. Rectal cancer patients with higher cytoplasmic PME-1 protein expression (above median) had less recurrences (P = 0.003, n = 195) and better disease-free survival (DFS) than the patients with low cytoplasmic PME-1 protein expression (below median). Analysis of PPME-1 mRNA expression from TCGA dataset of colon and rectal adenocarcinoma (COADREAD) patient cohort confirmed high PPME1 expression as an independent protective factor predicting favorable overall survival (OS) (P = 0.005, n = 396) compared to patients with low PPME1 expression. CRC cell lines were used to study the effect of PME-1 knockdown by siRNA on cell survival. Contrary to other cancer types, PME-1 inhibition in CRC cell lines did not reduce the viability of cells or the expression of active phosphorylated AKT and ERK proteins. In conclusion, PME-1 expression predicts for a favorable outcome of CRC patients. The unexpected role of PME-1 in CRC in contrast with the oncogenic role of PP2A inhibitor proteins in other malignancies warrants further studies of cancer-specific function for each of these proteins. PMID:26377365

  15. Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase.

    PubMed Central

    Klionsky, D J; Emr, S D

    1989-01-01

    The Saccharomyces cerevisiae PHO8 gene product, repressible alkaline phosphatase (ALP), is a glycoprotein enzyme that is localized to the yeast vacuole (lysosome). Using antibodies raised against synthetic peptides corresponding to two distinct hydrophilic sequences in ALP, we have been able to examine the biosynthesis, sorting and processing of this protein. ALP is synthesized as an inactive precursor containing a C-terminal propeptide that is cleaved from the protein in a PEP4-dependent manner. The precursor and mature protein are anchored in the membrane by an N-terminal hydrophobic domain that also appears to function as an uncleaved internal signal sequence. ALP has the topology of a type-II integral membrane protein containing a short basic N-terminal cytoplasmic tail that is accessible to exogenous protease when associated both with the endoplasmic reticulum and the vacuole. Similar to the soluble vacuolar hydrolases carboxypeptidase Y (CPY) and proteinase A (PrA), ALP transits through the early stages of the secretory pathway prior to vacuolar delivery. Two observations indicate, however, that ALP is localized to the vacuole by a mechanism which is in part different from that used by CPY and PrA: (i) maturation of proALP, which is indicative of vacuolar delivery, is less sensitive than CPY and PrA to the defects exhibited by certain of the vacuolar protein sorting (vps) mutants; and (ii) maturation of proALP proceeds normally in the presence of a potent vacuolar ATPase inhibitor, bafilomycin A1, which is known to block vacuole acidification and leads to the mis-sorting and secretion of precursor forms of CPY and PrA. These results indicate that ALP will be a useful model protein for studies of membrane protein sorting in yeast. Images PMID:2676517

  16. UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum*

    PubMed Central

    Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D.; Tremblay, Michel L.; Michalak, Marek

    2015-01-01

    Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. PMID:25586181

  17. A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation, Motility and Invasion

    PubMed Central

    Ferguson, David J. P.; Szöőr, Balázs; Wickstead, Bill; Carroll, Paula L.; Ramakrishnan, Chandra; Brady, Declan; Patzewitz, Eva-Maria; Straschil, Ursula; Solyakov, Lev; Green, Judith L.; Sinden, Robert E.; Tobin, Andrew B.; Holder, Anthony A.; Tewari, Rita

    2012-01-01

    Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl− mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first

  18. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function

    PubMed Central

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F.; Mori Sequeiros García, M. Mercedes; Maloberti, Paula M.; Orlando, Ulises D.; Mele, Pablo G.; Poderoso, Cecilia; Podesta, Ernesto J.

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the “classical” protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed. PMID:27375556

  19. Structure and Configuration of Phosphoeleganin, a Protein Tyrosine Phosphatase 1B Inhibitor from the Mediterranean Ascidian Sidnyum elegans.

    PubMed

    Imperatore, Concetta; Luciano, Paolo; Aiello, Anna; Vitalone, Rocco; Irace, Carlo; Santamaria, Rita; Li, Jia; Guo, Yue-W; Menna, Marialuisa

    2016-04-22

    A new phosphorylated polyketide, phosphoeleganin (1), has been isolated from the Mediterranean ascidian Sidnyum elegans. Its structure and configuration have been determined by extensive use of 2D NMR and microscale chemical degradation and/or derivatization. Phosphoeleganin (1) inhibited the protein tyrosine phosphatase 1B (PTP1B) activity. PMID:27064611

  20. Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation.

    PubMed

    Chen, Zan; Dempsey, Daniel R; Thomas, Stefani N; Hayward, Dawn; Bolduc, David M; Cole, Philip A

    2016-07-01

    PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380-385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-cross-linking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PMID:27226612

  1. The C. elegans sex-determining gene fem-2 encodes a putative protein phosphatase.

    PubMed Central

    Pilgrim, D; McGregor, A; Jäckle, P; Johnson, T; Hansen, D

    1995-01-01

    The genetic and molecular analysis of genes involved in the regulation of sex determination in Caenorhabditis elegans suggests that the gene fem-2 plays an important role in regulating a pathway transducing a non-cell-autonomous signal to a nuclear transcription factor. The wild-type fem-2 gene was cloned by identifying sequences from the C. elegans physical map that could restore normal Fem-2 function to homozygous mutant fem-2 transgenic animals. cDNA sequences mapping to the minimal rescuing region correspond to an open reading frame with a sequence similar to protein phosphatase 2C enzymes from systems as diverse as yeast, humans, and plants, but the alignments suggest that FEM-2 falls into a separate class of proteins than the canonical homologues. Several fem-2 mutant alleles were sequenced, and the mutations are predicted to cause protein changes consistent with their observed phenotypes, such as missense mutations in conditional alleles, and a nonsense mutation in a predicted null allele. This is the first evidence implicating phosphorylation and/or dephosphorylation as a control mechanism in C. elegans sex determination. Images PMID:8534913

  2. The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

    PubMed Central

    Luke, M M; Della Seta, F; Di Como, C J; Sugimoto, H; Kobayashi, R; Arndt, K T

    1996-01-01

    SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions. PMID:8649382

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

    PubMed

    Maertens, Goedele N

    2016-01-01

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

  4. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    PubMed

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  5. Probing the target-specific inhibition of sensitized protein tyrosine phosphatases with biarsenical probes

    PubMed Central

    Pomorski, Adam; Adamczyk, Justyna; Bishop, Anthony C.; Krężel, Artur

    2014-01-01

    Selective control of enzyme activity is critical for elucidating the roles of specific proteins in signaling pathways. One potential means for developing truly target-specific inhibitors involves the use of protein engineering to sensitize a target enzyme to inhibition by a small molecule that does not inhibit homologous wild-type enzymes. Previously, it has been shown that protein tyrosine phosphatases (PTPs) can be sensitized to inhibition by a biarsenical probe, FlAsH-EDT2, which inhibits PTP activity by specifically binding to cysteine residues that have been introduced into catalytically important regions. In the present study, we developed an array of biarsenical probes, some newly synthesized and some previously reported, to investigate for the first time the structure-activity relationships for PTP inhibition by biarsenicals. Our data show that biarsenical probes which contain substitutions at the 2′ and 7′ positions are more effective than FlAsH-EDT2 at inhibiting sensitized PTPs. The increased potency of 2′,7′-substituted probes was observed when PTPs were assayed with both para-nitrophenylphosphate and phosphopeptide PTP substrates and at multiple probe concentrations. The data further indicate that the enhanced inhibitory properties are the result of increased binding affinity between the 2′,7′-substituted biarsenical probes and sensitized PTPs. In addition we provide previously unknown physicochemical and stability data for various biarsenical probes. PMID:25460004

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

    PubMed

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

    2013-09-01

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

  7. Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

    PubMed Central

    Walaas, Sven Ivar; Hemmings, Hugh Caroll; Greengard, Paul; Nairn, Angus Clark

    2011-01-01

    Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington’s disease, and Parkinson’s disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly dopamine and adenosine 3′:5′-monophosphate-regulated phosphoprotein of 32 kDa (DARPP-32), regulator of calmodulin signaling (RCS), and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B, and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways. PMID:21904525

  8. Role of protein-tyrosine phosphatases in regulation of osteoclastic activity.

    PubMed

    Sheng, M H-C; Lau, K-H W

    2009-06-01

    Osteoclasts, the primary cell type mediating bone resorption, are multinucleated, giant cells derived from hematopoietic cells of monocyte-macrophage lineage. Osteoclast activity is, in a large part, regulated by protein-tyrosine phosphorylation. While information about functional roles of several protein-tyrosine kinases (PTK), including c-Src, in osteoclastic resorption has been accumulated, little is known about the roles of protein-tyrosine phosphatases (PTPs) in regulation of osteoclast activity. Recent evidence implicates important regulatory roles for four PTPs (SHP-1, cyt-PTP-epsilon, PTP-PEST, and PTPoc) in osteoclasts. Cyt-PTP-epsilon, PTP-PEST, and PTP-oc are positive regulators of osteoclast activity, while SHP-1 is a negative regulator. Of these PTPs in osteoclasts, only PTP-oc is a positive regulator of c-Src PTK through dephosphorylation of the inhibitory phosphotyrosine-527 residue. Although some information about mechanisms of action of these PTPs to regulate osteoclast activity is reviewed in this article, much additional work is required to provide more comprehensive details about their functions in osteoclasts. PMID:19189046

  9. Spinophilin directs Protein Phosphatase 1 specificity by blocking substrate binding sites

    PubMed Central

    Ragusa, Michael J.; Dancheck, Barbara; Critton, David A.; Nairn, Angus C.; Page, Rebecca; Peti, Wolfgang

    2010-01-01

    The serine/threonine Protein Phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets. PP1 associates with ≥200 regulatory proteins to form highly specific holoenzymes. These regulatory proteins target PP1 to its point of action within the cell and prime its enzymatic specificity for particular substrates. However, how they direct PP1’s specificity is not understood. Here we show that spinophilin, a neuronal PP1 regulator, is entirely unstructured in its unbound form and binds PP1, through a folding-upon-binding mechanism, in an elongated fashion, blocking one of PP1’s three putative substrate binding sites, without altering its active site. This mode of binding is sufficient for spinophilin to restrict PP1’s activity toward a model substrate in vitro, without affecting its ability to dephosphorylate its neuronal substrate GluR1. Thus, our work provides the molecular basis for the ability of spinophilin to dictate PP1 substrate specificity. PMID:20305656

  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. Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans.

    PubMed

    Mitchell, Christopher J; Kim, Min-Sik; Zhong, Jun; Nirujogi, Raja Sekhar; Bose, Anjun K; Pandey, Akhilesh

    2016-06-01

    The leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans - PTPRF, PTPRD and PTPRS - that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2. PMID:27067626

  12. Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

    PubMed Central

    Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

    2013-01-01

    A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens. PMID:23682679

  13. Striatal-Enriched Protein Tyrosine Phosphatase Controls Responses to Aversive Stimuli: Implication for Ethanol Drinking

    PubMed Central

    Legastelois, Rémi; Darcq, Emmanuel; Wegner, Scott A.; Lombroso, Paul J.; Ron, Dorit

    2015-01-01

    The STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase whose dysregulation in expression and/or activity is associated with several neuropsychiatric disorders. We recently showed that long-term excessive consumption of ethanol induces a sustained inhibition of STEP activity in the dorsomedial striatum (DMS) of mice. We further showed that down-regulation of STEP expression in the DMS, and not in the adjacent dorsolateral striatum, increases ethanol intake, suggesting that the inactivation of STEP in the DMS contributes to the development of ethanol drinking behaviors. Here, we compared the consequence of global deletion of the STEP gene on voluntary ethanol intake to the consumption of an appetitive rewarding substance (saccharin) or an aversive solution (quinine or denatonium). Whereas saccharin intake was similar in STEP knockout (KO) and wild type (WT) littermate mice, the consumption of ethanol as well as quinine and denatonium was increased in STEP KO mice. These results suggested that the aversive taste of these substances was masked upon deletion of the STEP gene. We therefore hypothesized that STEP contributes to the physiological avoidance towards aversive stimuli. To further test this hypothesis, we measured the responses of STEP KO and WT mice to lithium-induced conditioned place aversion (CPA) and found that whereas WT mice developed lithium place aversion, STEP KO mice did not. In contrast, conditioned place preference (CPP) to ethanol was similar in both genotypes. Together, our results indicate that STEP contributes, at least in part, to the protection against the ingestion of aversive agents. PMID:25992601

  14. Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1 [corrected].

    PubMed

    Takemiya, Atsushi; Shimazaki, Ken-ichiro

    2010-08-01

    Stomata open in response to blue light under a background of red light. The plant hormone abscisic acid (ABA) inhibits blue light-dependent stomatal opening, an effect essential for promoting stomatal closure in the daytime to prevent water loss. However, the mechanisms and molecular targets of this inhibition in the blue light signaling pathway remain unknown. Here, we report that phosphatidic acid (PA), a phospholipid second messenger produced by ABA in guard cells, inhibits protein phosphatase 1 (PP1), a positive regulator of blue light signaling, and PA plays a role in stimulating stomatal closure in Vicia faba. Biochemical analysis revealed that PA directly inhibited the phosphatase activity of the catalytic subunit of V. faba PP1 (PP1c) in vitro. PA inhibited blue light-dependent stomatal opening but did not affect red light- or fusicoccin-induced stomatal opening. PA also inhibited blue light-dependent H(+) pumping and phosphorylation of the plasma membrane H(+)-ATPase. However, PA did not inhibit the autophosphorylation of phototropins, blue light receptors for stomatal opening. Furthermore, 1-butanol, a selective inhibitor of phospholipase D, which produces PA via hydrolysis of phospholipids, diminished the ABA-induced inhibition of blue light-dependent stomatal opening and H(+) pumping. We also show that hydrogen peroxide and nitric oxide, which are intermediates in ABA signaling, inhibited the blue light responses of stomata and that 1-butanol diminished these inhibitions. From these results, we conclude that PA inhibits blue light signaling in guard cells by PP1c inhibition, accelerating stomatal closure, and that PP1 is a cross talk point between blue light and ABA signaling pathways in guard cells. PMID:20498335

  15. Chimeric proteins combining phosphatase and cellulose-binding activities: proof-of-concept and application in the hydrolysis of paraoxon.

    PubMed

    Gonçalves, Larissa M; Chaimovich, Hernan; Cuccovia, Iolanda M; Marana, Sandro R

    2014-05-01

    Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical Km values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification. PMID:24555432

  16. Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

    PubMed

    Boratkó, Anita; Péter, Margit; Thalwieser, Zsófia; Kovács, Előd; Csortos, Csilla

    2015-12-01

    TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements. PMID:26497934

  17. Colocalisation of the protein tyrosine phosphatases PTP-SL and PTPBR7 with beta4-adaptin in neuronal cells.

    PubMed

    Dilaver, Gönül; Schepens, Jan; van den Maagdenberg, Arn; Wijers, Mietske; Pepers, Barry; Fransen, Jack; Hendriks, Wiljan

    2003-01-01

    The mouse gene Ptprr encodes the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7. These proteins differ in their N-terminal domains, with PTP-SL being a cytosolic, membrane-associated phosphatase and PTPBR7 a type I transmembrane protein. In this study, we further explored the nature of the PTP-SL-associated vesicles in neuronal cells using a panel of organelle markers and noted a comparable subcellular distribution for PTP-SL and the beta4-adaptin subunit of the AP4 complex. PTP-SL, PTPBR7 and beta4-adaptin are localised at the Golgi apparatus and at vesicles throughout the cytoplasm. Immunohistochemical analysis demonstrated that PTP-SL, PTPBR7 and beta4-adaptin are all endogenously expressed in brain. Interestingly, coexpression of PTP-SL and beta4-adaptin leads to an altered subcellular localisation for PTP-SL. Instead of the Golgi and vesicle-type staining pattern, still observable for beta4-adaptin, PTP-SL is now distributed throughout the cytoplasm. Although beta4-adaptin was found to interact with the phosphatase domain of PTP-SL and PTPBR7 in the yeast two-hybrid system, it failed to do so in transfected neuronal cells. Our data suggest that the tyrosine phosphatases PTP-SL and PTPBR7 may be involved in the formation and transport of AP4-coated vesicles or in the dephosphorylation of their transmembrane cargo molecules at or near the Golgi apparatus. PMID:12548400

  18. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    SciTech Connect

    Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen; Wang, Yan Wei; Sakamoto, Akira; Zhang, Yan Fang; Naranmandura, Hua; Suzuki, Noriyuki

    2012-09-15

    Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly

  19. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits

    PubMed Central

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-01-01

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  20. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    PubMed

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  1. Activation of HIV-1 with Nanoparticle-Packaged Small-Molecule Protein Phosphatase-1-Targeting Compound

    PubMed Central

    Smith, Kahli A.; Lin, Xionghao; Bolshakov, Oleg; Griffin, James; Niu, Xiaomei; Kovalskyy, Dmytro; Ivanov, Andrey; Jerebtsova, Marina; Taylor, Robert E.; Akala, Emmanuel; Nekhai, Sergei

    2015-01-01

    Complete eradication of HIV-1 infection is impeded by the existence of latent HIV-1 reservoirs in which the integrated HIV-1 provirus is transcriptionally inactive. Activation of HIV-1 transcription requires the viral Tat protein and host cell factors, including protein phosphatase-1 (PP1). We previously developed a library of small compounds that targeted PP1 and identified a compound, SMAPP1, which induced HIV-1 transcription. However, this compound has a limited bioavailability in vivo and may not be able to reach HIV-1-infected cells and induce HIV-1 transcription in patients. We packaged SMAPP1 in polymeric polyethylene glycol polymethyl methacrylate nanoparticles and analyzed its release and the effect on HIV-1 transcription in a cell culture. SMAPP1 was efficiently packaged in the nanoparticles and released during a 120-hr period. Treatment of the HIV-1-infected cells with the SMAPP1-loaded nanoparticles induced HIV-1 transcription. Thus, nanoparticles loaded with HIV-1-targeting compounds might be useful for future anti-HIV-1 therapeutics. PMID:26839837

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

    PubMed Central

    Maertens, Goedele N.

    2016-01-01

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

  3. Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets.

    PubMed

    Stanley, William J; Litwak, Sara A; Quah, Hong Sheng; Tan, Sih Min; Kay, Thomas W H; Tiganis, Tony; de Haan, Judy B; Thomas, Helen E; Gurzov, Esteban N

    2015-07-01

    Type 1 diabetes (T1D) is the result of an autoimmune assault against the insulin-producing pancreatic β-cells, where chronic local inflammation (insulitis) leads to β-cell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T-cell invasion and activation. Cytokine-signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from NOD mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling compared with NOD mice that do not have insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and β-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation-induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis. PMID:25732191

  4. The polymorphism of protein phosphatase Z1 gene in Candida albicans.

    PubMed

    Kovács, László; Farkas, Ilona; Majoros, László; Miskei, Márton; Pócsi, István; Dombrádi, Viktor

    2010-12-01

    The gene of protein phosphatase Z1 (CaPPZ1) that codes a fungus specific regulatory enzyme was investigated in Candida albicans. After cloning and sequencing CaPPZ1 we revealed the heterozygous nature of the ATCC 10231 reference strain, and identified two new alleles termed CaPPZ1-2 and CaPPZ1-3. The genetic polymorphism in CaPPZ1 was extended by finding a fourth allele CaPPZ1-4 in a clinical isolate. Single nucleotide replacements and short in/del mutations were identified in the gene, some of which resulted in amino acid changes in the protein. The analysis of the hypervariable 3'-noncoding gene region in 27 DNA sequences obtained from reference strains and clinical samples confirmed the presence of four distinct DNA sequence-groups that correspond to the four main alleles of CaPPZ1. In addition to the allelic combinations, we detected individual mutations elevating genetic variability of the opportunistic pathogen. We utilized the hypervariable gene region for genotyping C. albicans in clinical isolates by sequencing the cloned amplified region, by direct sequencing of the PCR products, or by RFLP analysis. The comparison of the genotypes of the strains originating from different body parts of the same patient proved to be useful in delineating the origin of the infection. PMID:20473966

  5. Protein tyrosine phosphatase mu regulates glioblastoma cell growth and survival in vivo

    PubMed Central

    Kaur, Harpreet; Burden-Gulley, Susan M.; Phillips-Mason, Polly J.; Basilion, James P.; Sloan, Andrew E.; Brady-Kalnay, Susann M.

    2012-01-01

    Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM. PMID:22505657

  6. Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

    PubMed Central

    Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

    2015-01-01

    Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

  7. Mitogen-activated protein kinase phosphatase-1: function and regulation in bone and related tissues.

    PubMed

    Broome, David T; Datta, Nabanita S

    2016-05-01

    In this review, we have highlighted work that has clearly demonstrated that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), a negative regulator of MAPKs, is an important signaling mediator in bone, muscle, and fat tissue homeostasis and differentiation. Further, we examined recent studies with particular focus on MKP-1 overexpression or deletion and its impact on tissues connected to bone. We also summarized regulation of MKP-1 by known skeletal regulators like parathyroid hormone (PTH)/PTH-related peptide (PTHrP) and bone morphogenic proteins. MKP-1's integration into the pathophysiological state of osteoporosis, osteoarthritis, rheumatoid arthritis, obesity, and muscular dystrophy are examined to emphasize possible involvement of MKP-1 both at the molecular level and in disease complications such as sarcopenia- or diabetes-related osteoporosis. We predict that understanding the mechanism of MKP-1-mediated signaling in bone-muscle-fat crosstalk will be a key in coordinating their activities and developing therapeutics to improve clinical outcomes for diseases associated with advanced age. PMID:27031422

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

    PubMed Central

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

    2011-01-01

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

  9. Immunohistochemical Expression of Dual-Specificity Protein Phosphatase 4 in Patients with Colorectal Adenocarcinoma

    PubMed Central

    Sim, Jongmin; Yi, Kijong; Kim, Hyunsung; Ahn, Hyein; Chung, Yumin; Rehman, Abdul; Jang, Se Min; Lee, Kang Hong; Jang, Kiseok; Paik, Seung Sam

    2015-01-01

    The role of dual-specificity protein phosphatase 4 (DUSP4) appears to vary with the type of malignant tumors and is still controversial. The purpose of our study was to clarify the exact role of DUSP4 expression in colorectal adenocarcinoma. We constructed tissue microarrays and investigated DUSP4 expression by immunohistochemistry. DUSP4 was more frequently expressed in adenocarcinomas and lymph node/distant metastases compared to that in normal colorectal tissues and tubular adenomas (P < 0.001). Mean DUSP4 expression score was significantly higher in malignant tumors than in benign lesions (P < 0.001). DUSP4 expression was significantly correlated with older age (P = 0.017), male gender (P = 0.036), larger tumor size (P = 0.014), nonmucinous tumor type (P = 0.023), and higher T stage (P = 0.040). Kaplan-Meier survival curves revealed a significant effect of DUSP4 expression on both overall survival and disease-free survival in AJCC stage I (P = 0.008 and P = 0.003, resp., log-rank test) and male gender (P = 0.017 and P = 0.049, resp., log-rank test). DUSP4 protein is frequently upregulated in colorectal adenocarcinoma and may play an important role in carcinogenesis and cancer progression and may be a marker of adverse prognosis. PMID:25688264

  10. A novel antimicrobial protein isolated from potato (Solanum tuberosum) shares homology with an acid phosphatase.

    PubMed

    Feng, Jie; Yuan, Fenghua; Gao, Yin; Liang, Chenggang; Xu, Jin; Zhang, Changling; He, Liyuan

    2003-12-01

    The nucleotide and amino acids sequences for AP(1) will appear in the GenBank(R) and NCBI databases under accession number AY297449. A novel antimicrobial protein (AP(1)) was purified from leaves of the potato ( Solanum tuberosum, variety MS-42.3) with a procedure involving ammonium sulphate fractionation, molecular sieve chromatography with Sephacryl S-200 and hydrophobic chromatography with Butyl-Sepharose using a FPLC system. The inhibition spectrum investigation showed that AP(1) had good inhibition activity against five different strains of Ralstonia solanacearum from potato or other crops, and two fungal pathogens, Rhizoctonia solani and Alternaria solani from potato. The full-length cDNA encoding AP(1) has been successfully cloned by screening a cDNA expression library of potato with an anti-AP(1) antibody and RACE (rapid amplification of cDNA ends) PCR. Determination of the nucleotide sequences revealed the presence of an open reading frame encoding 343 amino acids. At the C-terminus of AP(1) there is an ATP-binding domain, and the N-terminus exhibits 58% identity with an/the acid phosphatase from Mesorhizobium loti. SDS/PAGE and Western blotting analysis suggested that the AP(1) gene can be successfully expressed in Escherichia coli and recognized by an antibody against AP(1). Also the expressed protein showed an inhibition activity the same as original AP(1) protein isolated from potato. We suggest that AP(1) most likely belongs to a new group of proteins with antimicrobial characteristics in vitro and functions in relation to phosphorylation and energy metabolism of plants. PMID:12927022

  11. Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

    PubMed Central

    Sagara, Junji; Arata, Toshiaki; Taniguchi, Shunichiro

    2009-01-01

    Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes. PMID:19158953

  12. Identification of putative negative regulators of yeast signaling through a screening for protein phosphatases acting on cell wall integrity and mating MAPK pathways.

    PubMed

    Sacristán-Reviriego, Almudena; Martín, Humberto; Molina, María

    2015-04-01

    The lack of signaling through MAPK pathways leads to a defective cellular response to the corresponding stimulus, but an improper hyperactivation of these routes results in deleterious effects as well. Protein phosphorylation is an activating modification for signal transmission through components of MAPK pathways and thus, protein phosphatases are key negative regulators of these cellular routes by limiting excessive signaling activity. However, in contrast to most of the protein kinases operating in MAPK pathways, protein phosphatases usually exhibit redundancy and promiscuity, which has limited the identification of their function. In order to identify new putative phosphatases operating in Saccharomyces cerevisiae MAPK signaling, we have taken advantage of growth inhibition promoted by overproduction of constitutively active components of the mating and cell wall integrity (CWI) pathways to perform a screen with a collection of 43 protein phosphatases or phosphatase-regulatory proteins. The phosphatases able to alleviate the induced growth inhibition when overproduced were further studied by testing their capacity to downregulate expression of mating and CWI responsive promoters and the consequences of their removal on MAPK signaling. Epistasis analysis placed the Ser/Thr protein phosphatase Ppq1 as a regulator of the mating MAPK module downstream the MAPKKK Ste11. The dual specificity phosphatase Yvh1 was found to be important for the maintenance of cell wall integrity and appropriate signaling through the CWI pathway. Moreover, we have found that Ptc2 and Ptc4 bind to the CWI MAPK Slt2. Together with known phosphatases of the mating and CWI pathway, as Msg5 or Ptp2, other putative negative regulators of both pathways that came up in the screening were Ptc2, Oca2 and Ptp1. We show that Ptp1 physically interacts with Slt2 and the mating MAPK Fus3. Elimination of Ptp1 results in increased signaling through these pathways, suggesting that this tyrosine

  13. Reactive Nitrogen Species and Hydrogen Sulfide as Regulators of Protein Tyrosine Phosphatase Activity

    PubMed Central

    2014-01-01

    Abstract Significance: Redox modifications of thiols serve as a molecular code enabling precise and complex regulation of protein tyrosine phosphatases (PTPs) and other proteins. Particular gasotransmitters and even the redox modifications themselves affect each other, of which a typical example is S-nitrosylation-mediated protection against the further oxidation of protein thiols. Recent Advances: For a long time, PTPs were considered constitutively active housekeeping enzymes. This view has changed substantially over the last two decades, and the PTP family is now recognized as a group of tightly and flexibly regulated fundamental enzymes. In addition to the conventional ways in which they are regulated, including noncovalent interactions, phosphorylation, and oxidation, the evidence that has accumulated during the past two decades suggests that many of these enzymes are also modulated by gasotransmitters, namely by nitric oxide (NO) and hydrogen sulfide (H2S). Critical Issues: The specificity and selectivity of the methods used to detect nitrosylation and sulfhydration remains to be corroborated, because several researchers raised the issue of false-positive results, particularly when using the most widespread biotin switch method. Further development of robust and straightforward proteomic methods is needed to further improve our knowledge of the full extent of the gasotransmitters-mediated changes in PTP activity, selectivity, and specificity. Further Directions: Results of the hitherto performed studies on gasotransmitter-mediated PTP signaling await translation into clinical medicine and pharmacotherapeutics. In addition to directly affecting the activity of particular PTPs, the use of reversible S-nitrosylation as a protective mechanism against oxidative stress should be of high interest. Antioxid. Redox Signal. 20, 2191–2209. PMID:24328688

  14. Phenylarsine oxide and vanadate: apparent paradox of inhibition of protein phosphotyrosine phosphatases in rat adipocytes.

    PubMed

    Li, J; Elberg, G; Shechter, Y

    1996-07-24

    Vanadate mimics, whereas phenylarsine oxide (PAO) antagonizes, the effects of insulin in rat adipocytes. Both vanadate and PAO are documented inhibitors of protein-phosphotyrosine phosphatases. The relationship between the inhibition of 'inhibitory' PTPase and 'stimulatory' PTPase has been studied here in primary rat adipocytes. Low concentrations of PAO (IC50 = 0.6-2.0 microM) blocked the stimulating effects of insulin, vanadate and pervanadate on hexose uptake and glucose metabolism. Inhibition of isoproterenol-mediating lipolysis by vanadate and insulin was not blocked by PAO. The activating effects of okadaic acid on hexose uptake and glucose metabolism, which occur at points downstream to tyrosine phosphorylation, were also not blocked by PAO. Subsequent studies suggested that the PAO-sensitive PTPase comprises a minute fraction of the total adipocytic PTPase activity. To identify its location we applied procedures involving fractionations and activation of non-receptor adipocytic protein tyrosine kinase by PAO and vanadate in cell free assays. We found that the 'inhibitory' PTPase is exclusively associated with the membrane fraction whereas the 'stimulatory' PTPases are present in both the cytosolic and plasma membrane compartments. We next searched for markers, possibly associated with PAO-dependent desensitization and found that several proteins became phosphorylated on tyrosine moieties in the supernatant of PAO but not in vanadate pretreated adipocytes. In summary, we propose the presence of a minute, plasma membrane associated PTPase in primary rat adipocytes, inhibition of which arrests the activation of glucose metabolism. In contrast, inhibition of all the other cellular adipose PTPases, ultimately activates rather than inhibits these same bioeffects. PMID:8703991

  15. Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

    PubMed

    Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

    2016-02-24

    Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. PMID:26911683

  16. The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

    PubMed

    Baldauf, Christina; Jeschke, Anke; Kanbach, Vincent; Catala-Lehnen, Philip; Baumhoer, Daniel; Gerull, Helwe; Buhs, Sophia; Amling, Michael; Nollau, Peter; Harroch, Sheila; Schinke, Thorsten

    2015-01-01

    Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells. PMID:26360410

  17. Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

    PubMed

    Bettaieb, Ahmed; Koike, Shinichiro; Chahed, Samah; Bachaalany, Santana; Griffey, Stephen; Sastre, Juan; Haj, Fawaz G

    2016-08-01

    Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis. PMID:27461362

  18. Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

    PubMed

    Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

    2014-11-01

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar

  19. The Mechanism of Allosteric Inhibition of Protein Tyrosine Phosphatase 1B

    PubMed Central

    Lu, Shaoyong; Huang, Wenkang; Geng, Lv; Shen, Qiancheng; Zhang, Jian

    2014-01-01

    As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors. PMID:24831294

  20. Activation of Protein Serine/Threonine Phosphatase PP2Cα Efficiently Prevents Liver Fibrosis

    PubMed Central

    Chen, Jing; Yang, Zhengyi; Yu, Liang; Hu, Lihong; Shen, Xu

    2010-01-01

    Background Over-activation of TGFβ signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2Cα was reported to negatively regulate TGFβ signaling pathway and cell cycle. Our study aimed to investigate the role of PP2Cα in liver fibrogenesis. Methodology/Principal Findings The effects of PP2Cα activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2Cα by overexpression or the new discovered small molecular activator NPLC0393 terminated TGFβ-Smad3 and TGFβ-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased α-smooth muscle actin (α-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl4- and BDL-induced mice. Conclusions/Significance Our findings suggested that PP2Cα activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease. PMID:21151953

  1. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress

    PubMed Central

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-01-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  2. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

    PubMed

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-03-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  3. Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

    PubMed

    Zhou, Yuehan; Skelton, Lara A; Xu, Lumei; Chandler, Margaret P; Berthiaume, Jessica M; Boron, Walter F

    2016-09-01

    Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis. PMID:26839367

  4. Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

    PubMed Central

    Chiaradia-Delatorre, Louise Domeneghini; Menegatti, Angela Camila Orbem; Monache, Franco Delle; Ferrari, Franco; Yunes, Rosendo Augusto; Nunes, Ricardo José; Terenzi, Hernán; Botta, Bruno; Botta, Maurizio

    2013-01-01

    Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with Ki = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors. PMID:24155919

  5. Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma.

    PubMed

    Bourgonje, Annika M; Verrijp, Kiek; Schepens, Jan T G; Navis, Anna C; Piepers, Jolanda A F; Palmen, Chantal B C; van den Eijnden, Monique; Hooft van Huijsduijnen, Rob; Wesseling, Pieter; Leenders, William P J; Hendriks, Wiljan J A J

    2016-01-01

    The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy. PMID:27586084

  6. Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

    PubMed Central

    Lee, Jihye; Adelmant, Guillaume; Marto, Jarrod A.; Lee, Dong-Hyun

    2015-01-01

    Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells. PMID:26194823

  7. Nitropropenyl Benzodioxole, An Anti-Infective Agent with Action as a Protein Tyrosine Phosphatase Inhibitor

    PubMed Central

    White, Kylie S; Nicoletti, Gina; Borland, Robert

    2014-01-01

    We report on the activities of a broad spectrum antimicrobial compound,nitropropenyl benzodioxole (NPBD) which are of relevance to its potential as an anti-infective drug. These investigations support the proposal that a major mechanism of NPBD is action as a tyrosine mimetic, competitively inhibiting bacterial and fungal protein tyrosine phosphatases (PTP). NPBD did not affect major anti-bacterial drug targets, namely, ATP production, cell wall or cell membrane integrity, or transcription and translation of RNA. NPBD inhibited bacterial YopH and human PTP1B and not human CD45 in enzyme assays. NPBD inhibited PTP-associated bacterial virulence factors, namely, endospore formation in Bacillus cereus, prodigiosin secretion in Serratia marcescens, motility in Proteus spp., and adherence and invasion of mammalian cells by Yersinia enterocolitica. NPBD acts intracellularly to inhibit the early development stages of the Chlamydia trachomatis infection cycle in mammalian cells known to involve sequestration of host cell PTPs. NPBD thus both kills pathogens and inhibits virulence factors relevant to early infection, making it a suitable candidate for development as an anti-infective agent, particularly for pathogens that enter through, or cause infections at, mucosal surfaces. Though much is yet to be understood about bacterial PTPs, they are proposed as suitable anti-infective targets and have been linked to agents similar to NPBD. The structural and functional diversity and heterogeneous distribution of PTPs across microbial species make them suitably selective targets for the development of both broadly active and pathogen-specific drugs. PMID:24976873

  8. Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions.

    PubMed

    Lee, Jihye; Adelmant, Guillaume; Marto, Jarrod A; Lee, Dong-Hyun

    2015-08-01

    Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells. PMID:26194823

  9. [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

  10. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.

    PubMed

    Couto, Daniel; Niebergall, Roda; Liang, Xiangxiu; Bücherl, Christoph A; Sklenar, Jan; Macho, Alberto P; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Maclean, Dan; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min; Zipfel, Cyril

    2016-08-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2010-08-01

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

  13. Protein tyrosine phosphatase SHP-1: resurgence as new drug target for human autoimmune disorders.

    PubMed

    Sharma, Yadhu; Bashir, Samina; Bhardwaj, Puja; Ahmad, Altaf; Khan, Farah

    2016-08-01

    Recognition of self-antigen and its destruction by the immune system is the hallmark of autoimmune diseases. During the developmental stages, immune cells are introduced to the self-antigen, for which tolerance develops. The inflammatory insults that break the immune tolerance provoke immune system against self-antigen, progressively leading to autoimmune diseases. SH2 domain containing protein tyrosine phosphatase (PTP), SHP-1, was identified as hematopoietic cell-specific PTP that regulates immune function from developing immune tolerance to mediating cell signaling post-immunoreceptor activation. The extensive research on SHP-1-deficient mice elucidated the diversified role of SHP-1 in immune regulation, and inflammatory process and related disorders such as cancer, autoimmunity, and neurodegenerative diseases. The present review focalizes upon the implication of SHP-1 in the pathogenesis of autoimmune disorders, such as allergic asthma, neutrophilic dermatosis, atopic dermatitis, rheumatoid arthritis, and multiple sclerosis, so as to lay the background in pursuance of developing therapeutic strategies targeting SHP-1. Also, new SHP-1 molecular targets have been suggested like SIRP-α, PIPKIγ, and RIP-1 that may prove to be the focal point for the development of therapeutic strategies. PMID:27216862

  14. Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

    PubMed

    De Smedt, Veronique; Poulhe, Robert; Cayla, Xavier; Dessauge, Frederic; Karaiskou, Anthi; Jessus, Catherine; Ozon, Rene

    2002-08-01

    Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. PMID:12036957

  15. NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

    PubMed Central

    Spalinger, Marianne R.; Kasper, Stephanie; Gottier, Claudia; Lang, Silvia; Atrott, Kirstin; Vavricka, Stephan R.; Scharl, Sylvie; Gutte, Petrus M.; Grütter, Markus G.; Beer, Hans-Dietmar; Contassot, Emmanuel; Chan, Andrew C.; Dai, Xuezhi; Rawlings, David J.; Mair, Florian; Becher, Burkhard; Falk, Werner; Fried, Michael; Rogler, Gerhard

    2016-01-01

    Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation. PMID:27043286

  16. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  17. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease

    PubMed Central

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-01

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  18. Dehydroepiandrosterone-enhanced dual specificity protein phosphatase (DDSP) prevents diet-induced and genetic obesity.

    PubMed

    Watanabe, Tetsuhiro; Ashida, Kenji; Goto, Kiminobu; Nawata, Hajime; Takayanagi, Ryoichi; Yanase, Toshihiko; Nomura, Masatoshi

    Dehydroepiandrosterone (DHEA) exerts a wide variety of therapeutic effects against medical disorders, such as diabetes and obesity. However, the molecular basis of DHEA action remains to be clarified. Previously, we reported that DHEA-enhanced dual specificity protein phosphatase, designated DDSP, is one of the target molecules of DHEA. To examine the role of DDSP in DHEA signaling, we generated mice that carry a DDSP transgene in which expression is driven by the CAG promoter (DDSP-Tg). DDSP-Tg mice weighed significantly less than wild-type (WT) control mice when a high fat diet was supplied (p < 0.01). No difference in food-intake or locomotor activity was found between DDSP-Tg and WT mice. Oxygen consumption of DDSP-Tg mice was higher than that of WT mice (p < 0.01), which suggested an increase in basal metabolism in DDSP-Tg mice. To further investigate the role of DDSP in genetic obese mice, DDSP-Tg mice with a db/db background were generated (DDSP-Tg db/db). We observed cancellation of obesity by the db/db mutation and development of a cachexic phenotype in DDSP-Tg db/db mice. In conclusion, our study shows that expression of DDSP leads to prevention of diet-induced and genetic (db/db) obesity. Anti-obese effects of DHEA might be mediated through DDSP, which might be a therapeutic target for intervention of obesity. PMID:26523513

  19. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1

    PubMed Central

    Liang, Xiangxiu; Bücherl, Christoph A.; Sklenar, Jan; Macho, Alberto P.; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min

    2016-01-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  20. Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling

    PubMed Central

    Holmes, Derek A.; Suto, Eric; Lee, Wyne P.; Ou, Qinglin; Gong, Qian; Smith, Hamish R.C.; Caplazi, Patrick

    2015-01-01

    The protein tyrosine phosphatase PTPN22(C1858T) allelic polymorphism is associated with increased susceptibility for development of systemic lupus erythematosus (SLE) and other autoimmune diseases. PTPN22 (also known as LYP) and its mouse orthologue PEP play important roles in antigen and Toll-like receptor signaling in immune cell functions. We demonstrate here that PEP also plays an important inhibitory role in interferon-α receptor (IFNAR) signaling in mice. PEP co-immunoprecipitates with components of the IFNAR signaling complex. Pep−/− hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep+/+ progenitors in response to IFN-α. In addition, Pep−/− mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α–treated Pep+/+ mice. As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias. PMID:26077719

  1. Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1

    PubMed Central

    Jerebtsova, Marina; Klotchenko, Sergei A.; Artamonova, Tatiana O.; Ammosova, Tatiana; Washington, Kareem; Egorov, Vladimir V.; Shaldzhyan, Aram A.; Sergeeva, Maria V.; Zatulovskiy, Evgeny A.; Temkina, Olga A.; Petukhov, Mikhail G.; Vasin, Andrei V.; Khodorkovskii, Mikhail A.; Orlov, Yuri N.

    2011-01-01

    Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription. PMID:20941529

  2. An Essential Role for Inhibitor-2 Regulation of Protein Phosphatase-1 in Synaptic Scaling

    PubMed Central

    Siddoway, Benjamin A.; Altimimi, Haider F.; Hou, Hailong; Petralia, Ronald S.; Xu, Bo; Stellwagen, David

    2013-01-01

    Protein phosphatase-1 (PP1) activity is important for many calcium-dependent neuronal functions including Hebbian synaptic plasticity and learning and memory. PP1 activity is necessary for the induction of long-term depression, whereas downregulation of PP1 activity is required for the normal induction of long-term potentiation. However, how PP1 is activated is not clear. Moreover, it is not known whether PP1 plays a role in homeostatic synaptic scaling, another form of synaptic plasticity which functions to reset the neuronal firing rate in response to chronic neuronal activity perturbations. In this study, we found that PP1 inhibitor-2 (I-2) is phosphorylated at serine 43 (S43) in rat and mouse cortical neurons in response to bicuculine application. Expression of I-2 phosphorylation-blocking mutant I-2 (S43A) blocked the dephosphorylation of GluA2 at serine 880, AMPA receptor trafficking, and synaptic downscaling induced by bicuculline application. Our data suggest that the phosphorylation of I-2 at S43 appears to be mediated by L-type calcium channels and calcium/calmodulin-dependent myosin light-chain kinase. Our work thus reveals a novel calcium-induced PP1 activation pathway critical for homeostatic synaptic plasticity. PMID:23825423

  3. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease.

    PubMed

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-21

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  4. Effect of chlorination on the protein phosphatase inhibition activity for several microcystins.

    PubMed

    Mash, H; Wittkorn, A

    2016-05-15

    Microcystins are of particular concern due to their toxicity to both humans and animals and may be the most prominent cyanotoxin observed in freshwater. Although a number of studies have investigated the fate of microcystins and other algal toxins through drinking water treatment facilities, measurement of their potential for toxic activity after chlorination, a popular form of treatment in the United States, has not been investigated. In this study, six microcystin variants are subjected to chlorine oxidation. The degradation of each microcystin variant is measured by liquid chromatography/mass spectrometry simultaneously with protein phosphatase inhibition (PPI) response over reaction time with chlorine. Results show that inhibition is dependent on the incorporated amino acid residues, their placement within the microcystin structure, as well as pH. This pH dependence may have practical implications to such activities such as drinking water treatment when the pH is usually adjusted to around 8. Namely, at this pH, even with chlorine addition for disinfection, PPI activity may not be totally eliminated even when the initial MYCs are eliminated. PMID:26999255

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

    PubMed

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

    2016-06-01

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

  6. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  7. Molecular Insights into the Fungus-Specific Serine/Threonine Protein Phosphatase Z1 in Candida albicans

    PubMed Central

    Chen, Emily; Choy, Meng S.; Petrényi, Katalin; Kónya, Zoltán; Erdődi, Ferenc; Dombrádi, Viktor; Peti, Wolfgang

    2016-01-01

    ABSTRACT The opportunistic pathogen Candida is one of the most common causes of nosocomial bloodstream infections. Because candidemia is associated with high mortality rates and because the incidences of multidrug-resistant Candida are increasing, efforts to identify novel targets for the development of potent antifungals are warranted. Here, we describe the structure and function of the first member of a family of protein phosphatases that is specific to fungi, protein phosphatase Z1 (PPZ1) from Candida albicans. We show that PPZ1 not only is active but also is as susceptible to inhibition by the cyclic peptide inhibitor microcystin-LR as its most similar human homolog, protein phosphatase 1α (PP1α [GLC7 in the yeast Saccharomyces cerevisiae]). Unexpectedly, we also discovered that, despite its 66% sequence identity to PP1α, the catalytic domain of PPZ1 contains novel structural elements that are not present in PP1α. We then used activity and pulldown assays to show that these structural differences block a large subset of PP1/GLC7 regulatory proteins from effectively binding PPZ1, demonstrating that PPZ1 does not compete with GLC7 for its regulatory proteins. Equally important, these unique structural elements provide new pockets suitable for the development of PPZ1-specific inhibitors. Together, these studies not only reveal why PPZ1 does not negatively impact GLC7 activity in vivo but also demonstrate that the family of fungus-specific phosphatases—especially PPZ1 from C. albicans—are highly suitable targets for the development of novel drugs that specifically target C. albicans without cross-reacting with human phosphatases. PMID:27578752

  8. Receptor protein-tyrosine phosphatase. gamma. is a candidate tumor suppressor gene at human chromosome region 3p21

    SciTech Connect

    LaForgia, S.; Cannizzaro, L.A.; Boghosian-Sell, L.; Croce, C.M.; Huebner, K. ); Morse, B. ); Levy, J.; Barnea, G.; Schlessinger, J. ); Li, F. ); Nowell, P.C.; Glick, J. ); Weston, A.; Harris, C.C. ); Drabkin, H. ); Patterson, D. )

    1991-06-01

    PTPG, the gene for protein-tyrosine phosphatase {gamma} (PTP{gamma}), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP {gamma} allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP {gamma} allele was lost in three lung tumors that had not lost flanking loci. PTP {gamma} mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP {gamma} gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.

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

    NASA Astrophysics Data System (ADS)

    Li, Xinghai

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

  10. Potent inhibition of protein-tyrosine phosphatase by phosphotyrosine-mimic containing cyclic peptides.

    PubMed

    Akamatsu, M; Roller, P P; Chen, L; Zhang, Z Y; Ye, B; Burke, T R

    1997-01-01

    In an effort to derive potent and bioavailable protein-tyrosine phosphatase inhibitors, we have previously reported hexameric peptides based on the epidermal growth factor receptor sequence EGFR988-993 (Asp-Ala-Asp-Glu-Xxx-Leu, where Xxx = Tyr), in which the tyrosyl residue has been replaced by the non-hydrolyzable phosphotyrosyl mimics phosphonomethylphenylalanine (Pmp), difluorophosphonomethylphenylalanine (F2Pmp) and O-malonyltyrosine (OMT). Inhibitory potencies (IC50 values) of these peptides against the tyrosine phosphatase PTP IB were 200, 0.2 and 10 microM, respectively. Since cellular penetration of peptides containing highly charged phosphonate residues is compromised, and good bioreversible protection strategies for the F2Pmp residue have not yet been reported, the OMT residue is of particular interest in that it affords potential new prodrug approaches. In the current study we have prepared cyclized versions of the OMT-containing EGFR988-993 peptide in order to increase its proteolytic stability and restrain conformational flexibility. Three different cyclic analogues were synthesized. Two of these were cyclized through the peptide backbone ('head to tail') using in one case a single glycine spacer (heptamer peptide) and in the second instance, two glycines (octamer peptide). In a PTPI-based assay the cyclic heptamer experienced a two-fold loss of potency (Ki = 25.2 +/- 3.9 microM) relative to the linear hexamer parent (Ki = 13 +/- 0.9 microM), while the cyclic octamer demonstrated a live-fold increase in potency (Ki = 2.60 +/- 0.11 microM). The third peptide was cyclized by means of a sulfide bridge between the side chain of a C-terminally added cysteine residue and the beta-carbon of a N-terminal acetyl residue. Although the overall size of this ring was identical to that exhibited by the preceding backbone-cyclized octamer, it displayed a three-fold enhancement in potency (Ki = 0.73 +/- 0.03 microM). The structural basis for the observed results are

  11. Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

    PubMed Central

    Prévilon, Miresta; Pezet, Mylène; Vinet, Laurent; Mercadier, Jean-Jacques; Rouet-Benzineb, Patricia

    2014-01-01

    Background Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. Methodology and Principal Findings Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. Conclusions/Significance Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts

  12. Regulation of yeast glycogen metabolism and sporulation by Glc7p protein phosphatase.

    PubMed Central

    Ramaswamy, N T; Li, L; Khalil, M; Cannon, J F

    1998-01-01

    Glc7p is an essential serine/threonine type 1 protein phosphatase (PP1) from the yeast Saccharomyces cerevisiae, which has a role in many processes including cell cycle progression, sporulation, glycogen accumulation, translation initiation, and glucose repression. Two hallmarks of PP1 enzymes are very high amino acid sequence conservation and association of the catalytic subunit with a variety of noncatalytic, regulatory subunits. We tested the hypothesis that PP1 sequence conservation was the result of each PP1 residue playing a role in multiple intermolecular interactions. Analysis of 24 glc7 mutants, isolated primarily by their glycogen accumulation traits, revealed that every mutated Glc7p residue altered many noncatalytic subunit affinities and conferred unselected sporulation traits to various degrees. Furthermore, quantitative analysis showed that Glc7p affinity for the glycogen-binding noncatalytic subunit Gac1p was not the only parameter that determines the glycogen accumulation by a glc7 mutant. Sds22p is one Glc7p noncatalytic subunit that is essential for mitotic growth. Surprisingly, several mutant Glc7p proteins had undetectable affinity for Sds22p, yet grew apparently normally. The characterization of glc7 diploid sporulation revealed that Glc7p has at least two meiotic roles. Premeiotic DNA synthesis was undetectable in glc7 mutants with the poorest sporulation. In the glc7 diploids examined, expression of the meiotic inducer IME1 was proportional to the glc7 diploid sporulation frequency. Moreover, IME1 hyperexpression could not suppress glc7 sporulation traits. The Glc7p/Gip1p holoenzyme may participate in completion of meiotic divisions or spore packaging because meiotic dyads predominate when some glc7 diploids sporulate. PMID:9584086

  13. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    PubMed

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

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

    PubMed

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

    2014-01-21

    Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr(307) and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr(307) in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr(307). These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5-PP2A complex, and its phosphorylation at Tyr(307) by Src. PMID:24395787

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

    PubMed Central

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

    2014-01-01

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

  16. Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

    PubMed Central

    van Eekelen, Mark; Overvoorde, John; van Rooijen, Carina; den Hertog, Jeroen

    2010-01-01

    Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs. PMID:20838449

  17. Substrate Specificity of Protein Tyrosine Phosphatases 1B, RPTPα, SHP-1, and SHP-2†

    PubMed Central

    Ren, Lige; Chen, Xianwen; Luechapanichkul, Rinrada; Selner, Nicholas G.; Meyer, Tiffany M.; Wavreille, Anne-Sophie; Chan, Richard; Iorio, Caterina; Zhou, Xiang; Neel, Benjamin G.; Pei, Dehua

    2011-01-01

    We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately two orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active towards multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY−1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY, but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY−1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme’s in vivo substrate specificity. PMID:21291263

  18. Unique drought resistance functions of the highly ABA-induced clade A protein phosphatase 2Cs.

    PubMed

    Bhaskara, Govinal Badiger; Nguyen, Thao Thi; Verslues, Paul E

    2012-09-01

    Six Arabidopsis (Arabidopsis thaliana) clade A protein phosphatase 2Cs (PP2Cs) have established abscisic acid (ABA) signaling roles; however, phenotypic roles of the remaining three "HAI" PP2Cs, Highly ABA-Induced1 (HAI1), AKT1-Interacting PP2C1/HAI2, and HAI3, have remained unclear. HAI PP2C mutants had enhanced proline and osmoregulatory solute accumulation at low water potential, while mutants of other clade A PP2Cs had no or lesser effect on these drought resistance traits. hai1-2 also had increased expression of abiotic stress-associated genes, including dehydrins and late embryogenesis abundant proteins, but decreased expression of several defense-related genes. Conversely, the HAI PP2Cs had relatively less impact on several ABA sensitivity phenotypes. HAI PP2C single mutants were unaffected in ABA sensitivity, while double and triple mutants were moderately hypersensitive in postgermination ABA response but ABA insensitive in germination. The HAI PP2Cs interacted most strongly with PYL5 and PYL7 to -10 of the PYL/RCAR ABA receptor family, with PYL7 to -10 interactions being relatively little affected by ABA in yeast two-hybrid assays. HAI1 had especially limited PYL interaction. Reduced expression of the main HAI1-interacting PYLs at low water potential when HAI1 expression was strongly induced also suggests limited PYL regulation and a role of HAI1 activity in negatively regulating specific drought resistance phenotypes. Overall, the HAI PP2Cs had greatest effect on ABA-independent low water potential phenotypes and lesser effect on classical ABA sensitivity phenotypes. Both this and their distinct PYL interaction demonstrate a new level of functional differentiation among the clade A PP2Cs and a point of cross talk between ABA-dependent and ABA-independent drought-associated signaling. PMID:22829320

  19. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    PubMed

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease. PMID:25581833

  20. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

    PubMed

    Luna, Sandra; Mingo, Janire; Aurtenetxe, Olaia; Blanco, Lorena; Amo, Laura; Schepens, Jan; Hendriks, Wiljan J; Pulido, Rafael

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest. PMID:27514801

  1. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

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

    PubMed

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

    2016-05-13

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

  3. Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae.

    PubMed

    Ruiz, Amparo; Xu, Xinjing; Carlson, Marian

    2013-10-25

    The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability. PMID:24019512

  4. Regulation of Protein Phosphatase 1I by Cdc25C-associated Kinase 1 (C-TAK1) and PFTAIRE Protein Kinase*

    PubMed Central

    Platholi, Jimcy; Federman, Anna; Detert, Julia A.; Heerdt, Paul; Hemmings, Hugh C.

    2014-01-01

    Protein phosphatase 1I (PP-1I) is a major endogenous form of protein phosphatase 1 (PP-1) that consists of the core catalytic subunit PP-1c and the regulatory subunit inhibitor 2 (I-2). Phosphorylation of the Thr-72 residue of I-2 is required for activation of PP-1I. We studied the effects of two protein kinases identified previously in purified brain PP-1I by mass spectrometry, Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE (PFTK1) kinase, for their ability to regulate PP-1I. Purified C-TAK1 phosphorylated I-2 in reconstituted PP-1I (PP-1c·I-2) on Ser-71, which resulted in partial inhibition of its ATP-dependent phosphatase activity and inhibited subsequent phosphorylation of Thr-72 by the exogenous activating kinase GSK-3. In contrast, purified PFTK1 phosphorylated I-2 at Ser-86, a site known to potentiate Thr-72 phosphorylation and activation of PP-1I phosphatase activity by GSK-3. These findings indicate that brain PP-1I associates with and is regulated by the associated protein kinases C-TAK1 and PFTK1. Multisite phosphorylation of the I-2 regulatory subunit of PP-1I leads to activation or inactivation of PP-1I through bidirectional modulation of Thr-72 phosphorylation, the critical activating residue of I-2. PMID:25028520

  5. Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α-mediated stress signaling.

    PubMed

    Santos, Celio X C; Hafstad, Anne D; Beretta, Matteo; Zhang, Min; Molenaar, Chris; Kopec, Jola; Fotinou, Dina; Murray, Thomas V; Cobb, Andrew M; Martin, Daniel; Zeh Silva, Maira; Anilkumar, Narayana; Schröder, Katrin; Shanahan, Catherine M; Brewer, Alison C; Brandes, Ralf P; Blanc, Eric; Parsons, Maddy; Belousov, Vsevelod; Cammack, Richard; Hider, Robert C; Steiner, Roberto A; Shah, Ajay M

    2016-02-01

    Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress. PMID:26742780

  6. Protein serine/threonine phosphatase PPM1A dephosphorylates Smad1 in the bone morphogenetic protein signaling pathway.

    PubMed

    Duan, Xueyan; Liang, Yao-Yun; Feng, Xin-Hua; Lin, Xia

    2006-12-01

    Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation. PMID:16931515

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

    PubMed Central

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

    2013-01-01

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

  8. 3D model for Cancerous Inhibitor of Protein Phosphatase 2A armadillo domain unveils highly conserved protein-protein interaction characteristics.

    PubMed

    Dahlström, Käthe M; Salminen, Tiina A

    2015-12-01

    Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A. PMID:26393783

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

    PubMed

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

    2014-12-01

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

  10. The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization.

    PubMed

    Stewart, Alan J; Roberts, Scott J; Seawright, Elaine; Davey, Megan G; Fleming, Robert H; Farquharson, Colin

    2006-11-01

    PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (P(i)) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization. PMID:16837257

  11. The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth

    NASA Astrophysics Data System (ADS)

    Kim, Hyunjoo; Lee, Ho-June; Oh, Yumin; Choi, Seon-Guk; Hong, Se-Hoon; Kim, Hyo-Jin; Lee, Song-Yi; Choi, Ji-Woo; Su Hwang, Deog; Kim, Key-Sun; Kim, Hyo-Joon; Zhang, Jianke; Youn, Hyun-Jo; Noh, Dong-Young; Jung, Yong-Keun

    2014-02-01

    Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADDSer194. Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2+/- mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADDSer191. These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

  12. PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures

    PubMed Central

    Huesa, Carmen; Yadav, Manisha C.; Finnilä, Mikko A.J.; Goodyear, Simon R.; Robins, Simon P.; Tanner, K. Elizabeth; Aspden, Richard M.; Millán, José Luis; Farquharson, Colin

    2011-01-01

    Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1−/− mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1−/− mice did not fracture during 3- point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1−/− tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1−/− tibia. The altered dihydroxylysinonorleucine/hydroxyllysinonorleucine and pyridoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1−/− mice and this we associated with increased bone remodelling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1−/− bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone. PMID:21272676

  13. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation. PMID:24040070

  14. N-Acetyl-beta-D-glucopyranosylamine 6-phosphate is a specific inhibitor of glycogen-bound protein phosphatase 1.

    PubMed Central

    Board, M

    1997-01-01

    Previous work has shown that the C-1-substituted glucose-analogue N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc) is a competitive inhibitor of glycogen phosphorylase (GP) and stimulates the inactivation of this enzyme by GP phosphatase. In addition to its effects on GP, 1-GlcNAc also prevents the glucose-led activation of glycogen synthase (GS) in whole hepatocytes. Such an effect on GS was thought to be due to the formation of 1-GlcNAc-6-P by the action of glucokinase within the hepatocyte [Board, Bollen, Stalmans, Kim, Fleet and Johnson (1995) Biochem. J. 311, 845-852]. To investigate this possibility further, a pure preparation of 1-GlcNAc-6-P was synthesized. The effects of the phosphorylated glucose analogue on the activity of protein phosphatase 1 (PP1), the enzyme responsible for dephosphorylation and activation of GS, are reported. During the present study, 1-GlcNAc-6-P inhibited the activity of the glycogen-bound form of PP1, affecting both the GSb phosphatase and GPa phosphatase activities. A level of 50% inhibition of GSb phosphatase activity was achieved with 85 microM 1-GlcNAc-6-P in the absence of Glc-6-P and with 135 microM in the presence of 10 mM Glc-6-P. At either Glc-6-P concentration, 500 microM 1-GlcNAc-6-P completely inhibited activity. The Glc-6-P stimulation of the GPa phosphatase activity of PP1 was negated by 1-GlcNAc-6-P but there was no inhibition of the basal rate in the absence of Glc-6-P. 1-GlcNAc-6-P inhibition was specific for the glycogen-bound form of PP1 and did not inhibit the GSb phosphatase activity of the cytosolic form of the enzyme. The present work explains our previous observations on the inactivating effects on GS of incubating whole hepatocytes with 1-GlcNAc. These observations have their basis in the inhibition of glycogen-bound PP1 by 1-GlcNAc-6-P. A novel inhibitor of PP1, specific for the glycogen-bound form of the enzyme, is presented. PMID:9371733

  15. A Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically Promiscuous Enzyme

    PubMed Central

    McWhirter, Claire; Lund, Elizabeth A.; Tanifum, Eric A.; Feng, Guoqiang; Sheikh, Qaiser; Hengge, Alvan C.; Williams, Nicholas H.

    2009-01-01

    The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKas of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted βlg of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are 18(V/K)bridge = 1.0170 and 15(V/K) = 1.0010 which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted βlg of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the βlg measured for monoester substrates, indicative of similar transition states. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction, and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates. PMID:18798625

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  18. Protein tyrosine phosphatase PTPN22 +1858C/T polymorphism is associated with active vitiligo

    PubMed Central

    GARCIA-MELENDEZ, MARTHA ELENA; SALINAS-SANTANDER, MAURICIO; SANCHEZ-DOMINGUEZ, CELIA; GONZALEZ-CARDENAS, HUGO; CERDA-FLORES, RICARDO M.; OCAMPO-CANDIANI, JORGE; ORTIZ-LÓPEZ, ROCÍO

    2014-01-01

    Vitiligo is characterized by a skin depigmentation disorder resulting from an autoimmune response targeting melanocytes. Within the genetic factors involved in the development of the vitiligo immune response, various genes in the major histocompatibility complex (MHC) and non-MHC loci have been considered to be risk factors. The PTPN22 gene encodes for a lymphoid protein tyrosine phosphatase, a regulator of the activation and development of T-cells. The +1858C/T polymorphism has been associated to autoimmune disease susceptibility in different populations and could be implicated in the onset of vitiligo. To assess the possible association between the presence of PTPN22 +1858C/T and vitiligo, 187 patients with vitiligo and 223 control subjects were analyzed in the study. Genomic DNA was isolated using the salting-out method and samples were subjected to polymerase chain reaction-restriction fragment length polymorphism in order to detect the PTPN22 +1858C/T polymorphism. Causal associations were determined by χ2 test and their respective odds ratio (OR) was assessed in a 2×2 contingency table. The results showed an association between active vitiligo and the allele T load [P=0.0418; OR, 2.5706; 95% confidence interval (CI), 1.0040–6.5816], and active vitiligo-CT genotype (P=0.0389, OR, 2.6548; 95% CI, 1.0191–6.9156). In conclusion, the present data indicates a possible association between the PTPN22 +1858C/T genotype and a significant susceptibility of developing an active form of vitiligo. PMID:25289035

  19. Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection.

    PubMed

    Sassolas, Audrey; Catanante, Gaëlle; Hayat, Akhtar; Marty, Jean-Louis

    2011-09-30

    Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample. In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L(-1) using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L(-1) and 0.123 μg L(-1) with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC(50) value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L(-1). This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish. PMID:21839207

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

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2015-11-01

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

  2. Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

    PubMed Central

    Phillips, Ryan M.; Dailey, Lisa A.; Bair, Eric; Samet, James M.; Allbritton, Nancy L.

    2014-01-01

    We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min−1 mg−1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min−1 mg−1, and was also effectively inhibited by pre-incubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2- naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min−1 mg−1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34–36 pmol min−1 mg−1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. PMID:24380370

  3. Regulatory protein phosphorylation in Mycoplasma pneumoniae. A PP2C-type phosphatase serves to dephosphorylate HPr(Ser-P).

    PubMed

    Halbedel, Sven; Busse, Julia; Schmidl, Sebastian R; Stülke, Jörg

    2006-09-01

    Among the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr. PMID:16857667

  4. The Streptococcus pyogenes orphan protein tyrosine phosphatase, SP-PTP, possesses dual specificity and essential virulence regulatory functions.

    PubMed

    Kant, Sashi; Agarwal, Shivani; Pancholi, Preeti; Pancholi, Vijay

    2015-08-01

    Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP-PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP-PTP is, therefore, questionable. Here, we demonstrate that SP-PTP possesses dual phosphatase specificity for Tyr- and Ser/Thr-phosphorylated GAS proteins, such as Ser/Thr kinase (SP-STK) and the SP-STK-phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP-PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP-PTP displayed increased Ser-/Thr-/Tyr-phosphorylation. SP-PTP also differentially regulates the expression of ∼50% of the total GAS genes, including several virulence genes potentially through the two-component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP-PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr-phosphorylation in GAS and the relevance of SP-PTP as an important therapeutic target. PMID:25939957

  5. Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography-mass spectrometry.

    PubMed

    Houser, Whitney M; Butterer, Annika; Addepalli, Balasubrahmanym; Limbach, Patrick A

    2015-06-01

    Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2',3'-cyclic phosphates and 3'-linear phosphates. To generate a homogeneous 3'-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3'-phosphate-containing products that could be characterized by LC-MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce (18)O labels within the 3'-phosphate of digestion products when incubated in the presence of H2(18)O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2. PMID:25797349

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

    PubMed Central

    Puustinen, Pietri; Junttila, Melissa R.; Vanhatupa, Sari; Sablina, Anna A.; Hector, Melissa E.; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C.; Westermarck, Jukka

    2010-01-01

    ERK/MAPK pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies, however the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A (PP2A) promotes basal ERK pathway activity, and is required for efficient growth factor response. Mechanistically PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and PKC. In malignant glioblastoma, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (N=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells, and suggest important functional role for PME-1 in the disease progression of human astrocytic gliomas. PMID:19293187

  7. Mechanism of phospho-ubiquitin-induced PARKIN activation.

    PubMed

    Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

    2015-08-20

    The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal-recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy. PINK1 is stabilized on the outside of depolarized mitochondria and phosphorylates polyubiquitin as well as the PARKIN ubiquitin-like (Ubl) domain. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in patients with AR-JP. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilization of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens up new avenues to identify small-molecule PARKIN activators. PMID:26161729

  8. Mechanism of phospho-ubiquitin induced PARKIN activation

    PubMed Central

    Wauer, Tobias; Simicek, Michal; Schubert, Alexander; Komander, David

    2016-01-01

    Summary The E3 ubiquitin ligase PARKIN (encoded by PARK2) and the protein kinase PINK1 (encoded by PARK6) are mutated in autosomal recessive juvenile Parkinsonism (AR-JP) and work together in the disposal of damaged mitochondria by mitophagy1–3. PINK1 is stabilised on the outside of depolarised mitochondria, and phosphorylates poly-ubiquitin (polyUb)4–8 as well as the PARKIN Ub-like (Ubl) domain9,10. These phosphorylation events lead to PARKIN recruitment to mitochondria, and activation by an unknown allosteric mechanism4–12. Here we present the crystal structure of Pediculus humanus PARKIN in complex with Ser65-phosphorylated ubiquitin (phosphoUb), revealing the molecular basis for PARKIN recruitment and activation. The phosphoUb binding site on PARKIN comprises a conserved phosphate pocket and harbours residues mutated in AR-JP patients. PhosphoUb binding leads to straightening of a helix in the RING1 domain, and the resulting conformational changes release the Ubl domain from the PARKIN core; this activates PARKIN. Moreover, phosphoUb-mediated Ubl release enhances Ubl phosphorylation by PINK1, leading to conformational changes within the Ubl domain and stabilisation of an open, active conformation of PARKIN. We redefine the role of the Ubl domain not only as an inhibitory13 but also as an activating element that is restrained in inactive PARKIN and released by phosphoUb. Our work opens new avenues to identify small molecule PARKIN activators. PMID:26161729

  9. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles.

    PubMed

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  10. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    PubMed Central

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  11. Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.

    PubMed

    Naessan, Cecilia L; Egge-Jacobsen, Wolfgang; Heiniger, Ryan W; Wolfgang, Matthew C; Aas, Finn Erik; Røhr, Asmund; Winther-Larsen, Hanne C; Koomey, Michael

    2008-01-01

    The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae. PMID:17951381

  12. Quantitative proteomics reveals protein kinases and phosphatases in the individual phases of contextual fear conditioning in the C57BL/6J mouse.

    PubMed

    Šmidák, Roman; Mayer, Rupert Laurenz; Bileck, Andrea; Gerner, Christopher; Mechtcheriakova, Diana; Stork, Oliver; Lubec, Gert; Li, Lin

    2016-04-15

    A series of protein kinases and phosphatases (PKPs) have been linked to contextual fear conditioning (cFC) but information is mainly derived from immunochemical studies. It was therefore decided to use an explorative label-free quantitative proteomics approach to concomitantly determine PKPs in hippocampi of mice in the individual phases of cFC. C57BL/6J mice were divided into four groups: three training groups representing the acquisition, consolidation and retrieval phases of cFC and a foot shock control group. Using this approach we identified 32 protein kinases or phosphatases/phosphatase subunits with significantly changed protein levels in one or more training groups as compared to foot shock control. These include members of PKP signalling modules of mitogen-activated protein kinase (MAP3K10, RAF1, KSR2), Ca2+/calmodulin-dependent protein kinase (CaMKIIα, DAPK1), protein kinase C (PRKCD) and protein phosphatases 1, 2A, 2B(3) previously implicated in various learning paradigms. In addition, our analysis showed protein kinases WNK1, LYN, VRK1, ABL1, CDK4, CDKL3, SgK223 and ADCK1, and protein phosphatases PTPRF, ACP1, DNAJC6, SSH2 and UBASH3B that have not been directly linked to fear memory processes so far. Determination of PKPs in the individual cFC phases represents a valuable resource for interpretation of previous and design of future studies on PKPs in memory mechanisms. PMID:26748257

  13. NMR structure of the forkhead-associated domain from the Arabidopsis receptor kinase-associated protein phosphatase

    PubMed Central

    Lee, Gui-in; Ding, Zhaofeng; Walker, John C.; Van Doren, Steven R.

    2003-01-01

    Forkhead-associated (FHA) domains are phosphoprotein-binding modules found in diverse signaling proteins that bind partners phosphorylated on threonine or serine. Kinase-associated protein phosphatase from Arabidopsis employs its FHA domain for negative regulation of receptor-like kinase signaling pathways, which are important in plant development. The solution structure of the free state of kinase-interacting FHA domain (KI-FHA) of kinase-associated protein phosphatase has been determined with high precision and accuracy using residual dipolar couplings. KI-FHA is a sandwich of a five-stranded mixed β-sheet with a six-stranded antiparallel β-sheet. Despite homology only in the recognition loops, this fold is shared with FHA domains from checkpoint proteins from yeast and humans, as well as with nonhomologous MH2 domains of Smad tumor suppressors. A shared pattern of hydrophobicity throughout FHA domains and Smad MH2 domains may stabilize the core of the β-sandwich. Evolutionary trace analysis of FHA domains suggests class-specific residues in the recognition loops that could tune their phosphoprotein-binding specificity. This surface agrees with that of KI-FHA in contact with a phosphothreonine peptide ligand. Evolutionary trace analysis also predicts an unexpected swath of class-specific residues on another face of FHA domains. Protein interactions with these faces may affect assembly of transmembrane signaling complexes in plants, and in other FHA domain-containing assemblies. PMID:14500786

  14. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

    PubMed

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-08-01

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity. PMID:26195794

  15. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium

    PubMed Central

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-01-01

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer–associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10–deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1–deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity. PMID:26195794

  16. Novel Ser/Thr Protein Phosphatase 5 (PP5) Regulated Targets during DNA Damage Identified by Proteomics Analysis

    SciTech Connect

    Ham, Bryan M.; Jayachandran, Hemalatha; Yang, Feng; Jaitly, Navdeep; Polpitiya, Ashoka D.; Monroe, Matthew E.; Wang, Ling; Zhao, Rui; Purvine, Samuel O.; Livesay, Eric A.; Camp, David G.; Rossie, Sandra S.; Smith, Richard D.

    2010-02-05

    The DNA damage response is a global phosphorylation signaling cascade process involved in sensing the damaged DNA condition and coordinating responses to cope with and repair the perturbed cellular state. We utilized a label-free liquid chromatography-mass spectrometry approach to evaluate changes in protein phosphorylation associated with PP5 activity during the DNA damage response. Biological replicate analyses of bleomycin-treated HeLa cells expressing either WT-PP5 or mutant inactive PP5 lead to the identification of six potential target proteins of PP5 action. Four of these putative targets are known to be involved in DNA damage responses. Using phospho-site specific antibodies, we confirmed that phosphorylation of one target, ribosomal protein S6, was selectively decreased in cells overexpressing catalytically inactive PP5. Our findings also suggest that PP5 may play a role in controlling translation and in regulating substrates for proline-directed kinases, such as MAP kinases and cyclin-dependent protein kinases that are involved in response to DNA damage.

  17. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  18. Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components

    PubMed Central

    Wynne, David J.

    2015-01-01

    It is widely accepted that the kinetochore is built on CENP-A–marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A–free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment. PMID:26347137

  19. Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components.

    PubMed

    Wynne, David J; Funabiki, Hironori

    2015-09-14

    It is widely accepted that the kinetochore is built on CENP-A-marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A-free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment. PMID:26347137

  20. Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1.

    PubMed

    Abbott, Karen L; Archambault, Jacques; Xiao, Hua; Nguyen, Bao D; Roeder, Robert G; Greenblatt, Jack; Omichinski, James G; Legault, Pascale

    2005-03-01

    FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HIV-1 Tat protein, CK2, TFIIB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1(562)(-)(738)). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCP1 are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74(436)(-)(517) interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif. HIV-1 Tat inhibits the binding of RAP74(436)(-)(517) to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1(562)(-)(619) by CK2 at this site increases binding to RAP74(436)(-)(517), but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions. PMID:15723517

  1. Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

    PubMed Central

    Sato, Hiroki; Yoneda, Misako; Honma, Reiko; Ikeda, Fusako; Watanabe, Shinya

    2015-01-01

    ABSTRACT Measles virus (MeV) causes several unique syndromes, including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase, which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions. IMPORTANCE Measles virus (MeV) is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by cellular protein phosphatase 5 (PP5) and DNA-dependent protein kinase. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV. PMID:26157124

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

    SciTech Connect

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

    2007-01-01

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

  3. The protein tyrosine phosphatase DEP-1/PTPRJ promotes breast cancer cell invasion and metastasis.

    PubMed

    Spring, K; Fournier, P; Lapointe, L; Chabot, C; Roussy, J; Pommey, S; Stagg, J; Royal, I

    2015-10-29

    DEP-1/PTPRJ is a receptor-like protein tyrosine phosphatase mainly known for its antiproliferative and tumor-suppressive functions. Many identified substrates are growth factor receptors, and DEP-1 is deleted and/or mutated in human cancers including that of the breast. However, DEP-1 was also identified as a promoter of Src activation and proinvasive functions in the endothelium, suggesting it could perhaps mediate breast cancer invasiveness that is likewise driven by Src family kinases. We show here that DEP-1 expression was greater in highly invasive breast cancer cells (MDA-MB-231, Hs578T, BT-549) than in the less invasive or untransformed cell lines tested (MCF-7, T47D, SK-BR3 and MCF10A). DEP-1 silencing experiments in invasive cells demonstrated that moderately expressed and catalytically active DEP-1 was required, in collaboration with basal epidermal growth factor receptor activity, for Src activation and the phosphorylation of its substrate Cortactin, and for their colocalization at the cell's leading edge. This correlated with an increased number of cell protrusions, and an enhanced capacity of the cells to migrate and invade. Similarly, moderate overexpression of DEP-1 in the low-invasive cells resulted in the promotion of their invasiveness in an Src-dependent manner. Consistent with these data, the expression of endogenous DEP-1 was elevated in a bone metastatic cell line derived from MDA-MB-231 cells, and promoted increased Src Y418 and Cortactin Y421 phosphorylation, as well as pro-MMP9 secretion and Matrigel invasion. Importantly, the silencing of DEP-1 in MDA-MB-231 cells greatly decreased their ability to metastasize, despite having no effect on tumor growth or angiogenesis. Hence, we found that moderate expression of DEP-1 was associated with the increased relapse and decreased survival of breast cancer patients. These results therefore identify a new and unsuspected role for DEP-1 as a mediator of an invasive cell program implicating Src

  4. Archaeal Signal Transduction: Impact of Protein Phosphatase Deletions on Cell Size, Motility, and Energy Metabolism in Sulfolobus acidocaldarius*

    PubMed Central

    Reimann, Julia; Esser, Dominik; Orell, Alvaro; Amman, Fabian; Pham, Trong Khoa; Noirel, Josselin; Lindås, Ann-Christin; Bernander, Rolf; Wright, Phillip C.; Siebers, Bettina; Albers, Sonja-Verena

    2013-01-01

    In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius. PMID:24078887

  5. Negative regulation of HER2 signaling by the PEST-type protein-tyrosine phosphatase BDP1.

    PubMed

    Gensler, Miriam; Buschbeck, Marcus; Ullrich, Axel

    2004-03-26

    Signaling by receptor tyrosine kinases (RTK) mediates a variety of complex cellular functions and in case of deregulation can contribute to pathophysiological processes. A tight and finely tuned control of RTK activity is therefore critical for the cell. We investigated the role of the PEST-type protein-tyrosine phosphatase BDP1 in the regulation of HER2, a member of the epidermal growth factor receptor (EGFR) family of RTKs. Here we demonstrate that HER2 signaling is highly sensitive to BDP1 activity. Overexpression of BDP1 inhibited ligand-induced activation of HER2 but not that of the closely related EGFR. On the other hand, suppression of endogenous BDP1 expression increased the phosphorylation state of HER2. In addition, BDP1 was able to interfere with downstream signaling events by inhibiting the phosphorylation of the adaptor protein Gab1 and reducing mitogen-activated protein kinase activation. Supported by the finding that BDP1 is coexpressed with HER2 in breast cancer cells, we suggest that BDP1 is an important regulator of HER2 activity and thus the first protein-tyrosine phosphatase shown to be involved in HER2 signal attenuation. PMID:14660651

  6. Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection.

    PubMed

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-04-13

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating

  7. Phosphatidylinositol 5-Phosphatase Oculocerebrorenal Syndrome of Lowe Protein (OCRL) Controls Actin Dynamics during Early Steps of Listeria monocytogenes Infection*

    PubMed Central

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-01-01

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P2) and trisphosphate (PI[3,4,5]P3) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5′-phosphatase OCRL dephosphorylates PI(4,5)P2 and to a lesser extent PI(3,4,5)P3, we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P2, and PI(3,4,5)P3 levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin

  8. Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    SciTech Connect

    Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki; Sugiyama, Yasunori; Numano, Takamasa; Shigeri, Yasushi; Taniguchi, Takanobu; Kameshita, Isamu Ishida, Atsuhiko

    2007-11-23

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.

  9. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    PubMed

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

  10. Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

    PubMed Central

    Gupta, Vandana A.; Hnia, Karim; Smith, Laura L.; Gundry, Stacey R.; McIntire, Jessica E.; Shimazu, Junko; Bass, Jessica R.; Talbot, Ethan A.; Amoasii, Leonela; Goldman, Nathaniel E.; Laporte, Jocelyn; Beggs, Alan H.

    2013-01-01

    X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., “MTMRs”). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of

  11. Organization and alternate splice products of the gene encoding nuclear inhibitor of protein phosphatase-1 (NIPP-1).

    PubMed

    Van Eynde, A; Pérez-Callejón, E; Schoenmakers, E; Jacquemin, M; Stalmans, W; Bollen, M

    1999-04-01

    Nuclear inhibitor of protein phosphatase-1 (NIPP-1) is one of two major regulatory subunits of protein phosphatase-1 in mammalian nuclei. We report here the cloning and structural characterization of the human NIPP-1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature. PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situ hybridization to chromosome 1p33-32. PPP1R8 is an authentic NIPP-1 gene and was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed of seven exons and encodes four different transcripts, as determined from cDNA library screening, reverse transcriptase-PCR (RT-PCR) and/or EST (expressed sequence tag) database search analysis. NIPP-1alpha mRNA represents the major transcript in human tissues and various cell lines, and encodes a polypeptide of 351 residues that only differs from the previously cloned calf thymus NIPP-1 by a single residue. The other transcripts, termed NIPP-1beta, gamma and delta, are generated by alternative 5'-splice site usage, by exon skipping and/or by alternative polyadenylation. The NIPP-1beta/delta and NIPP-1gamma mRNAs are expected to encode fragments of NIPP-1alpha that differ from the latter by the absence of the first 142 and 224 residues, respectively. NIPP-1gamma corresponds to 'activator of RNA decay-1' (Ard-1) which, unlike NIPP-1alpha, displays in vitro and endoribonuclease activity and lacks an RVXF consensus motif for interaction with protein phosphatase-1. While the NIPP-1alpha/beta/delta-transcripts were found to be present in various human tissues, the NIPP-1gamma transcript could only be detected in human transformed B-lymphocytes. PMID:10103062

  12. Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

    SciTech Connect

    Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha; Lou, Quanzhou; Monroe, Matthew E.; Du, Xiuxia; Gritsenko, Marina A.; Zhang, Rui; Anderson, David J.; Purvine, Samuel O.; Adkins, Joshua N.; Moore, Ronald J.; Mottaz, Heather M.; Ding, Shi-Jian; Lipton, Mary S.; Camp, David G.; Udseth, Harold R.; Smith, Richard D.; Rossie, Sandra S.

    2007-10-12

    To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.

  13. The Biological Buffer, Bicarbonate/CO2, Potentiates H2O2-Mediated Inactivation of Protein Tyrosine Phosphatases

    PubMed Central

    Zhou, Haiying; Singh, Harkewal; Parsons, Zachary D.; Lewis, Sarah M.; Bhattacharya, Sanjib; Seiner, Derrick R.; LaButti, Jason N.; Reilly, Thomas J.; Tanner, John J.; Gates, Kent S.

    2012-01-01

    Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H2O2-mediated cellular signal transduction processes but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer, bicarbonate/CO2, potentiates the ability of H2O2 to inactivate PTPs. The results of biochemical experiments and high resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H2O2 with HCO3 −/CO2. PMID:21913686

  14. New Hippolide Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activity from the Marine Sponge Hippospongia lachne

    PubMed Central

    Piao, Shu-Juan; Jiao, Wei-Hua; Yang, Fan; Yi, Yang-Hua; Di, Ying-Tong; Han, Bing-Nan; Lin, Hou-Wen

    2014-01-01

    Five new sesterterpenoids, compounds 1–5, have been isolated from the sponge Hippospongia lachne off Yongxing Island in the South China Sea. The structures of compounds 1–5 were elucidated through extensive spectroscopic analysis, including HRMS, 1D, and 2D NMR experiments. The stereochemistry, including absolute configurations of these compounds, was determined by spectroscopic, chemical, and computational methods. Compounds 1 and 5 showed moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activities with IC50 values of 5.2 μM and 8.7 μM, respectively, more potent than previously reported hippolides. PMID:25007159

  15. MicroRNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

    PubMed Central

    Sheng, Wei-Zhong; Chen, Yu-Sheng; Tu, Chuan-Tao; He, Juan; Zhang, Bo; Gao, Wei-Dong

    2016-01-01

    AIM: To explore the regulatory mechanism of the target gene of microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells. METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated. RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05). CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC. PMID:27350731

  16. Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

    PubMed Central

    Reyes, Gloria; Niederst, Matt; Cohen-Katsenelson, Ksenya; Stender, Joshua D.; Kunkel, Maya T.; Chen, Muhan; Brognard, John; Sierecki, Emma; Gao, Tianyan; Nowak, Dawid G.; Trotman, Lloyd C.; Glass, Christopher K.; Newton, Alexandra C.

    2014-01-01

    Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs. PMID:25201979

  17. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

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

    PubMed

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

    2015-01-01

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

  19. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    PubMed

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods. PMID:15797237

  20. Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum.

    PubMed

    Valjent, Emmanuel; Pascoli, Vincent; Svenningsson, Per; Paul, Surojit; Enslen, Hervé; Corvol, Jean-Christophe; Stipanovich, Alexandre; Caboche, Jocelyne; Lombroso, Paul J; Nairn, Angus C; Greengard, Paul; Hervé, Denis; Girault, Jean-Antoine

    2005-01-11

    Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d-amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d-amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Delta(9)-tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32). The effects of d-amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse. PMID:15608059

  1. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library.

    PubMed

    Huang, Ziliang; Li, Gang; Zhang, Chong; Xing, Xin-Hui

    2016-02-01

    Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes. PMID:26777244

  2. The role of serine/threonine protein phosphatase type 5 (PP5) in the regulation of stress-induced signaling networks and cancer.

    PubMed

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

    2008-06-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-09-01

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

  5. The neural receptor protein tyrosine phosphatase DPTP69D is required during periods of axon outgrowth in Drosophila.

    PubMed Central

    Desai, Chand; Purdy, Joy

    2003-01-01

    We have isolated and characterized a series of 18 chemically induced alleles of Ptp69D ranging in strength from viable to worse than null, which represent unique tools for probing the structure, function, and signaling pathway of DPTP69D. Three alleles are strongly temperature sensitive and were used to define the developmental periods requiring DPTP69D function; adult health requires DPTP69D during the mid- to late-pupal stage, eclosion requires DPTP69D during the early to mid-larval stage, and larval survival requires DPTP69D during embryogenesis. Mutations predicted to abolish the phosphatase activity of the membrane proximal D1 domain severely reduce but do not abolish DPTP69D function. Six alleles appear null; only 20% of null homozygotes pupate and <5% eclose, only to fall into the food and drown. One allele, Ptp69D(7), confers axon and viability defects more severe than those of the null phenotype. Sequence analysis predicts that Ptp69D(7) encodes a mutant protein that may bind but not release substrate. Like mutations in the protein tyrosine phosphatase gene Dlar, strong Ptp69D alleles cause the ISNb nerve to bypass its muscle targets. Genetic analysis reveals that the bypass defect in Dlar and Ptp69D mutants is dependent upon DPTP99A function, consistent with the hypothesis that DPTP69D and DLAR both counteract DPTP99A, allowing ISNb axons to enter their target muscle field. PMID:12807778

  6. Loss of the Protein Tyrosine Phosphatase PTPN22 Reduces Mannan-Induced Autoimmune Arthritis in SKG Mice

    PubMed Central

    Sood, Shatakshi; Brownlie, Rebecca J.; Garcia, Celine; Cowan, Graeme; Salmond, Robert J.; Sakaguchi, Shimon

    2016-01-01

    The cytoplasmic phosphatase, protein tyrosine phosphatase nonreceptor type 22 (PTPN22), is a negative regulator of T cell signaling. Genome-wide association studies have shown that single-nucleotide polymorphisms in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how the variant protein contributes to autoimmunity is not well understood. To address this issue, we investigated the effect of PTPN22 deficiency on disease susceptibility in a mouse model of autoimmune arthritis. The SKG mouse expresses a hypomorphic mutant allele of ZAP70, which, upon exposure to fungal Ags, predisposes the mice to a CD4+ T cell–mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. Surprisingly, SKG Ptpn22−/− mice developed less severe mannan-induced arthritis compared with SKG mice. Diminution of disease was not due to significant alterations in thymocyte development or repertoire selection in SKG Ptpn22−/− mice, even though T cell–mediated signal transduction was improved. Instead, Ptpn22 deficiency appeared to bias CD4 Th cell differentiation away from the Th17 lineage, which is pathogenic in this setting, to a more Th1/T regulatory–focused response. These data show that even small perturbations in TCR signal transduction pathways can have profound consequences on the differentiation of T cell lineages and thus for the development of autoimmune diseases. PMID:27288531

  7. 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.

  8. Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

    SciTech Connect

    Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.; Ulrich, Robert G.; Burke, Terrence R. Jr Waugh, David S.

    2011-07-01

    The first X-ray crystal structure of the Y. pestis protein tyrosine phosphatase YopH in complex with an isothiazolidinone-based lead-fragment compound is reported. Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors.

  9. Domains involved in calcineurin phosphatase inhibition and nuclear localisation in the African swine fever virus A238L protein

    SciTech Connect

    Abrams, Charles C.; Chapman, Dave A.G.; Silk, Rhiannon; Liverani, Elisabetta; Dixon, Linda K.

    2008-05-10

    The African swine fever virus A238L protein inhibits calcineurin phosphatase activity and activation of NF-{kappa}B and p300 co-activator. An 82 amino acid domain containing residues 157 to 238 at the C-terminus of A238L was expressed in E. coli and purified. This purified A238L fragment acted as a potent inhibitor of calcineurin phosphatase in vitro with an IC{sub 50} of approximately 70 nM. Two putative nuclear localisation signals were identified between residues 80 to 86 (NLS-1) and between residues 203 to 207 overlapping with the N-terminus of the calcineurin docking motif (NLS-2). Mutation of these motifs independently did not reduce nuclear localisation compared to the wild type A238L protein, whereas mutation of both motifs significantly reduced nuclear localisation of A238L. Mutation of the calcineurin docking motif resulted in a dramatic increase in the nuclear localisation of A238L provided an intact NLS was present. We propose that binding of calcineurin to A238L masks NLS-2 contributing to the cytoplasmic retention of A238L.

  10. Loss of the Protein Tyrosine Phosphatase PTPN22 Reduces Mannan-Induced Autoimmune Arthritis in SKG Mice.

    PubMed

    Sood, Shatakshi; Brownlie, Rebecca J; Garcia, Celine; Cowan, Graeme; Salmond, Robert J; Sakaguchi, Shimon; Zamoyska, Rose

    2016-07-15

    The cytoplasmic phosphatase, protein tyrosine phosphatase nonreceptor type 22 (PTPN22), is a negative regulator of T cell signaling. Genome-wide association studies have shown that single-nucleotide polymorphisms in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how the variant protein contributes to autoimmunity is not well understood. To address this issue, we investigated the effect of PTPN22 deficiency on disease susceptibility in a mouse model of autoimmune arthritis. The SKG mouse expresses a hypomorphic mutant allele of ZAP70, which, upon exposure to fungal Ags, predisposes the mice to a CD4(+) T cell-mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. Surprisingly, SKG Ptpn22(-/-) mice developed less severe mannan-induced arthritis compared with SKG mice. Diminution of disease was not due to significant alterations in thymocyte development or repertoire selection in SKG Ptpn22(-/-) mice, even though T cell-mediated signal transduction was improved. Instead, Ptpn22 deficiency appeared to bias CD4 Th cell differentiation away from the Th17 lineage, which is pathogenic in this setting, to a more Th1/T regulatory-focused response. These data show that even small perturbations in TCR signal transduction pathways can have profound consequences on the differentiation of T cell lineages and thus for the development of autoimmune diseases. PMID:27288531

  11. Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

    PubMed

    Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

    2015-05-01

    Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness. PMID:25672645

  12. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors

    PubMed Central

    Bachovchin, Daniel A.; Mohr, Justin T.; Speers, Anna E.; Wang, Chu; Berlin, Jacob M.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S.; Schürer, Stephan C.; Nomura, Daniel K.; Rosen, Hugh; Fu, Gregory C.; Cravatt, Benjamin F.

    2011-01-01

    National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets. PMID:21398589

  13. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.

    PubMed

    Bachovchin, Daniel A; Mohr, Justin T; Speers, Anna E; Wang, Chu; Berlin, Jacob M; Spicer, Timothy P; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S; Schürer, Stephan C; Nomura, Daniel K; Rosen, Hugh; Fu, Gregory C; Cravatt, Benjamin F

    2011-04-26

    National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets. PMID:21398589

  14. Molecular Investigations of the Structure and Function of the Protein Phosphatase 1-Spinophilin-Inhibitor 2 Heterotrimeric Complex

    SciTech Connect

    Dancheck, B.; Allaire, M.; Ragusa, M.J.; Nairn, A.C.; Page, R.; Peti, W.

    2011-01-06

    Regulation of the major Ser/Thr phosphatase protein phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry, and small-angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and inhibitor 2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1-spinophilin-I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1-spinophilin and PP1-I-2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1-I-2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multifunctional PP1 holoenzymes.

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

    PubMed Central

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

    1996-01-01

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

  16. Identification of a regulatory subunit of protein phosphatase 1 which mediates blue light signaling for stomatal opening.

    PubMed

    Takemiya, Atsushi; Yamauchi, Shota; Yano, Takayuki; Ariyoshi, Chie; Shimazaki, Ken-ichiro

    2013-01-01

    Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase comprised of a catalytic subunit (PP1c) and a regulatory subunit that modulates catalytic activity, subcellular localization and substrate specificity. PP1c positively regulates stomatal opening through blue light signaling between phototropins and the plasma membrane H(+)-ATPase in guard cells. However, the regulatory subunit functioning in this process is unknown. We identified Arabidopsis PRSL1 (PP1 regulatory subunit2-like protein1) as a regulatory subunit of PP1c. Tautomycin, a selective inhibitor of PP1c, inhibited blue light responses of stomata in the single mutants phot1 and phot2, supporting the idea that signals from phot1 and phot2 converge on PP1c. We obtained PRSL1 based on the sequence similarity to Vicia faba PRS2, a PP1c-binding protein isolated by a yeast two-hybrid screen. PRSL1 bound to Arabidopsis PP1c through its RVxF motif, a consensus PP1c-binding sequence. Arabidopsis prsl1 mutants were impaired in blue light-dependent stomatal opening, H(+) pumping and phosphorylation of the H(+)-ATPase, but showed normal phototropin activities. PRSL1 complemented the prsl1 phenotype, but not if the protein carried a mutation in the RVxF motif, suggesting that PRSL1 functions through binding PP1c via the RVxF motif. PRSL1 did not affect the catalytic activity of Arabidopsis PP1c but it stimulated the localization of PP1c in the cytoplasm. We conclude that PRSL1 functions as a regulatory subunit of PP1 and regulates blue light signaling in stomata. PMID:22585556

  17. Molecular Investigations of the Structure and Function of the Protein Phosphatase 1:Spinophilin:Inhibitor-2 Heterotrimeric Complex

    PubMed Central

    Dancheck, Barbara; Ragusa, Michael J.; Allaire, Marc; Nairn, Angus C.; Page, Rebecca; Peti, Wolfgang

    2011-01-01

    Regulation of the major ser/thr phosphatase Protein Phosphatase 1 (PP1) is controlled by a diverse array of targeting and inhibitor proteins. Though many PP1 regulatory proteins share at least one PP1 binding motif, usually the RVxF motif, it was recently discovered that certain pairs of targeting and inhibitor proteins bind PP1 simultaneously to form PP1 heterotrimeric complexes. To date, structural information for these heterotrimeric complexes, and, in turn, how they direct PP1 activity is entirely lacking. Using a combination of NMR spectroscopy, biochemistry and small angle X-ray scattering (SAXS), we show that major structural rearrangements in both spinophilin (targeting) and Inhibitor-2 (I-2, inhibitor) are essential for the formation of the heterotrimeric PP1:spinophilin:I-2 (PSI) complex. The RVxF motif of I-2 is released from PP1 during the formation of PSI, making the less prevalent SILK motif of I-2 essential for complex stability. The release of the I-2 RVxF motif allows for enhanced flexibility of both I-2 and spinophilin in the heterotrimeric complex. In addition, we used inductively coupled plasma atomic emission spectroscopy to show that PP1 contains two metals in both heterodimeric complexes (PP1:spinophilin and PP1:I2) and PSI, demonstrating that PSI retains the biochemical characteristics of the PP1:I2 holoenzyme. Finally, we combined the NMR and biochemical data with SAXS and molecular dynamics simulations to generate a structural model of the full heterotrimeric PSI complex. Collectively, these data reveal the molecular events that enable PP1 heterotrimeric complexes to exploit both the targeting and inhibitory features of the PP1-regulatory proteins to form multi-functional PP1 holoenzymes. PMID:21218781

  18. Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor.

    PubMed Central

    Chinkers, M

    1994-01-01

    Protein kinase-related domains of unknown function are present in the JAK family of protein tyrosine kinases and in receptor/guanylyl cyclases. I used the yeast two-hybrid system to screen for proteins interacting with the kinase-like domain of the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase. A yeast strain was constructed expressing a fusion of this kinase-like domain to the lexA DNA-binding domain and containing a HIS3 gene under the control of lexA upstream activating sequences. These yeast cells were transformed with a plasmid library of mouse embryo cDNA fragments fused to the VP16 transcriptional activation domain. Cells containing VP16-fusion proteins interacting with the lexA-kinase-like domain fusion protein were selected by growth in the absence of histidine. A partial-length cDNA clone isolated by using this approach encoded a protein that interacted specifically with the ANP-receptor protein kinase-like domain both in yeast cells and in vitro. Tissue-specific expression of a 2.2-kb mRNA hybridizing to this cDNA paralleled the known pattern of ANP-receptor mRNA expression. A full-length cDNA clone isolated from a rat lung library was predicted to encode a 55-kDa protein containing at its amino terminus a targeting domain that binds to the ANP-receptor kinase-like domain and containing at its carboxyl terminus a putative protein-serine phosphatase domain. This protein is a possible candidate for the phosphatase involved in desensitizing the ANP receptor. Targeting of regulatory proteins may be an important function of protein kinase-like domains. Images PMID:7972012

  19. Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

    PubMed

    Trappanese, Danielle M; Sivilich, Sarah; Ets, Hillevi K; Kako, Farah; Autieri, Michael V; Moreland, Robert S

    2016-06-01

    Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1. PMID:27053523

  20. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    PubMed

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

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

    SciTech Connect

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

    1986-05-01

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

  2. The anti-inflammatory compound BAY 11-7082 is a potent inhibitor of Protein Tyrosine Phosphatases

    PubMed Central

    Krishnan, Navasona; Bencze, Gyula; Cohen, Philip; Tonks, Nicholas K.

    2013-01-01

    Summary The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signalling events, but also may have therapeutic implications. BAY 11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY 11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY 11-7082 inactivated PTPs by forming a covalent adduct with the active site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY 11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY 11-7082 that have been reported to date. PMID:23578302

  3. The anti-inflammatory compound BAY-11-7082 is a potent inhibitor of protein tyrosine phosphatases.

    PubMed

    Krishnan, Navasona; Bencze, Gyula; Cohen, Philip; Tonks, Nicholas K

    2013-06-01

    The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date. PMID:23578302

  4. The 1.35 A resolution structure of the phosphatase domain of the suppressor of T cell receptor signaling protein in complex with sulfate

    SciTech Connect

    Jakoncic, J.; Sondgeroth, B.; Carpino, N.; Nassar, N.

    2010-04-19

    The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis-Menten constants at acidic pH.

  5. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    NASA Technical Reports Server (NTRS)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

  6. Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

    PubMed Central

    2011-01-01

    Background The mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS). P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely. Methods We have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro. Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3). In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR. This study is supported by the Declaration of Helsinki and subsequent ethical guidelines. Results The combination of these techniques allowed for: (1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer

  7. Dual-specificity phosphatase 26 (DUSP26) stimulates Aβ42 generation by promoting amyloid precursor protein axonal transport during hypoxia.

    PubMed

    Jung, Sunmin; Nah, Jihoon; Han, Jonghee; Choi, Seon-Guk; Kim, Hyunjoo; Park, Jaesang; Pyo, Ha-Kyung; Jung, Yong-Keun

    2016-06-01

    Amyloid beta peptide (Aβ) is a pathological hallmark of Alzheimer's disease (AD) and is generated through the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Hypoxia is a known risk factor for AD and stimulates Aβ generation by γ-secretase; however, the underlying mechanisms remain unclear. In this study, we showed that dual-specificity phosphatase 26 (DUSP26) regulates Aβ generation through changes in subcellular localization of the γ-secretase complex and its substrate C99 under hypoxic conditions. DUSP26 was identified as a novel γ-secretase regulator from a genome-wide functional screen using a cDNA expression library. The phosphatase activity of DUSP26 was required for the increase in Aβ42 generation through γ-secretase, but this regulation did not affect the amount of the γ-secretase complex. Interestingly, DUSP26 induced the accumulation of C99 in the axons by stimulating anterograde transport of C99-positive vesicles. Additionally, DUSP26 induced c-Jun N-terminal kinase (JNK) activation for APP processing and axonal transport of C99. Under hypoxic conditions, DUSP26 expression levels were elevated together with JNK activation, and treatment with JNK inhibitor SP600125, or the DUSP26 inhibitor NSC-87877, reduced hypoxia-induced Aβ generation by diminishing vesicle trafficking of C99 to the axons. Finally, we observed enhanced DUSP26 expression and JNK activation in the hippocampus of AD patients. Our results suggest that DUSP26 mediates hypoxia-induced Aβ generation through JNK activation, revealing a new regulator of γ-secretase-mediated APP processing under hypoxic conditions. We propose the role of phosphatase dual-specificity phosphatase 26 (DUSP26) in the selective regulation of Aβ42 production in neuronal cells under hypoxic stress. Induction of DUSP26 causes JNK-dependent shift in the subcellular localization of γ-secretase and C99 from the cell body to axons for Aβ42 generation. These findings provide a

  8. The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

    PubMed

    Shree, Sonal; Singh, Abhishek Kumar; Saxena, Richa; Kumar, Harish; Agarwal, Aparna; Sharma, Vijay Kumar; Srivastava, Kanchan; Srivastava, Kishore Kumar; Sanyal, Sabyasachi; Ramachandran, Ravishankar

    2016-09-01

    Mycobacterium tuberculosis codes for a HAD-phosphatase, Rv3042c (MtSerB2), tha